United States
Environmental Protection
Agency
EPA 6CX 8-82 OObT
Jtilv 19fa
F null Re}' it
Research and Development
vvEPA
Health Assessment Final
Document for Report
Trichloroethylene
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EPA/600/8-82/006F
July 1985
Final Report
Health Assessment Document
for
Trichloroethylene
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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NOTICE
This document has been reviewed in accordance with the U.S. Environmental
Protection Agency's peer and administrative review policies and approved for
presentation and publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
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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 is placed in perspective with observed environmental levels.
Any information regarding sources, emissions, ambient air concentrations,
and public exposure has been included only to give the reader a preliminary
indication of the potential presence of this substance in the ambient air.
While the available information is presented as accurately as possible, it is
acknowledged to be limited and dependent in some instances on assumption
rather than specific data. This information is not intended, nor should it be
used, to support any conclusions regarding risks to public health.
If a review of the health information indicates that the Agency should
consider regulatory action for this substance, a considerable effort will be
undertaken to obtain appropriate information regarding sources, emissions, and
ambient air concentrations. Such data will provide additional information for
drawing regulatory conclusions regarding the extent and significance of public
exposure to this substance.
iii
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CONTENTS
LIST OF TABLES vii
LIST OF FIGURES x
AUTHORS AND REVIEWERS x1i
1. EXECUTIVE SUMMARY 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 3-24
3.5 CRITERIA, STANDARDS, AND REGULATIONS 3-26
3.6 REFERENCES 3-27
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 4-5
4.1.4 Tissue Distributions and Concentrations 4-10
4.2 EXCRETION 4-14
4.2.1 Pulmonary Elimination in Man 4-14
4.2.2 Urinary Metabolite Excretion in Man 4-17
4.2.3 Excretion Kinetics in the Rodent 4-18
4.3 MEASURES OF EXPOSURE AND BODY BURDEN 4-23
4.4 METABOLISM 4-26
4.4.1 Known Metabolites 4-26
4.4.2 Magnitude of TCI Metabolism: Evidence of Dose-
Dependent Metabol ism 4-29
4.4.3 Enzyme Pathways of Biotransformation 4-39
4.4.4 Metabolism and Covalent Binding 4-48
4.4.5 TCI Metabolism: Drug and Other Interactions 4-53
4.4.6 Metabolism and Cellular Toxicity 4-58
4.5 REFERENCES 4-63
iv
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CONTENTS (continued)
5. TOXICOLOGICAL EFFECTS IN MAN AND EXPERIMENTAL ANIMALS 5-1
5.1 INTRODUCTION 5-1
5.2 NEURAL AND BEHAVIORAL EFFECTS 5-1
5.2.1 Human Studies 5-1
5.2.2 Laboratory Animals 5-5
5.3 EFFECTS ON THE CARDIOVASCULAR AND RESPIRATORY SYSTEMS ... 5-10
5.3.1 Cardiovascular Effects 5-10
5.3.2 Respiratory Effects 5-13
5.4 HEPATIC AND RENAL TOXICITY 5-14
5.4.1 Human Studies 5-14
5.4.2 Animal Studies 5-15
5.5 IMMUNOLOGICAL AND HEMATOLOGICAL EFFECTS 5-21
5.6 DERMAL EFFECTS 5-23
5.7 MODIFICATION OF TOXICITY: SYNERGISM AND ANTAGONISM 5-23
5.8 SUMMARY OF TOXICOLOGICAL EFFECTS 5-27
5.9 REFERENCES 5-28
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 6-12
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 7-9
7.2 CHROMOSOME ABERRATION STUDIES 7-20
7.2.1 Experimental Animals 7-21
7.2.2 Humans 7-24
7.3 OTHER STUDIES INDICATIVE OF MUTAGENIC ACTIVITY 7-26
7.3.1 Gene Conversion in Yeast 7-26
7.3.2 Sister Chromatid Exchange (SCE) Formation 7-27
7.3.3 Unscheduled DNA Synthesis (UDS) 7-29
7.4 EVIDENCE THAT TCI REACHES THE GONADS 7-33
7.5 MUTAGENICITY OF METABOLITES 7-34
7.6 BINDING TO DNA 7-36
7.7 SUMMARY AND CONCLUSIONS 7-36
7.8 REFERENCES 7-44
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CONTENTS (continued)
8. CARCINOGENICITY 8-1
8.1 DESCRIPTION AND ANALYSIS OF 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 8-48
8.1.6 Other Carcinogenicity Evaluations of TCI 8-55
8.1.7 TCI Oxide 8-55
8.1.8 Cell Transformation Studies 8-61
8.1.9 Summary of Animal and Cell Transformation
Studies 8-69
8.2 DESCRIPTION AND ANALYSIS OF EPIDEMIOLOGIC STUDIES 8-72
8.2.1 Axel son et al. (1978) 8-72
8.2.2 Tola et al. (1980) 8-75
8.2.3 Malek et al. (1979) 8-78
8.2.4 Novotna et al. (1979) 8-78
8.2.5 Hardel 1 et al. (1981) 8-79
8.2.6 Paddle (1983) 8-80
8.3 RISK ESTIMATES FROM ANIMAL DATA 8-81
8.3.1 Selection of Animal Data Sets 8-82
8.3.2 Interspecies Dose Conversion 8-84
8.3.3 Choice of Risk Model 8-108
8.3.4 Calculation of Unit Risk 8-115
8.4 RISK ESTIMATION FROM EPIDEMIOLOGIC DATA 8-126
8.4.1 Selection of Epidemiologic Data Sets 8-126
8.4.2 Description of Risk Model 8-127
8.4.3 Calculation of Upper Limits of Risk 8-128
8.5 RELATIVE CARCINOGENIC POTENCY 8-131
8.5.1 Derivation 8-131
8.5.2 Potency Index 8-137
8.6 SUMMARY 8-137
8.6.1 Qualitative Assessment 8-137
8.6.2 Quantitative Assessment 8-143
8.7 CONCLUSIONS 8-144
8.8 REFERENCES 8-147
vi
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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 3-4
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 1 eve!s of TCI in surface waters 3-22
4-1 Recovery of radioactivity for 72 hours after a single
oral dose of 14C-TCI (200 mg/kg) to female Wistar rats and
NMRI mice 4-3
4-2 Disposition of single doses of 14C-TCI administered by
gavage to Osborne-Mendel rats and B6C3F1 mice 4-4
4-3 Pulmonary uptake of TCI for 25 volunteers exposed to TCI for
four consecutive 30-minute periods at rest and during
exercise 4-5
4-4 Pulmonary uptake of TCI for 4 subjects at rest exposed to
70 and 140 ppm TCI for 4-hour periods and 3-hour periods ... 4-8
4-5 Partition coefficients of TCI for various body tissues and
components of rat and man 4-9
4-6 Recovery of radioactivity for 50-hour postinhalation
exposure of male B6C3F1 mice to 10 or 600 ppm 14C-TCI for
6 hours 4-11
4-7 Recovery of radioactivity for 50-hour postinhalation
exposure of male Osborne-Mendel rats to 10 or 600 ppm
14C-TCI for 6 hours 4-11
4-8 Organ contents of TCI after daily inhalation exposure of
200 ppm for 6 hours per day 4-14
4-9 Proportion of TCI metabolites (TCA and TCE) in urine of
rats and mice after single oral doses of 14C-TCI 4-21
4-10 Effect of chronic daily oral dosing of TCI (1000 mg/kg)
on metabolism and metabolite excretion in B6C3F1 mice 4-24
4-11 Identification by GC/MS of TCI metabolites in exhaled
air and urine of Wistar rats and NMRI mice after a
single oral dose of 200 mg/kg of 14C-TCI 4-28
4-12 Demonstrated metabol ites of TCI 4-33
4-13 Jji vitro covalent binding of TCI 4-50
4-14 Microsomal bioactivation and covalent binding of aliphatic
halides to calf thymus DMA 4-51
4-15 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 4-54
4-16 jLn 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 . 4-54
5-1 Effects of TCI exposure on experimental animals 5-17
Vll
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LIST OF TABLES (continued)
Table Page
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 6-6
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
7-1 Mutagenicity of technical-grade TCI in Salmonel1 a/mammalian
mi crosome assay 7-4
7-2 Mutagenicity of trichloroethylene and epichlorohydrin in
S. pombe by means of the host-mediated assay in B6C3F1 mice. 7-11
7-3 Mutagenicity of technical-grade TCI at the his 1-7 locus of
Saccharomyces cerevisiae 7-13
7-4 Mutagemcity of reagent-grade TCI at the ilv locus in
Saccharomyces cerevisiae 7-15
7-5 Mutagenicity of TCI at the ilv locus in Saccharomyces
cerevisiae D7 7-17
7-6 Mutagenicity of TCI in the dominant lethal assay using NMRI
mice 7-23
7-7 Numerical chromosome aberrations in TCI workers 7-25
7-8 Unscheduled DMA synthesis in WI-38 cells 7-30
7-9 Summary of tests for mutagenicity of TCI 7-38
8-1 TCI carcinogenicity 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 mice
receiving TCI by oral gavage 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 HA: ICR Swiss mice 8-40
8-15 Estimation of probability of survival of rats 8-43
8-16 Estimation of probability of survival of hamsters 8-48
8-17 Estimation of probability of survival of mice 8-49
viii
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LIST OF TABLES (continued)
Table page
8-18 Statistical analysis of the incidence of primary hepato-
cellular neoplasms in control and TCI-treated B6C3F1 mice .. 8-54
8-19 Summary of negative carcinogenic!ty data for TCI 8-56
8-20 Chronic skin application in mice 8-58
8-21 Chronic subcutaneous injection in mice 8-59
8-22 Jji vitro transformation of Fischer rat embryo cell cultures
by TCI 8-63
8-23 Transformation and survival of Syrian hamster embryo cells
treated with diverse chloroalkene oxides 8-70
8-24 A cohort study of mortality among men exposed to TCI in the
1950s and 1960s and followed through December 1975 8-74
8-25 Comparison of workers exposed to TCI with the total Finnish
population for mortality from all causes and for cancer
mortality 8-77
8-26 Incidence of hepatocellular carcinomas in male and female
B6C3F1 mice in the NTP (1982) and NCI (1976) gavage
studi es 8-83
8-27 Disposition of 14C-TCI 72 hours after single oral doses to
male Osborne-Mendel and Wistar-derived rats and to male
B6C3F1 and Swiss mice 8-88
8-28 Metabolism of TCI in B6C3F1 mice: Effect of chronic dosing . 8-90
8-29 Disposition of 14C-TCI radioactivity for 71 hours after
single oral dose (200 mg/kg) to rats and mice (NMRI) 8-92
8-30 Disposition of 14C-TCI 50 hours after inhalation exposure
of male B6C3F1 mice 8-96
8-31 Disposition of 14C-TCI 50 hours after inhalation exposure
of male Osborne-Mendel rats 8-96
8-32 Covalent binding to liver and kidney macromolecules of
14C-TCI in male B6C3F1 mice and Osborne-Mendel rats 8-101
8-33 Dose conversion for the NTP bioassay (1982) in B6C3F1 mice .. 8-105
8-34 Dose conversion for the NCI bioassay (1976) in B6C3F1 mice .. 8-107
8-35 Slope estimates (qf) based on extrapolation from data in
male and female mice 8-116
8-36 Number of workers with cancer deaths 8-129
8-37 Relative carcinogenic potencies among 54 chemicals evaluated
by the Carcinogen Assessment Group as suspect human car-
cinogens 8-133
ix
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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-hr exposure of
100 ppm 4-6
4-2 The relationship between the concentration of TCI in
arterial blood and alveolar air at the end of inhalation
exposures 4-8
4-3 Predicted partial pressure of TCI in fatty tissue for a
repeated exposure to 100 ppm, 5 days, 6 hr per day 4-13
4-4 Elimination curves of TCI and kinetic model for transfer
of TCI in the body 4-15
4-5 Whole blood levels (ug/ml) of TCI and metabolites after
single gavage dose of TCI (1000 mg/kg) in corn
oil to male Osborne-Mendel rats and B6C3F1 mice 4-20
4-6 Dose-TCI metabolite excretion relationship in male
B6C3F1 mice and Osborne-Mendel rats given single
intragastric doses 14C-TCI in corn oil 4-22
4-7 Relationship between TCI dose and the amount of total
urinary metabolite excreted per day by mice in each
group 4-25
4-8 Relationship between environmental TCI concentration
and urinary excretion of TCI metabolites in human urine 4-31
4-9 Relationship between administered single oral doses of
14C-TCI to rats and mice and amount of dose metabolized
in 24 hr, expressed as mg/kg b.w., as calculated from
14C-radioactivity excreted in urine, feces, and
expired air 4-38
4-10 Postulated scheme for the metabolism of TCI to chloral
and to trichloroethylene epoxide and its metabolites 4-40
4-11 Metaboli sm of chloral hydrate 4-41
4-12 Postulated scheme for the metabolism of TCI based on
urinary metabolite profiles of rats and mice 4-42
4-13 Dose-effect relationship between chronic daily oral TCI
dose and increase in liver weight of mice after six
weeks. Relationship between liver weight increase with
chronic dosing at increasing levels and total urinary
metabolite (TCA and TCE) excreted per day by mice
at various dose 1 evel s 4-60
4-14 Dose-effect relationship between chronic daily oral TCI
dose and inhibition of liver glucose-6-phosphatase
activity (G6P) of mice after six weeks. Relationship
between inhibition of liver G6P activity with
chronic dosing at increasing levels and total urinary
metabolite (TCA and TCE) excreted per day by mice
at various dose levels 4-61
8-1 Growth curves for rats administered TCI in corn oil by
gavage
8-2 Survival curves for rats administered TCI in corn oil by
gavage 8-7
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LIST OF FIGURES (continued)
Figure Page
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 T-l
(100 ppm) 8-44
8-10 TCI histogram of chamber concentration measurements T-l
(300 ppm) 8-45
8-11 TCI histogram of chamber concentration measurements T-3
(600 ppm) 8-46
8-12 Incidence of malignant lymphomas in female mice 8-51
8-13 Cell transformation assays on vinyl chloride and TCI in
BHK-21/C1 13 IQ vitro 8-66
8-14 Structures and stability of chloroalkene oxides 8-71
8-15 Histogram representing the frequency distribution of the
potency indices of 54 suspect carcinogens evaluated by the
Carcinogen Assessment Group 8-132
xi
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AUTHORS AND REVIEWERS
The principal 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.
Vernon Benignus, Health Effects Research Laboratory, U.S. Environmental
Protection Agency, Research Triangle Park, N.C.
Chao W. Chen, Carcinogen Assessment Group, U.S. Environmental Protection
Agency, Washington, D.C.
I. W. F. Davidson, Department of Physiology/Pharmacology, The Bowman Gray
School of Medicine, Wake Forest University, Winston-Salem, North Carolina.
John R. Fowle III, Reproductive 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, Environmental Criteria and Assessment Office, U.S. Environ-
mental Protection Agency, Research Triangle Park, North Carolina.
Jean C. Parker, Carcinogen Assessment Group, U.S. Environmental Protection
Agency, Washington, D.C.
The following individuals reviewed earlier drafts of this document and
submitted valuable comments.
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-Salem, North Carolina 27103
Dr. John Egle
Dept. of Pharmacology
Virginia Commonwealth University
Richmond, Virginia 23298
xii
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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
Dr. Norman Trieff
Dept. of Preventive Medicine
University of Texas Medical Branch
Galveston, Texas 77550
Dr. Benjamin Van Duuren
Institute of Environmental Medicine
New York University Medical Center
New York, New York 10016
Dr. James Withey
Food Directorate
Bureau of Food Chem.
Tunney's Pasture
Ottawa, Canada
Participating Members of the Carcinogen Assessment Group
Roy E. Albert, M.D., Chairman
Elizabeth L. Anderson, Ph.D.
Larry D. Anderson, Ph.D.
Steven Bayard, Ph.D.
David L. Bayliss, M.S.
Robert P. Beliles, Ph.D.
James Cogliano, Ph.D.
Chao W. Chen, Ph.D.
Margaret M.L. Chu, Ph.D.
I.W.F. Davidson, Ph.D. (consultant)
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.
Jean C. Parker, Ph.D.
Charles H. Ris, M.S., P.E.
Dharm V. Singh, D.V.M., Ph.D.
Todd W. Thorslund, Sc.D.
Participating Members of the Reproductive Effects Assessment Group
John R. Fowle III, Ph.D.
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.
Vicki Vaughan-Dellarco, Ph.D.
Peter E. Voytek, Ph.D.
xm
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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.
Members of the Agency Work Group on Solvents
Elizabeth L. Anderson
Charles H. Ris
Jean C. Parker
Mark M. Greenberg
Cynthia Sonich
Steve Lutkenhoff
Arnold Edelman
James A. Stewart
Paul Price
William Lappenbush
Hugh Spitzer
David R. Patrick
Lois Jacob
Josephine Brecher
Mike Ruggiero
Charles Delos
Jan Jablonski
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 Toxic Substances
Office of Drinking Water
Consumer Product Safety Commission
Office of Air Quality Planning and Standards
Office of General Enforcement
Office of Water Regulations and Standards
Office of Water Regulations and Standards
Office of Water Regulations and Standards
Office of Solid Waste
Office of Pesticide Programs
Office of Emergency and Remedial Response
xiv
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1. EXECUTIVE SUMMARY
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 TCI 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 concentra-
tion 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 transposition 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 radi-
cals is the principal mechanism by which TCI is scavenged from the atmosphere.
TCI 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.
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 consequences of TCI in the envi-
ronment.
The pharmacokinetics and metabolism of TCI have been studied in man as
well as in animals. Inhalation is the principal route of concern by which TCI
enters the body. Ingestion of drinking water contaminated with TCI is another
important concern. The extent of TCI absorption after oral ingestion is virtual'
ly complete; with air exposure, the amount of TCI absorbed increases in propor-
tion to its concentration in inspired air, the respiratory rate, and duration
of exposure. TCI distributes widely into body tissues. TCI is eliminated by
two major processes, pulmonary excretion of unchanged TCI and liver metabolism
to urinary metabolites. At air concentrations of 500 ppm (2690 mg/m3) or less,
humans are estimated to metabolize between 60 and 90 percent of absorbed TCI.
The proportion of the TCI dose metabolized in rodents has been demonstrated to
1-1
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decrease at higher doses; that is, metabolism becomes nonlinear and approaches
saturation. However, metabolism is linearly proportional to the inhaled concen-
tration dose in man, at least up to about 300 ppm (1614 mg/m^). There is no
evidence that TCI metabolism in man is saturation-dependent. Studies have not
been made of TCI metabolism after oral exposure in man. At the levels found or
expected in drinking water, virtually all TCI is expected to be absorbed and
metabolized. There is persuasive experimental evidence that the metabolic
pathways for TCI are qualitatively similar in mice, rats, and humans. In these
species, the principal urinary metabolites of TCI that have been identified
are trichloroethanol (TCE) and its glucuronide, and trichloroacetic acid (TCA).
Minor metabolites have also been identified for each of these species. In the
liver, TCI is first metabolized to chloral hydrate and to a reactive epoxide
(TCI oxide) by a microsomal P45Q system. Reactive intermediate metabolites such
as TCI epoxide covalently bind to cellular macromolecules, principally protein,
and to a much lesser extent, DNA. Hepatotoxicity of TCI has been shown to cor-
relate with the amount of metabolism. Metabolism of TCI is enhanced by drugs
(e.g., barbiturates, oral antidiabetic agents) and by other xenobiotics (e.g.,
PCBs) which induce the hepatic microsomal P^Q metabolizing system. Heightened
toxicity can result from drug interactions known to occur with ethanol, barbi-
turates, disulfiram, and Warfarin.
Excluding carcinogenicity as an end point, toxicity testing in experimen-
tal animals, coupled with limited human data derived principally from excessive
exposure, suggests that long-term exposure of humans to environmental (ambient)
levels of TCI is not likely to represent a health concern. Behavioral and psy-
chological effects, particularly as they affect psychomotor performance, have
been reported at levels of 200 ppm (1076 mg/m^) in some, but not all, experi-
mental and epidemiologic studies. There is no information that such impair-
ment, representative of dysfunction of the central nervous system (CNS), would
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 or as a result of environ-
mental exposures.
Teratogenicity is another health end point for which available data from
experimental animals suggest that the conceptus is not uniquely susceptible to
TCI. Exposure of rats, mice, and rabbits, during gestation, to levels (300 ppm
1-2
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or 1614 mg/m^) greatly in excess of those generally found in the environment,
has not been observed to result in any teratogenic effects. The teratogenic
potential of TCI for humans cannot be directly extrapolated from the observa-
tions in the animal studies. However, the animal studies suggest that at low
ambient levels that do not cause maternal toxicity, TCI would not pose a signi-
ficant hazard to the developing conceptus.
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. Thus, commercial TCI may have the poten-
tial to cause weak or borderline increases above the spontaneous level of muta-
genic effects in exposed human tissue. Adverse effects in the testes of mice
exposed to commercial TCI suggest that TCI could cause similar effects in
humans. A conclusion about the mutagenic potential of pure TCI cannot be made.
If TCI is mutagenic, the available data suggest that it would be a very weak,
indirect mutagen.
The evidence reviewed in this document for the carcinogenicity of TCI
in experimental animals includes: increases in the incidence of hepatocellu-
lar carcinomas in male and female B6C3F1 mice (three studies); malignant lym-
phomas in female HantNMRI mice; and renal adenocarcinomas in male Fischer 344
rats. Statistically significant increases of malignant liver tumors were
observed in both male and female B6C3F1 mice. A gavage study of purified TCI
in both sexes of B6C3F1 mice was conducted with basically the same experimental
design as a gavage study of technical grade TCI in male and female B6C3F1 mice
to evaluate the role of epoxide stabilizers in TCI in the induction of hepato-
cellular carcinomas. Similar carcinogenic responses were observed for the
purified epoxide-free and for stabilized TCI. The other studies provide some
additional support to the overall body of evidence, particularly since two of
them were carried out in a different species or strain. However, the third
study in B6C3F1 mice, an inhalation study in which an increase in liver tumors
was seen, was weakened by deficiencies in its conduct. In the inhalation study
in Han:NMRI mice, a 30 percent incidence of spontaneous lymphoma in control
mice, and the possibility of an indirect effect in treated mice, made these
results difficult to interpret. A borderline response was observed in the
gavage study of Fischer 344 rats.
1-3
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A gavage study with male and female Osborne-Mendel rats was negative;
however, this study may be considered inconclusive because of high mortality
in the treatment groups. Exposure to airborne concentrations of TCI did not
result in a carcinogenic effect in Han:Wist rats and Syrian hamsters, although
higher dose levels of TCI probably could have been given to the test animals.
Other long-term animal studies did not suggest a carcinogenic potential
for TCI. Most of these studies, however, were not specifically designed to
evaluate carcinogenicity, involved only small treatment groups, lacked suffi-
cient doses of TCI, or were of insufficient duration in view of the expected
latency period for cancer induction.
TCI induced malignant tumors of the liver in both male and female B6C3F1
mice in multiple studies. This constitutes a signal that TCI might be carcino-
genic in man. While kidney tumors observed in the NTP rat study are not strong
indications 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, the
statistical significance after mortality corrections suggests that a carcino-
genic effect may be taking place.
The U.S. Environmental Protection Agency's Proposed Guidelines for Carci-
nogen Assessment (U.S. EPA, 1984), take the position that the mouse-liver-
tumor-only response, when other conditions for a classification of "sufficient"
evidence in animal studies are met, should be considered as "sufficient" evi-
dence of carcinogenicity. Thus, based on EPA's proposed cancer guidelines,
the overall evidence for TCI would result in a classification of B2, i.e., a
probable human carcinogen.
The TCI carcinogenicity results could be classified under the criteria of
the International Agency for Research on Cancer (IARC) as either "sufficient"
or "limited" depending on which of the differing current scientific views
about chlorinated organic compound induction of liver tumors in mice is chosen.
Since there are no adequate epidemiologic data in humans, the overall ranking
of TCI under the criteria of the IARC depends primarily upon the position taken
regarding the mouse liver tumor. Thus, the overall ranking could be either
Group 2B or Group 3. The more conservative public health view would regard
TCI as a probable human carcinogen (Group 2B), but there is also scientific
sentiment for regarding TCI as an agent that cannot be classified as to its
carcinogenicity for humans (Group 3).
1-4
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Additional carcinogenicity studies on purified TCI (no detectable epox-
ides) are currently in progress. These studies and others published recently
will be incorporated into any updates of this Health Assessment Document.
Four sets of gavage bioassay data on hepatocellular carcinomas in male and
female mice provide a basis to calculate an upper-bound carcinogenic slope
estimate for TCI using a linearized multistage model. The development of these
risk estimates is for the purpose of evaluating the "what-if" question: If TCI
is carcinogenic in humans, what is the possible magnitude of the public health
impact? Since upper-bound potency estimates calculated on the basis of these
data sets are comparable, ranging from 5.8 x 10~3 to 1.9 x 10"2 mg/kg/day,
the geometric mean, 1.1 x 10~2 mg/kg/day is used to calculate the incremental
lifetime cancer risk (i.e., unit risk) due to .a unit.exposure of TCI in drinking
water and in air. The upper-bound estimate of the cancer risk due to 1 yg/L
of TCI in drinking water is 3.2 x 10~?. The upper-bound estimate of the cancer
risk due to 1 pg/m3 of TCI in air is 1.3 x 10~6. The upper-bound nature of
these estimates is such that the true risk is not likely to exceed this value
and may be lower. The carcinogenic potential of TCI is generally considered to
reside in cellular reactive intermediate metabolites, and therefore metabolic
and pharmacokinetic factors have been used in the calculation of the drinking
water and air unit risks.
None of the epidemiologic studies reviewed in this report provide positive
evidence from which to estimate a unit risk for exposure to TCI. As an alter-
native, an upper-bound estimate has been calculated from one negative study.
The calculation on the basis of human data results in a greater risk estimate
than the estimate from animal data, and thus does not appear to contradict the
risk estimate calculated from the animal data.
Expressed in terms of relative potency, TCI ranks in the lowest quartile
among the 54 suspect or known human carcinogens evaluated by EPA's Carcinogen
Assessment Group.
1-5
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RECOMMENDATIONS FOR FURTHER STUDIES
Areas for which incomplete information is available, and which 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 end points in
humans should serve as a stimulus for future research.
2. Neurobehavioral Toxicity. There have been few animal studies of the
effects of TCI on the nervous system and behavior. Most end points studied
have been relatively insensitive. Further studies are warranted on more sen-
sitive end points.
1-6
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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 invol-
ving individuals occupationally or accidentally exposed. During such exposures,
the concentrations 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 CMS, effects on clinical chemistries, and pharmacokinetic
parameters of TCI exposure.
The available epidemiologic studies have not been able to adequately
assess the overall impact of TCI on human health. It has therefore 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 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. On-going literature searches
have been conducted through 1985, resulting in the inclusion of selected refer-
ences in this document. However, the basic literature search for Chapter 8 is
current through 1982; the basic literature search for Chapter 7 is current
through 1983. The Agency is aware of additional carcinogenicity and mutagenicity
information that has subsequently become available. This information will be
considered for incorporation in any future updates of this document.
2-1
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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 proper-
ties are shown in Table 3-1.
TABLE 3-1. PHYSICAL PROPERTIES OF TRICHLOROETHYLENE
Molecular weight 131.39
Boiling point 87°C
Vapor pressure 94 mm Hg @ 30°C
Vapor specific gravity 4.55 at boiling point
(air = 1)
Solubility @ 20°C in water 0.107 g percent
At a temperature of 25°C and a pressure of 1 atmosphere, one part-per-
3
million (ppm) of the vapor is equivalent to 5.38 mg/m in air.
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 (McNeill, 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 (McNeill, 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 25°C when the TCI concentration is between 8 to 10 percent
in air (McNeill, 1979).
To inhibit its decomposition, stabilizers are added to commercial formu-
lations of TCI (McNeill, 1979). Commonly used stabilizers in vapor degreasing
grades of TCI are neutral inhibitors and free radical scavengers (McNeill, 1979).
3-1
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Epoxides (including epichlorohydrin, a known animal carcinogen) are added to
scavenge free HC1 or A1C1,. Antioxidants are usually added in quantities less
O
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)
Amyl alcohol
Propanol
Diethyl amine
Triethyl amine
Dipropylamine
Diisopropylamine
Diethanolamine
Morpholine
N-Methy1morphol i ne
Aniline
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
Methyl ethyl 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.
3-2
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Trichloroethylene is not readily hydrolyzed by water (McNeil!, 1979). In
the presence of strong alkali (e.g., sodium hydroxide) TCI can decompose to
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 are 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 muta-
genicity 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. 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 coeffici-
ents is shown in Table 3-3 (Sato and Nakajima, 1979). These values indicate
that TCI is highly lipophilic.
3.2 ENVIRONMENTAL FATE AND TRANSPORT
3.2.1 Production
Trichloroethylene has been declining in production in recent years because
of restrictions on emissions (Federal Register, 1977) and substitution by other
solvents. According to statistics published by the U.S. International Trade
Commission (1982), 117,363 metric tons of TCI were produced in 1981.
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 four American manufacturers (SRI, 1982). The
producers and production capacities are shown in Table 3-4.
Most of the TCI produced commercially is derived from ethylene or
dichloroethane (EDC) (McNeill, 1979). Dichloroethane, produced by
chlorination of ethylene, can be further chlorinated to TCI and
3-3
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TABLE 3-3. PARTITION COEFFICIENTS OF TRICHLOROETHYLENE AND OTHER
CHLORINATED HYDROCARBONS (Sato and Nakajima, 1979)
Solvent
Dichloromethane
Chloroform
Carbon tetrachloride
1,2-Dichloroethane (EDC)
Methyl chloroform
Tetrachl oroethyl ene
Trichloroethylene
Oil /water
21
115
1444
40
383
4458
552
Oil /blood
16
39
150
23
108
146
76
Log P
(partition
coefficient
1.32
2.06
3.16
1.60
2.58
3.65
2.74
TABLE 3-4. U.S. PRODUCTION CAPACITY FOR TRICHLOROETHYLENE (SRI, 1982)
Producer
Site
Annual capacity, Jan. 1981
(metric tons)
PPG Industries
Dow Chemical
Diamond Shamrock
Ethyl Corporation
Lake Charles, LA
Freeport, TX
Deer Park, TX
Baton Rouge, LA
91,000
54,000
23,000
20,000
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 (McNeill, 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).
3-4
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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 1, 1979).
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 may 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
There are no known natural sources of TCI. Graedel and Allara (1976) con-
cluded that atmospheric chemical production of TCI was negligible. Because of
its dispersive use pattern in degreasing operations, most of the TCI used is
expected to be emitted to the atmosphere. 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/hr can be
emitted, based on 200,000 pounds of metal cleaned per day (Bucon et al., 1978).
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.
3-5
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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 hydroxyl 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 -OH in a reaction cell, calculated an atmospheric lifetime for TCI of
about 54 hours. An -OH concentration in the troposphere of 10 molecules/cm
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 10
molecules/cm 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
6 3
•OH concentration of 1 x 10 molecules/cm , 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 year (^ 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 sur-
vival of TCI. Altshuller (1980) calculated that in January (when -OH levels
and solar flux are low) 0.6 day 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 day is required.
Trichloroethylene was observed to have a slow decomposition rate in dilute
aqueous solution (Billing 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.
3-6
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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. Oil ling
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 minutes.
Scherb (1978) measured the volatilization of TCI from an aeration channel in a
wastewater treatment plant and found the half-life to be 3 hours. 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 hours, for rapidly moving shallow streams,
to 10 days or longer for ponds and lakes (Table 3-6).
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 NO,,, 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
N0 did not alter the consumption rate of TCI above TCI controls.
3-7
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Edney et al. (1983) observed a number of degradation products when «OH
radicals reacted with TCI in a 700 liter cell coupled to an FTIR spectrometer
and scanning interferometer. Reaction products observed were phosgene, dichlo-
roacetyl chloride, formyl chloride, carbon monoxide, and nitric acid. The rate
-12 3
constant of reaction was 3.6 x 10 cm /sec.
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 N02, TCI (10 ppm) was very short-lived (less than 3.5 hours) (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 photo-
chemical smog have been discussed (U.S. EPA, 1978a; National Academy of
Sciences, 1977). Free radicals (e.g., -RO,,), are formed when TCI is photo-
decomposed, and these in turn react with NO by oxidizing it to NO^. Ac-
cording 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 quan-
titating halogenated hydrocarbons present in ambient air, water, soil, foods,
etc., two systems are commonly used: gas chromatography/mass spectrometry
3-8
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(GC-MS) and gas chromatography with electron capture detection (GC-ECD). Each
system has the capability of detecting TCI concentrations as low as a few parts
per trillion by volume. The usefulness of GC-ECD is that the apparatus can be
used in the field 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 GC-EC metho-
dology 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
deviation was 23.0 by electronic area integration (+ 5 percent by peak height
measurement). The system, which employs a freezeout concentration loop, has
the capability of measuring various halocarbons in 500-ml aliquots of air.
Total analysis time is 40 minutes. The GC column consisted of a 6-mm x 3-m
stainless steel column packed with 10 percent SF-96 on 100/120 mesh-
Chromosorb 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 stan-
dard deviation) 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 percent 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.
3-9
-------
The applicability of GC-ECD 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
ozone was oxidizing the Tenax monomer and producing electron-absorbing oxy-
genated 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. An extremely low detection limit of <0.2
ppt (<1.36 x 10 mg/m ) was reported. Sampling canisters were made of stain-
less 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 preconcen-
tration 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 ug/1 was reported. An OV225 capillary column was used.
Dietz and Singley (1979) 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 from 0.1 ppb to several ppm of TCI were detec-
ted. 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 chromato-
grams from the purge-trap method were of poorer quality. The headspace method
was recommended for routine sample analyses of volatile halocarbons.
Wang and Lenahan (1984) described a closed-loop system that combines the
techniques of both gas stripping and headspace, with a reported detection limit
of about 0.2 H9/1-
3.3.1.2 Gas Chromatography-Mass Spectrometry. While GC-ECD 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
3-10
-------
of GC-MS in field measurements. Often, GC-MS is used to verify results obtained
by GC-ECD.
Comparisons between the precision afforded by GC-MS versus GC-ECD 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 stan-
dard 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 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 halogena-
ted 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 10 mg/m ). Accuracy was
reported at ±30 percent. Included among the inherent analytical errors were
(1) the ability to accurately determine the breakthrough volume, (2) the percent
recovery from the sampling cartridge after a period of storage, and (3) the
reproducibility of thermal desorption from the cartridge and its introduction
into the analytical system. To minimize loss of sample, cartridge samplers
should be enclosed in cartridge holders and placed in a second container that
can be sealed, protected from light, and stored at 0°C. The advantages repor-
ted 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 breakthrough volumes at various temperatures suggests that Tenax GC is not
a particularly 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 ionization was used in detection. Interference between TCI and ethylene
dichloride was reported with Porapak T.
3-11
-------
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 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 accom-
plished at a water temperature of about 22°C. In general, the method is appli-
cable 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/L range.
Desorption onto the head of the column can be accomplished in one step by heat-
ing.
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 al., 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
presented below (National Academy of Sciences, 1978).
(1) Pump-pressure samples: A mechanical pump is used to fill a stain-
less steel or glass container to a positive pressure relative to the surround-
ing atmosphere.
3-12
-------
(2) Ambient pressure samples: An evacuated chamber is opened and allowed
to fill until it has reached ambient pressure at the sampling location. If
filling is conducted at high altitude, the container may easily become contami-
nated when returned to ground level.
(3) Adsorption on molecular sieves, activated charcoal, or other adsor-
bents.
(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 electropolished stainless steel vessels
in their sampling of tropospheric 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,
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 proce-
dure 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 pres-
ence of chlorine in some drinking water supplies and the chlorination of organic
materials, an increase in the concentration of some halocarbons may occur also
during storage (Kopfler et al., 1976).
3-13
-------
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 ug TCI/L 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, ace-
tone rinse, distilled water rinse, and oven-drying at 150°C. Vials were com-
pletely 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 hours. 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 alter
greatly vapor retention. For TCI, tubes stored at 37°C had high vapor reten-
tion rates. When breath was exhaled into the tubes at 37°C, water vapor was
not condensed from 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 adsor-
bents 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 technique has been successfully applied to an actual water pollution case
for tetrachloroethylene. The detection limit for TCI was reported as 1 umol/£
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
3-14
-------
TABLE 3-7. AMBIENT AIR-MIXING RATIOS OF TRICHLOROETHYLENE
Location
ARIZONA
Phoenix
Grand Canyon*
ARKANSAS
Helena
El Dorado*
CALIFORNIA
£ Stanford Hills
Ul
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 ±
0
< 0.03
3.1 ± 3.
0.093 ±
0.315 ±
0.399 ±
0.024 ±
0.035 ±
0.015 ±
0.026 ±
0.266 ±
0.080 ±
0.012 ±
.5869
8
0.32
.310
.302
0.014
0.050
0.003
0.094
0.094
0.22
0.002
Reference
Singh et al. , 1981
Pellizzari, 1979a
Battell e, 1977
Pellizzari, 1979a
Singh et al., 1979a
Singh et al. , 1979a
Singh et al., 1981
Singh et al. , 1979
Singh et al., 1979
Singh et al., 1979
Singh et al., 1979
Singh et al., 1979
Singh et al., 1979
Pellizzari, 1979a
Pellizzari, 1979a
-------
TABLE 3-7. (continued)
Location
Point Arena
Point Arena
San Jose
Oakland
La Jolla*
DELAWARE
Delaware City
£ LOUISIANA
CT)
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
Mixing ratio
Maximum Minimum
0.012
0.034
2.192
1.5582
3.9
0.56
0.65
2.1
< 0.05
8.6
0.28
8.5
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
Pellizzari, 1979a
Pellizzari, 1979a
Pellizzari, 1979a
Singh et al., 1981
Su and Goldberg, 1976
Lillian et al., 1975
Pellizzari et al., 1979
Battelle, 1977
>
Battelle, 1977; Pellizzari,
1979b
Pellizzari et al., 1979
Bozzelli and Kebbekus,
Bozzelli and Kebbekus,
Bozzelli and Kebbekus,
1983
1979
1983
-------
TABLE 3-7. (continued)
co
Location
Batso
Seagirt
Newark
Sandy Hook
Elizabeth
Fords*
Middlesex*
Edison*
Carlstadt*
Camden
Bayonne
Boundbrook*
Bridgewater*
Rahway*
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 1978
12/29 1979
03/26 1976
09/27 1978
07/23-07/28 1978
03/24 1976
09/24 1978
09/28-09/30 1978
04/03-10/24 1979
March-Dec. 1973
03/26 1976
09/20 1978
07/17 1978
08/05 1978
09/20-09/22 1978
Mixing ratio (ppb)
Maximum Minimum Average
1.3
2.8
1.9
0.8
6.4
3.1
0.36
6.1
9
3.5
8.8
4.1
6.9
18
0
< 0.05
0
< 0.05
0
0
0
0.54
3
0
< 0.05
0
0
3.4
0.08 ± 0.28
0.26
0.25
0.34
0.76
1.9 ± 1.1
0.22 ± 0.11
2.7 ± 2.1
6.4 ± 3.1
0.42 ± 0.87
0.92
2.4 ± 1.8
0.35 ± 0.29
11 ± 7.8
Reference
Bozzelli and Kebbekus, 1983
Lillian et al., 1975
Bozzelli and Kebbekus, 1983
Lillian et al. , 1975
Bozzelli and Kebbekus, 1983
Pellizzari, 1977;
Pellizzari et al., 1979
Bozzelli and Kebbekus, 1979
Pellizzari, 1978;
Pellizzari, 1979;
Pellizzari, 1977b
Pellizzari et al. , 1979
Bozzelli and Kebbekus, 1983
Bozzelli and Kebbekus, 1983
Pellizzari, 1977;
Pellizzari et al. , 1979
Bozzelli and Kebbekus, 1979
Pellizzari et al. , 1979
-------
TABLE 3-7. (continued)
co
i
00
Location
NEW YORK
NYC
Whiteface Mtns.
Niagara Falls/Buffalo
OHIO
Wilmington
NEVADA
Reese River
KANSAS
Jetmar
WASHINGTON
Pullman
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
0.0124
Reference
Lillian et
Lillian et
Pellizzari
Pellizzari
Singh et al
Singh et al
al., 1975
al., 1975
et al . , 1979
et al., 1979
., 1979
., 1979
Grimsrud and
Rasmussen, 1975
Cronn et al
Cronn et al
., 1977a
., 1977b
(lat 9°N)
July 1977
0.015
Cronn and Robinson,
1978
-------
TABLE 3-7. (continued)
Location
NORTH ATLANTIC OCEAN
WESTERN IRELAND
ENGLAND
JAPAN
Tokyo
Various sites
WESTERN EUROPE
£ COLORADO
VO
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 ratio
Maximum Minimum
.005 4 x 10"4
47.1 0.8
0.038 0.005
8.5 < 0.02
0.41 0.028
0.24 0.019
0.029 0
0.3 0
0.029 0
0.33 0
(ppb)
Average
< 0.005 ± 0.0657
0.015 ± 0.0121
.002
3.5 ± 3.7
0.014 ± 0.010
0.2 ± 0.11
0.11 ± 0.063
0.009 ± 0.017
0.15 ± 0.11
0.001 ± 0.004
0.12 ± 0.098
Reference
Lovelock, 1974a
Lovelock, 1974a
Murray and Riley, 1973a
Tada et al . , 1976
Makide, et al. , 1980
Correia et al. , 1977
Singh et al. , 1980
Singh et al., 1980
Pellizzari et al., 1979
Pellizzari, 1979a
Battelle, 1977b;
Pellizzari et al. , 1979
Pellizzari et al., 1979;
Singh et al. , 1980
-------
TABLE 3-7. (continued)
u>
i
ro
O
Location
WASHINGTON
Auburn*
WEST VIRGINIA*
Nitro
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
Battelle, 1977b
Pellizzari, 1978
Pellizzari, 1978
Pellizzari, 1978
*Data obtained from summary report of Brodzinsky and Singh, 1982.
-------
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. Trichloroethylene 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 reviewed by the
U.S. Environmental Protection Agency (1979).
3.3.3.2.1 Drinking water. Dowty et al. (1975) detected TCI in finished drink-
ing 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.
In a comprehensive surface water monitoring effort for TCI, Ewing et al.
(1977) have shown TCI to be a common low-level (ppb) contaminant of surface
waters in the vicinity of urban/industrial areas. TCI was reported at concen-
trations of 1 |jg/L (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.
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 quantifi-
cation 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/L. The average concentration
in positive water supplies was 0.47 ug/L. Thirty percent of all supplies con-
tained a level of TCI which was below I ug/L. The remaining two percent of
the supplies contained a concentration between 1 to 4 ug/L.
3-21
-------
TABLE 3-8. MEAN LEVELS OF TCI IN SURFACE WATERS'
Surface water samples
State
A1 abama
Delaware
Illinois
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/L)
All b
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
aSource: Ewing et al. (1977); drinking water samples not reported in these data.
bMean levels were calculated by substituting a value of one-half the reporting
limit of 1 ppb (1 ug/L) for samples reported as having non-detectable (i.e.,
less than 1 ug/L).
GLevels of TCI in sample from the following states (number of sites per state
in parentheses) were reported as not detectable (i.e., less than 1 ug/L):
California (9) North Carolina (1)
Iowa (2) Oregon (3)
Mississippi (1) Washington (2)
Missouri (3) Wisconsin (6)
3-22
-------
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 pg/L with a range from
0.11 to 53.0 ug/L.
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 detec-
table levels of TCI were between 0.5 to 5 ug/L. TCI was found in finished water
from small and large systems alike.
In analyses of drinking waters from sites near producer and user facili-
ties and from a background site, Battelle (1977) found the following concen-
trations 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).
Boateng et al. (1984) have described the geology, hydrology, and chemistry
of both shallow and deep aquifers in relation to the occurrence of TCI in two
production wells.
3.3.3.2.2 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).
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
chlorination, the concentration detected by GC-MS was 8.6 ppb; after chlorina-
tion, 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
receive effluent from production and user sites. Values ranged from 0.02 to
74 ug/L (ppb w/w), showing the effect of these additions on background levels.
Dilution effects and losses to the surrounding air were noted.
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3.3.3.3 Soil and Sediment. Battelle (1977) reported the presence of TCI in
soil samples in the vicinity of producers and users at levels up to 5.6 ppb.
In freshwater sediment taken near the Hooker Chemical facility at Taft,
Louisiana, up to 300 ppb TCI was detected (Battelle, 1977). Trichloroethy-
lene 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.
Rogers and McFarlane (1981) reported that aluminum (Al )-saturated mont-
morillonite 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
absorption when calcium-saturated montmorillonite clay was used. With two silty
clay loams (organic carbon content = 2 percent), less than 6 percent of the
TCI available was sorbed. When soil sorption was normalized, based on the soil
organic carbon contents, a correlation was found between water solubility,
sorption constant, 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 susceptibil-
ity of various lifestages to the test substance may vary considerably.
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 organism(s) continuously to a constant concentration of TCI while oxygen
is continuously replenished and waste products are removed. A static test, on
the other hand, exposes the organism(s) to the added initial concentration only.
Both types of tests are commonly used as initial indications of the potential
of substances to cause adverse effects.
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Alexander et al. (1978) used both flow-through and static methods to in-
vestigate 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 (LCcn) 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-hr LC50 concentration for TCI was 40.7
mg/L in the flow-through test. The 95 percent confidence band was between 31.4
and 71.8 mg/L. Of four solvents tested (perchloroethylene, methylene chloride,
methyl chloroform, and TCI), TCI had the second highest toxicity (after perchlo-
roethylene).
In acute static experiments with bluegills, TCI was reported to have a
96-h LC5Q value of 44.7 mg/L (U.S. EPA, 1978b). With Daphnia magna as the test
organism in a 48-hr static experiment, the LC5Q for TCI was 85.2 mg/L. In a
chronic test with this organism, no adverse effects were found at the highest
test concentrations of 10 mg/L.
Lay et al. (1984) evaluated the effect of TCI on Daphnia magna and phyto-
plankton species in a natural pond. The pond was compartmentalized and two
compartments each were used for a single high dose of 110 ng/L, a single low
dose of 25 ng/L, and controls. Complete elimination of Daphnia magna in the
high dose groups was noted at 24 and 72 hours after dosing. In the low dosage
groups, TCI was less toxic to Daphnia. However, a significant decrease in pop-
ulation was noted in both treated compartments from day 3 to day 28 of the ex-
periment. The relative abundance and absolute number of all phytoplankton
individuals counted increased with the increase of TCI concentrations.
Investigation of the bioconcentration potential of TCI for bluegill indi-
cated 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 suggest that residues of TCI at exposure concentrations below
those directly toxic to aquatic life are not likely (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/L, respectively, and noted erratic swimming, uncontrolled movement, and
loss of equilibrium after several minutes.
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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,
3
1978) is 100 ppm (525 mg/m ) as a time-weighted average (TWA) over an 8-hr
period. More recently, the National Institute for Occupational Safety and
Health (NIOSH) (Page and Arthur, 1978) has noted that the current standard ap-
pears to be inadequate for protection of workers from the carcinogenic poten-
tial of TCI. A level of 25 ppm, as a TWA, was the recommended standard proposed
by NIOSH, based on engineering control capabilities. The American Council of
Governmental Industrial Hygienists has established a TLV® of 50 ppm.
3-26
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4. PHARMACOKINETICS AND METABOLISM
4.1 ABSORPTION AND DISTRIBUTION
Trichloroethylene is a highly volatile liquid whose vapors exert pharma-
cologic 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.
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 impor-
tant route of absorption. Although sparingly soluble in water (1.0 gm/£,
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 immer-
sion, 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 j_n vivo and T_n vitro techniques. The percutaneous absorption rate of
2
TCI in mice was reported to be between 59.8 and 92.4 umol/min/cm or between
2
7.82 and 12.1 ug/min/cm . From this study, Tsuruta concluded that direct
contact with TCI, even if the percutaneous absorption rate of TCI is different
between human skin and mouse skin, may have the potential to contribute impor-
tantly 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 minutes after immersion was
only 0.5 ppm,indicating that the rate of absorption through the skin is very
4-1
-------
slow, even after allowing for the possibility of lipid storage and for metab-
olism to urinary metabolites (see below). In addition, Sato and Nakajima
(1978) demonstrated that for skin exposure in man, a large proportion 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 hours and solvent concentration was monitored in blood during and
following dermal applications. Blood concentration (reflecting absorption
rate) increased rapidly, peaking at 0.5 hour (0.8 ug/ml blood), and then
decreased despite continuing exposure (0.46 ug/ml blood after 6 hours). This
pattern was characteristic of halogenated 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 LD5Q in mice, rats and dogs are similar, being 3.2, 4.9 and 2.8 g/kg,
respectively (Klaasen and Plaa, 1966, 1967; Smyth et al., 1969).
Daniel (1963) dosed rats by stomach-tube with Cl-labeled TCI liquid (40
to 60 mg/kg b.w.) and recovered 90 to 95 percent of the label in expired air
and urine, suggesting virtually complete absorption by the route. Similar
results have been reported recently by Dekant et al. (1984) and Prout et al.
14
(1984). Dekant et al. dosed by gavage both rats and mice with 200 mg C-TCI/kg
in corn oil vehicle and recovered 93 to 98 percent of the radiolabel in expired
14
air and urine (Table 4-1). Prout et al. administered C-TCI in corn oil
vehicle as single intragastric doses of 10, 500, 100, and 2000 mg/kg to both
rats and mice and recovered 91 to 98 percent of the doses in expired air and
urine (Table 4-2). Peak blood levels occurred at about 1 hour in mice and 3
hours in rats, indicating rapid absorption from the GI tract.
4-2
-------
TABLE 4-1. RECOVERY OF RADIOACTIVITY FOR 72 HOURS AFTER A SINGLE ORAL DOSE OF
14C-TCI (200 mg/kg) TO FEMALE WISTAR RATS AND NMRI MICE
As % Recovered Radioactivity*
Exhaled air
Unchanged, 14C-TCI
14C02
Urine
Cage rinse
Feces
Carcass, tissues
Time
(hr)
0-72
0-72
0-12
12 - 24
24 - 48
48 - 72
0-72
0-72
Rats
52.0
1.9
53.9
22.0
17.0
2.0
0.2
0.2
41.4
1.8
2.9
Mice
11.0
6.0
17.0
30.0
39.0
6.0
1.0
0.1
76.3
4.9
2.0
*N = 2 rats and 3 mice; total recovered radioactivity was 93 to 98% of the
administered radioactivity given the rats and mice. The average weight
of the rats was 240 gm and the mice 25.5 gm.
Source: DeKant et al. (1984).
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 vehicle. 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 minutes
for aqueous solution) averaged 15 times higher for water than for corn oil
solution (14.7 versus 1.0 ug/ml); however, the peak blood concentration was
reached faster for water solution than for oil solution, which exhibited a
second delayed peak 80 minutes 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 hours after dosing
was 218, water:corn oil. These results illustrate the effect of solution
4-3
-------
TABLE 4-2. DISPOSITION OF SINGLE DOSES OF 14C-TCI ADMINISTERED BY GAVAGE TO
OSBORNE-MENDEL RATS AND B6C3F1 MICE
% Dose Excreted **
Dose, Unchanged
mg/kg 14C-TCI
Osborne-Mendel
10
500
1000
f 2000
B6C3F1 Mice*
10
500
1000
2000
Rats*
1.3
42.7
56.2
77.8
3.9
6.0
17.5
13.6
Metabolized
14C02
10.2
3.7
2.9
1.4
11.7
8.9
7.6
7.8
Urine
69.4
43.6
31.1
14.0
59.4
63.7
46.4
47.5
Feces
8.3
4.3
3.1
3.4
17.2
14.5
15.0
19.2
Carcass
8.4
3.8
2.4
1.3
4.8
4.4
8.6
3.8
Total
96.3
55.4
39.5
20.1
93.1
91.5
77.6
78.3
Recovery
97.6
98.1
95.7
97.9
97.0
97.5
95.1
91.9
*Means of groups of 4 animals; rat weights 180 to 200 gm and mouse 25 to 32 gm.
**During 72-hr period after dosing.
Source: Prout et al. (1984).
-------
medium on TCI intestinal absorption; the much slower absorption of TCI dis-
solved in corn oil is probably due to the high lipid solubility (and poor
aqueous solubility) of this compound which remains as a depot 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. Inhaled TCI rapidly equilibrates across the lung alveolar endo-
thelium. 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 4-3).
TABLE 4-3. PULMONARY UPTAKE OF TCI FOR 25 VOLUNTEERS EXPOSED TO TCI FOR FOUR
CONSECUTIVE 30-MIN PERIODS AT REST AND DURING EXERCISE (50 TO 150 WATTS)
Exposure
concentration, Amount inhaled, Amount taken up,*
mg/m3
540
1080
540
1080
Conditions
rest
rest
Exercise (50w)
Exercise (50w)
mg
149 ± 6
313 ± 15
395 ± 6
803 ± 12
mg
79 ± 4
156 ± 9
160 ± 5
308 ± 16
540 and 1080 mg/m3 = 100 and 200 ppm, respectively.
Calculated from difference in inspired air and expired air x ventilation volume
x time. Retention value averaged = 45 percent.
Source: Astrand and Ovrum (1976).
4-5
-------
Pulmonary uptake is proportional to the magnitude of the gas partial
pressure difference between alveoli and pulmonary venous blood. This differ-
ence diminishes when all body tissues achieve equilibrium with the alveolar
(arterial) partial pressure. Initially, uptake is rapid, but about 8 hours of
exposure is required 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
hours) with the rich blood-perfused tissues. The poorly perfused fat tissues
(FG) require longer than 8 hours for equilibrium.
FG
— MG
VRG —
ALV. AIR AND ART. BLOOD
246
EXPOSURE, hours
246
POST EXPOSURE, hours
Figure 4-1. Predicted partial pressure of TCI in alveolar air and tissue groups during and
after an 8 hour exposure of 100 ppm.
Source: Fernandez et al. (1977).
4-6
-------
At equilibrium with a given concentration of TCI in inspired air, the
difference between the inspired air concentration of TCI and alveolar concen-
tration of TCI provides a measure of gas uptake. With appropriate accounting
for ventilation (about 4 to 8 L/min at rest) and duration of exposure, the
amount taken up by the body, or retention (dose; body burden), can be calcu-
lated. At alveolar and tissue equilibrium, the retention reflects disposition
within the body by tissue storage, metabolism, or excretion by routes other
than the lungs. Retention 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 4-3 and 4-4). 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 concentration 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
4-3 and 4-4. Astrand and Gamberale (1978) exposed volunteers to 100 and 200
ppm (ventilation volume 9.3 L/min) for 70 minutes and observed a linear rela-
tionship between pulmonary uptake (Q) and alveolar concentration (A) divided
by inspired air concentration (I)
Q = 0.72 + 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
concentration, the blood flow/tissue mass, and the relative solubilities of
TCI in tissues (Table 4-5). Therefore, TCI most rapidly attains equilibrium
4-7
-------
TABLE 4-4. 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)
Exposure concentration
and conditions
70 ppm,
70 ppm,
140 ppm,
140 ppm,
rest
rest + work
rest
rest + work
A
320
500
740
1050
Dose, mg
Subject
B
430
450
790
1100
C
330
470
710
970
D
470
660
790
1100
Mean
mg
390
520
755
1055
Retention value average = 38 percent.
Source: Monster (1976).
12
10
E 8
u
I 6
oc
<
I I I I
TRICHLOROETHYLENE
I
100
300 500 700
AVERAGE CONC. mg/m3
900
Figure 4-2. 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 exposure
period for a subject at rest, and at various levels of exercise. The
regression line is y = —0.489 + 0.014 X.
Source: Astrand and Ovrum (1976).
4-8
-------
TABLE 4-5. 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/air
Fat/air
Lecithin/air
Cholesterol/air
Cholesterol oleate/air
Triolein/air
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
Conversion factors:
1 ppm vapor in air = 5.38 mg/m3 at 25°C, 760 torr.
Source: Adapted from Sato et al. (1977).
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 (t, , 2.4 min) for VRG, 1.7 hr'1 (t^, 25 min) for MG, and
0.2 hr"1 (tj,, 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 percent, saturation and desaturation proceed more rapidly
from the MG compartment (tt , 25 min) than from the FG compartment (tj , 3.4 hr)
because of the considerably 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).
4-9
-------
4.1.3.2 Animals. Stott et al. (1982) have determined the pulmonary uptake of
o
TCI in mice and rats exposed to 10 and 600 ppm (54 and 3228 mg/m ). The
14
animals were exposed in a 30-L glass inhalation chamber to OKI, the concen-
tration of which was maintained within 10 percent of target. At termination
of exposure (6 hours), the cumulative pulmonary uptake was estimated by deter-
mining the radioactivity of carcass, and of TCI and metabolites in expired
air, urine and feces. The data for mice and rats are given in Tables 4-6 and
4-7, respectively. For mice, the body burdens for 6-hr exposure to 10 and
600 ppm (54 and 3228 mg/m3) were 78.5 and 3138 umol equivalents/kg (10.31 and
412.3 mg/kg; or 0.24 and 9.48 mg/mouse), respectively. For rats, amounts were
35.8 and 1075 umol equivalents/kg (4.7 and 141.3 mg/kg; or 1.03 and 31.09
mg/rat), respectively. For these species the 6-hr pulmonary uptake was not
directly proportional to inspired air concentration level, as observed in man.
Furthermore, total uptake of TCI, rat versus mouse, is more closely related to
p/o
their relative body surface areas—(220/23) ' = 4.5-fold--than it is to their
relative body weights, 9.6-fold. Presumably, this difference is primarily
related to the overriding influence of metabolism of TCI on uptake rather than
to differences in other factors such as ventilation, minute volume, and body
weight.
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 placental barrier. Helliwell and Mutton (1950), investigating the
distribution in pregnant sheep and goats, found that TCI appeared in the fetal
circulation within 2 minutes of exposure; at equilibrium, the fetal/maternal
blood concentration ratio was unity. The partition coefficients for various
tissues of rat and man are given in Table 4-5 (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, distribution, and excretion, based on experimental data, have pre-
dicted 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-hr inhalation exposure of 100 ppm (538 mg/m ). Figure 4-2
4-10
-------
TABLE 4-6. RECOVERY OF RADIOACTIVITY FOR 50-HOUR POSTINHALATION EXPOSURE OF
MALE B6C3F1 MICE TO 10 OR 600 PPM 14C-TCI FOR 6 HOURS1
10 ppm
umol-eq TCI/
kg body weight
Expired
1,1,2-TCI
C02
Urine
Feces
Cage wash3
Skin
Liver
Ki dney
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)
%2
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
1Average of four animals (±SD)
Percentage of recovered radioactivity.
3Primarily due to urine.
Source: Stott et al., 1982.
TABLE 4-7. RECOVERY OF RADIOACTIVITY FOR 50-HOUR POSTINHALATION EXPOSURE OF
MALE OSBORNE-MENDEL RATS TO 10 OR 600 PPM 14C~TCI FOR 6 HOURS1
Expired
1,1,2-TCI
C02
Urine
Feces
Cage wash3
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
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)
%2
21.1
2.9
55.3
3.6
1.4
7.1
1.6
0.16
7.0
78.9
1Average of four animals (±SD)
Percentage of recovered radioactivity.
Primarily due to urine.
Source: Stott et al. , 1982.
4-11
-------
shows that VRG and MG are nearly always in equilibrium with arterial blood
concentration, but the concentration of TCI in FG increases only very slowly
and continues to increase beyond the 8-hr exposure. During desaturation, TCI
in FG exhibits a long residence (about 40 hours) for disappearance with detec-
table FG concentrations remaining even after 70 hours. Because of the long
residence of TCI in FG, repeated daily exposure (6 hr/day; 100 ppm) results in
an accumulated concentration, as TCI from new exposures 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 relatively constant. For
a 6-hr/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.
4.1.4.2 Rodents. Following 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 minutes (dependent on dose) but a disappearance half-time of
215 minutes 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/day), found that blood
serum levels of TCI were not detectable (analyzed 2 hours after last dosing);
but adipose tissue levels averaged 0.28 and 20.0 ug 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 hours per day for 5 days to 200 ppm (1076 mg/m ) TCI. Table 4-8 gives
the values found. Seventeen hours after exposure on day four, blood and
adipose tissue levels were 0.35 and 0.23 mol/g, respectively, indicating long
storage in adipose tissue, though other tissues contained virtually no TCI.
With exposure on day five, tissue levels in brain, lungs, liver, fat, and
blood reached a steady-state within 2 to 3 hours. 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.
4-12
-------
oo
REPEATED EXPOSURE 100 ppm
(5 days, 5 hr. day)
Mo
Th FT
EXPOSURE, days
Figure 4-3. Predicted partial pressure of TCI in fatty tissue for a repeated exposure to
100 ppm, 5 days, 6 hours per day.
Source: Fernandez et al. (1977).
-------
TABLE 4-8. ORGAN CONTENTS OF TCI AFTER DAILY INHALATION EXPOSURE OF 200 PPM
FOR 6 HOURS PER DAY
Time (hr of
exposure) on
fifth day
0 [17 hr after
day 4- expo-
sure]
2
3
4
6
Cerebellum
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
Liver
nmol/g
0.08 0.04
4.9
± 0.3
5.5
± 1.4
5.8
± 1.1
5.6
± 0.5
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.
Source: Savolainen et al. (1977).
4.2 EXCRETION
The total elimination of absorbed TCI involves two major processes,
pulmonary excretion of unchanged TCI and hepatic biotransformation to urinary
metabolites. 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 concentration parallels blood concentration; both follow exponential
concentration decay curves showing three major components with approximate
half-times of 2 to 3 minutes, 30 minutes, and 3.5 to 5 hours (Figures 4-1 and
4-4), usually represented by VRG, MG, and FG compartments, respectively
4-14
-------
0.5
0.1
0.05
0.01
0.006
0.001
3 5
1
0.5
0.1
0.05
0.01
T I I \ I T
EXPIRED AIR
I I I I
0.0126»-°-1795t _
I I I I
I I I
8 9 10
I T I I I I I
I I
4 6B
HOURS
10
*Aax
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 concentration; V-), N/2, V"3 volumes of VRG, MG and FG com-
partments respectively.
Source: Sato et al. (1977).
-------
(Fernandez et al., 1977; Sato et al., 1977; Muller et al., 1974; Nomiyama and
Nomiyama, 1971; Monster et al., 1979).
Sato et al. (1977) developed a physiological kinetic model for TCI excre-
tion in man, based on experimental findings from controlled exposures of
volunteers. Four male medical students (avg. b.w.: 61.6 kg) inhaled 100 ppm
3
(538 mg/m ) for 4 hours. 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-hr 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+ -1.484- .0.184-
C . = 0.213E + 0.029E + 0.0126E
air
-16.7t -1.714- -0.204-
Cblood = °-115E + °-449E + 0.255E
These reflect first-order excretion from three compartments (VRG, MG, and FG,
respectively) of the model (Figure 4-4). As indicated in the model, C -
al T*
represents pulmonary clearance only, but C. , . represents whole-body clearance
by the pulmonary system and metabolism. The kinetic parameters were determined
as follows: V (volume of FRG, MG, and FG compartments), 8, 31, and 10/£,
respectively; metabolic clearance, 104 £/hr; partition coefficients (blood/air,
VRG/ blood, MG/blood, FG/blood), 10, 1.5, 1.0, 67; ta of FG, 3.4 hours.
Similar tri-exponential curves for decay of blood and exhaled air concentra-
tions 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 hours)
explains why, after a single short exposure (e.g., 4 to 6 hours), exhaled air
contains a notable concentration of TCI 18 hours later, and also why body
burden of TCI occurs with repeated, fluctuating daily exposures until steady-
state is reached (Section 4.1.4). Several investigators (Fernandez et al.,
1977; Monster et al., 1979) have shown that, for volunteers exposed to 70 to
3
100 ppm (377 to 538 mg/m ) 4 hours daily for 5 days, the concentration of TCI
in blood and exhaled air was two to three times higher 18 hours 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.
4-16
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4.2.2 Urinary Metabolite Excretion in Man
The urinary metabolites of TCI (total amount, excretion ratios, and
excretion time-course) have always been of considerable interest over the
years as quantitative indices of exposure and body burden. Recent investiga-
tions have determined that the half-time for renal elimination of TCE and of
TCE-glucuronide approximates 10 hours (range 7 to 14 hours) 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 concentration 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-time of TCE elimination, the half-time of
renal elimination of TCA produced during TCI exposure approximates 52 hours
(range 35 to 70 hours) (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 hours,
Paykoc and Powell, 1945; 51 hours, 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-time 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 hours postexposure before declining exponentially
for 10 to 15 days. The blood concentration 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 retained 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 hours after the last exposure (Muller et al., 1974;
Fernandez et al., 1977; Monster et al., 1979). Consequently the daily amount
4-17
-------
of TCA excreted into the urine increases during repeated 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+ hours) 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 hours after a single TCI
exposure or after repeated daily exposures (Soucek and Vlachova, 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 hours 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 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
elimination of TCI from blood of Wistar rats after intravenous (i.v.) admini-
stration 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 disappearance and best fitted a first-order two-compartment model,
with average kinetic parameters of K , 0.18 min ; V., 158 ml; k,„ and k~,,
0.059 and 0.39, respectively. For the highest dose (15 mg/kg), these investi-
gators 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, metabolism, and elimination, or that
three-compartment kinetics were followed at all dose levels, but with lower
doses, the third exponential component, representing the third compartment,
4-18
-------
was obscured by the limit of analytical sensitivity. The kinetic values for
the three-compartment model (using a dose of 15 mg/kg) were k , 0.174 min ;
Vd, 111 ml; k12, k21, k13: 0.183, 0.164, and 0.051, respectively. The half-
time, tj , for disappearance of TCI from peri renal fat was 217 minutes. Withey
and Collins (1980) state that they found no evidence in their kinetic analysis
of the disposition of i.v. 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.
However, other investigators have found clear evidence of dose-dependent
Michaelis-Menton metabolism kinetics in rats at higher doses (Section 4.4.2.2).
Prout et al. (1984) also have made observations on the overall rate of
elimination of TCI from the blood of rats and of mice. After single intra-
gastric doses of 1000 mg/kg in corn oil vehicle to rats and to mice, the blood
levels of TCI and principal metabolites were monitored by GC analysis. Figure
4-5 shows the results of these experiments. Peak blood TCI occurred at 1 hour
in the mouse, but rats exhibited a minor peak at 1.5 hours and a major peak at
3 hours. This absorption behavior in the rat has been previously reported by
Withey et al. (1983) and is characteristic of corn oil vehicle but not of
water solutions of TCI (Section 4.1.2). Following absorption and distribution
(post blood peak), blood TCI disappeared (pulmonary elimination plus metabolism)
in an exponential first-order manner with a half-life of about 1.75 hours for
the mouse and 2.25 hours for the rat, i.e., with nearly the same rate constant
in the two species. The primary hepatic metabolite, chloral (see Figure 4-5),
appears in mouse blood rapidly with a major peak at 2 hours, but in the rat
the peak was delayed to 10 hours; furthermore, the blood levels of chloral
were 2 to 4-fold higher in the mouse than rat. These investigators suggest
that these results indicate the metabolism of TCI in the mouse in considerably
more rapid than that of the rat. A further difference between the mouse and
rat was an apparent difference in the conversion of chloral to TCE and TCA.
Prout et al. (1984) stress that the more rapid conversion of chloral to TCA in
the mouse leads to an overall sustained elevation of blood levels of TCA in
the mouse (~7-fold; Figure 4-5, Panel D) than in the rat, although the relative
proportion of TCA to other metabolites (7 to 15 percent TCA) in urine, bile,
and feces was not greater than that of the rat (Table 4-9); indeed, the metab-
olite proportion appears independent of dose, species, or animal strain (Green
4-19
-------
Figure 4-5. Whole blood levels (jug/ml) of TCI and metabolites after single gavage dose of TCI
(1000 mg/kg) in corn oil to male Osborne-Mendei rats (triangles) and B6C3F1 mice (circles).
Source: Prout et al. (1984).
4-20
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TABLE 4-9. PROPORTION OF TCI METABOLITES (TCA AND TCE)
IN URINE OF RATS AND MICE AFTER SINGLE ORAL DOSES OF 14C-TCI
Dose
mg/kg
10
500
1000
2000
TCA
5.8
6.3
8.3
7.0
% of urine
Rat
TCE
91.5
90.1
88.4
91.9
radioactivity
Mouse
TCA
12.2
7.2
7.0
7.2
TCE
84.5
91.1
88.5
90.1
Urine collected for 24 hours from Osborne-Mendel rats and B6C3F1 mice.
Similar results from Alderly Park (Wistar derived) rats and Swiss-Webster
mice.
Source: Green and Prout (1984).
and Prout, 1984). However, as seen in Figure 4-5, Panel D, the area under the
TCA blood curve (AUC), as a measure of TCA production in the mouse, is 10-fold
greater than the AUC for the rat, with the implication of a considerably
greater production of TCA per unit dose in the mouse than rat.
Prout et al. (1984) also evaluated the excretion of TCA and of CO,, (as
measures of TCI), as a function of dose in both the rat and the mouse, respec-
14
tively. Animals were dosed with a solution of C-TCI in corn oil given as a
single intragastric administration at levels of 10, 500, 1000, and 2000 mg/kg.
14 14 14
C-radioactivity excreted in urine as C-TCA, and in expired air as CO^
(from TCA metabolism as one source) from 0 to 24 hours, was measured. Figure
14 14
4-6 shows the results. The sum of C-TCA and COp radioactivity excreted by
mice (B6C3F1) over the dose range 10 to 2000 mg/kg was linear with dose. In
contrast, the excretion of these metabolites by rats (Osborne-Mendel) was non-
linear and rate-limited, indicating in the rat (but not mouse) dose-dependent
kinetics for TCI metabolism to TCA.
Green and Prout (1984) have investigated the kinetics of urinary excretion
of TCI metabolites in mice (B6C3F1) during chronic administration of TCI.
Single intragastric doses of TCI in corn oil (1000 mg/kg) were administered
14
daily for 180 days. On days 10 and 180, C-TCI was incorporated into the
14 14
dose. The 24-hr excretion of unchanged C-TCI and of CO,, in the exhaled
4-21
-------
250r
200
i
O
u
Q
150
CO
Q
100
cc
u
X
CO
O
Q
50
O MOUS
-------
14 14
breath, and of total C-radioactivity in urine expressed as C-TCA and
14
C-TCE, were measured. Control animals (untreated) were given single doses
14
of C-TCI on days 10 and 180. The results are shown in Table 4-10. Chronic
dosing of TCI in mice for up to six months had no overall effect on the extent
of metabolism; a similar proportion of the TCI dose was excreted through the
14
lungs unchanged, and a similar proportion was metabolized ( C02 in exhaled
air plus total radioactivity in urine) after six months to that metabolized
after a single dose. These observations indicate that at this relatively high
dose (1000 mg/kg), induction of TCI metabolism in response to chronic admini-
stration did not occur, although TCI was undergoing nearly maximal (80 percent)
metabolism in any case. Hence, these observations might not pertain to "satur-
ating" doses of TCI (>2000 mg/kg; Figure 4-7). However, although there was no
overall increase in the proportion of TCI metabolized, a change in the relative
amounts of the major metabolites (TCA and TCE) in urine was observed. After
10 days of dosing and thereafter, the amount of TCA excreted in the urine was
double that found after a single dose (Table 4-5), while TCE (as the glucuro-
nide) decreased in proportion. These observations suggest that the oxidative
metabolism of chloral to TCA is increased at the expense of the reductive path-
way of TCE with chronic dosing as suggested by Green and Prout, or alternatively,
the kinetics of urinary excretion of TCA and TCE do not achieve a steady-state
with daily dosing of TCI until after 10 days, because of a relatively long half-
time for renal excretion of TCA. A similar phenomenon has been observed in the
human (Section 4.2.2) and in the rat (Muller et al., 1972, 1974).
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 concentra-
tions are likely to vary, air analysis does not necessarily provide a reliable
indication 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 retained as predicted by the known pharmacokinetics and metabolism of TCI.
Therefore, monitoring TCI levels in blood or expired air and measuring urinary
metabolites 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;
4-23
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TABLE 4-10. EFFECT OF CHRONIC DAILY ORAL DOSING OF TCI (1000 mg/kg) ON METABOLISM AND
METABOLITE EXCRETION IN B6C3Fa MICE*
Day
Unchanged
TPT rn~
i \* x *•**"' 2
Uri ne
Feces
As % of Dose (24-hr Collection)
1
10
Control**
180
Control
17.5
13.6
15.4
23.7
12.5
± 8
±8.6
± 8.6
± 6.2
± 2.0
6.0
6.3
5.3
2.3
4.2
±0.7
± 0.6
± 1.0
±1.0
± 0.6
41.8
52.3
46.9
48.3
50.7
± 6.5
± 7.8
± 10
± 6
± 5.1
13.1 ±9.5
16.4 ± 3.3
12.3 ± 1.9
1.9 ± 0.5
1.3 ± 0.5
Urine
TCA
As
i
7.
15.
8.
19.
10.
Urine
TCE
% of 14C-TCI Radioactivity
n 24-hr urine collection
0
6
4
9 ± 3.1
5 ± 2.4
88.5
81.9
88.1
77.4 ±
86.9 ±
2.8
1.8
*N = 4 test, N = 3 control.
**Test and untreated control mice received single doses of 14C-TCI (1000 mg/kg: 10 uCi) on day 10 and day 180.
Source: Green and Prout (1984).
-------
700
600
500
300
oc
D
200
100
1 I I I I
I I | I I I I I I I
MEANS±SEM
r2 . 0.995
a • 0.318
I
I
400 800 1200 1600 2000
TCI DOSAGE, mg/kg
2400
2800
3200
Figure 4-7. Relationship between TCI dose and the amount of total urinary metabolite excreted
Sr day by mice in each group. Values represent means ± SEM. N = 7-9 mice per group except
r the 100 and 3200 TCI/kg groups where N = 3 and 4 respectively. The slope (0.318) and r2 value
coefficient (0.996) are for linear regression fit of the 100 -1600 mg/kg data points.
Source: Buben and O'Flahery (1984).
4-25
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Pfaffli and Blackman, 1972; Kimmerle and Eben, 1973a,b; Lowry et a!., 1974;
Vesterberg et al., 1976). These methods, however, are subject to high inter-
personal 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 xenobiotics, 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-hr 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) con-
cluded 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 exposure 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
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 TCE, TCE-glucuronide ("urochloralic acid"), and 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 anesthe-
tized with TCI. Powell (1945, 1947) demonstrated the presence of TCA in both
4-26
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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
previously discovered that the hypnotic, chloral hydrate, was metabolized to
TCE, TCE-glucuronide, and TCA (Butler, 1948), hypothesized that this widely
used hypnotic 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 and TCA. However, Butler could not demonstrate
the presence of chloral hydrate in either plasma or urine. Nonetheless, this
demonstration of biotransformation of TCI to TCE as well as to TCA was the
first proof that TCI anesthetic was converted to a metabolite with hypnotic
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) and with
isolated hepatocytes (Costa and Ivanetich, 1984).
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 metab-
olize TCI to TCE, and TCA. Other organ tissues, such as kidney, spleen and
small intestine, may also metabolize TCI, as these 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).
In addition to the major end-product metabolites of TCI, chloral, TCE,
TCE-glucuronide and TCA, a number of other metabolites have been found in
urine or exhaled breath (CO, C02, monochloro- and dichloroacetic acids, TCA-
glucuronide, oxalic acid, N-(hydroxyacetyl) aminoethanol (HAEE), chloroform)
or ir\ vitro with microsomal systems (TCI-epoxide, glyoxylic acid, CO). These
metabolites are listed in Table 4-11. Except for HAEE, oxalic acid, TCA-CoA
conjugate, 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 4-11) to formic acid, dichloroacetic acid,
glyoxylic acid, and CO. These metabolites may, therefore, be formed at cellular
extra-microsomal sites of TCI-epoxide formation under aqueous conditions.
4-27
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TABLE 4-11. IDENTIFICATION BY GC/MS OF TCI METABOLITES IN EXHALED AIR
AND URINE OF WISTAR RATS AND NMRI MICE AFTER A SINGLE ORAL DOSE
OF 200 mg/kg OF 14C-TCI
Rat
Mouse
% of dose
radioactivity
% of dose
radioactivity
Exhaled air
Unchanged 14C-TCI
14C02
Urine
52.0
1.9
41.2
% of
11.0
6.0
76.2
urine radioactivity
Oxalic acid3
Dichloroacetic acid
N-(Hydroxyacetyl)-
aminoethanol (HAAE)
Trichloroacetic acid
Trichloroethanol (free)
Trichloroethanol
(conjugated)
1.3 0.7
2.0 0.1
7.2
15.3
11.7
61.9
4.1
0.1
0.1
94.2
Metabolites identified by GC-MS; female Wistar rats and NMRI mice; collec-
tions for 72 hr.
Source: DeKant et al. (1984).
Until recently, a thorough chemical isolation and identification of TCI
metabolites by rigorous methodologies (GS-MS, etc.), in one or more species,
had not been attempted. Dekant et al. (1984) have now reported their results
14
of a comparative study in rats and mice of C-TCI metabolites in breath and
in urine, where metabolite isolation was accomplished by reverse-phase HPLC
and identification by radio-GC and GC-MS. The metabolites identified and
their relative abundance are given in Table 4-11. All metabolites found in
the rat were also found in the mouse. The urine radioactivity represented 41
and 76 percent of the total radioactivity given to rats and mice, respectively,
4-28
-------
14
from a 200-mg C-TCI/kg dose. The radioactivity in feces represented less
than 5 percent of the dose with total recovery in urine, feces, and breath of
93 to 98 percent. Two new urinary metabolites were found in both species,
oxalic acid and HAAE. Dekant et al. also identified significant amounts of
HAAE in the urine of human volunteers exposed to 200 ppm (1096 mg/kg) TCI for
6 hours. However, the occurrence of oxalic acid in urine and of COp in breath
have not as yet been identified in man as specific metabolites of TCI; to do
14
so would require administration of labeled TCI (radioactive C-TCI or stable
14
isotope C-TCI) since both compounds are also normal endogenous compounds of
intermediary metabolism.
The data of Dekant et al. (Table 4-11) would indicate that TCA is not a
prominent urinary metabolite of TCI in NMRI mice, in comparison to Wistar rats
(or man, Section 4.2.2). Green and associates (1982, 1984) have postulated a
TCA-CoA conjugate in the mouse, which is slowly excreted via the biliary
system with the feces. Dekant et al. (1984) have also suggested that an
intense enterohepatic recirculation of TCA (as an alkali-labile conjugate) may
occur in this species. However, the small percentage of dose radioactivity
appearing in the feces of the mice (<5 percent), would not indicate this route
is important in the excretion of TCA.
In contrast to the observations of Dekant et al., Green and Prout (1984)
found a much greater relative proportion of TCA to TCE in mice (Table 4-9),
and also they found no difference in the proportion (TCA/TCE) between mouse
and rat species, or between different strains of these species. The propor-
tions were unaffected after single oral doses between 10 and 2000 mg/kg.
Furthermore, the proportion of TCA/TCE increased with chronic exposure to TCI
in the mouse (Green and Prout, 1984), an observation which has been shown also
for the rat and for man (Section 4.2.2). Green and Prout (1984) found that
not only were the relative proportions of the major metabolites TCA and TCE in
urine very similar in rats and mice but also minor metabolites (dichloroacetic
acid, monochloroacetic acid, free TCE) were also similar, and they therefore
concluded that there is not a major difference in pathways of TCI metabolism
in these two species.
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
4-29
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retained dose after vapor exposure by measuring pulmonary elimination of TCI
and metabolites 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 4-8) 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/m ) for 3 hours (equivalent to 25 mg TCI/kg
b.w.). Furthermore, the alveolar to inspired air concentration ratio (25 per-
cent; 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 anesthe-
tic 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-hr
exposure.
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 (274 to 2082
mg/m ), an average of 11 percent of retained TCI is eliminated unchanged by
the lungs (tj , 5 hr), 2 percent of the dose is eliminated as TCE by the lungs
"i
(t^, 10 to 12 hr), and 58 percent is eliminated as urinary metabolites (Fernandez
et al., 1975, 1977; Muller et al., 1977; Monster et al., 1976, 1979). Unac-
counted for is nearly 30 percent of the TCI dose. These investigators suggest
additional pathways or routes of elimination of one or more unknown metabolites.
This suggestion is particularly apropos in light of the recent demonstration
4-30
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600
100
200
300
ENVIRONMENTAL TRICHLOROETHYLENE CONCENTRATION, ppm
Figure 4-8. Relationship between environmental TCI concentration and urinary
excretion of TCI metabolites in human urine. Urine was collected
for 6 days after exposure. (Adapted from Nomiyama and Nomiyama,
1977).
by Dekant et al. (1984) of two new metabolites of TCI excreted in the urine of
rats and mice, oxalic acid and HAEE. 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 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 in humans 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 ubiqui-
tous 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.
4-31
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4.4.2.2 Animals. There is an increasing body of experimental evidence that
the magnitude of metabolism of TCI by rodents (rat and mouse) is dose-dependent
and possibly best described by Michaelis-Menten metabolism kinetics. Thus, at
very high doses, TCI metabolism exhibits saturation kinetics in both the mouse
and the rat, but differently, in that evidence of approaching saturation
occurs in mice at about 2000 mg/kg (absolute dose—60 mg/animal), and in rats
at about 500 to 1000 mg/kg (absolute dose--125-250 mg/animal). Hence, both
species have a considerable capacity to metabolize TCI. Furthermore, the
maximal capacities of the rat versus the mouse appear to be more closely
related to relative body surface areas (on an absolute dose/animal basis) than
to body weight.
The types of studies that have been done to investigate dose-metabolism
relationships are fourfold: 1) single-dose studies across species using
14
C-TCI and urinary radioactivity and/or other methods as measures of metab-
olism, 2) kinetic models based on inhalation kinetics, 3) comparative balance
14
studies between rat and mouse with C-TCI at 1 or 2 dose levels, 4) multiple
dose levels studies in rat and/or mouse.
Across species: Early studies by Butler (1948) demonstrated the extensive
metabolism of TCI in dogs for which he estimated the extent of metabolism (as
a percentage of the administered dose) to be 60 to 90 percent. Muller et al.
(1982) recently compared the metabolism of TCI in chimpanzees, baboons, and
14
rhesus monkeys. C-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 chimpanzee excreted only from 2 to 5 percent of
the total eliminated TCI metabolites in the feces, baboon and rhesus monkey
excreted 18 to 35 percent of the radioactivity in the feces. Rodents have
been reported to excrete in feces 3 to 8 and 17 to 19 percent for the rat and
mouse, respectively, of a radioactive dose of TCI (Table 4-12, Prout et al.,
1984). Green and Prout (1984) have directly demonstrated by bile duct cannula-
tion that TCI metabolite(s) are excreted in bile by both rats and mice.
4-32
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TABLE 4-12. DEMONSTRATED METABOLITES OF TCI (OTHER THAN
CHLORAL AND CHLORAL DERIVATIVES: TCE, TCE-GLUCURONIDE, AND TCA)
Metabolite
TCI-epoxide
Glyoxylic acid
Carbon monoxide
System
rat, mouse
microsomes
rat, mouse
microsomes
rat, mouse
microsomes
Investigator
Miller and Guengerich,
Miller and Guengerich,
Miller and Guengerich,
Traylor et al. , 1977
Fetz et al . , 1978
1982
1982
1982
Carbon dioxide
Monochloroacetic acid
Dichloroacetic acid
Oxalic acid
N-(Hydroxyacetyl)-
aminoethanol (HAAE)
TCA-glucuronide
TCA-CoA
Chloroform
exhaled air
rat and mouse
human and
rabbit urine
mouse and rat
urine
rat and mouse
urine
rat and mouse
urine
rat, mouse,
human urine
chimpanzee urine
mouse feces,
bile
rat adipose
tissue, serum,
urine
Parchman and Magee, 1982
Stott et al., 1982
Dekant et al., 1984
Green and Prout, 1984
Soucek and Vlachova, 1960
Ogata and Saeki, 1974
Green and Prout, 1984
Hathaway, 1980
Dekant et al., 1984
Green and Prout, 1984
Dekant et al., 1984
Dekant et al., 1984
Muller et al., 1982
Green et al., 1982
Green and Prout, 1984
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, 1978
Kline and Van Duuren, 1977
Bonse et al., 1975
Miller and Guengerich, 1982
4-33
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Inhalation kinetics: Filser and Bolt (1979) have exposed rats (Wistar)
to TCI in a closed system (desiccator jar chamber) and determined, from disap-
pearance of TCI from the chamber the inhalation pharmacokinetics, in accordance
with the following model:
vi k12 , K
V2 metabolism.
Chamber rat
atmosphere
They found that the metabolism of TCI was dose-dependent in the rat, with the
o
saturation point occurring at 65 ppm (350 mg/m ). Zero order V was 210
fllCL/\
umol/hr/kg and first-order clearance (below 65 ppm) was 77 L/hr/kg b.w.
Andersen et al. (1980) also analyzed the pharmacokinetic behavior of TCI
in rats (Fischer 344) using a Michaelis-Menten approach to demonstrate dose-
dependent metabolism. Exposures were conducted in a 31-L battery jar chamber,
and the rate of depletion of atmosphere TCI was determined for 30, 100, 1000,
3500, and 8000 ppm (116, 538, 5380, 18,830 and 43,040 mg/m3). TCI uptake was
fitted to a four-compartment model as follows:
TCI Blood, Fat, bone,
atmosphere < _ liver, etc. < _ etc.
ffl 32
Metabolites
The metabolism of TCI was found to be dose-dependent, with a saturation point
of about 1000 ppm (5380 mg/m ) (Km, 463 ppm). The kinetic parameters, calcu-
lated as molar equivalents TCI, were Vmav, 185 umoles/kg/hr; K, 292 umoles/
MlaX III
hr/kg for uptake from 31-L chamber. A "whole animal "/air partition coefficient
of 15.4 (as compared to 9.0 for blood/air) was calculated and suggested to be
a more accurate estimate of microsomal-to-gas distribution than a blood/gas
constant.
Balance studies: For rodents (rat and mouse), the extent of metabolism
after single doses of TCI has been estimated by balance studies using isotopi-
cally labeled TCI. Daniel (1963) carried out the earliest study in rats. The
4-34
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36
rats were dosed by stomach tube with C£-1abe1ed 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 hours; urinary metabolites TCE, TCE-glucuronide, and
TCA accounted for only 15 percent of the dose. More recent studies show
considerably different results.
Dekant et al. (1984) have compared the metabolism of TCI by balance
studies, with female rats (Wistar) and with female mice (NMRI). These results
14
are shown in Table 4-9. C-TCI was administered as a single dose by gavage
in corn oil at 200 mg/kg. The absolute dose given to the rats, based on their
experimental weights, was about 48 mg and to the mice about 5.1 mg. Radioac-
tivity was determined in urine, feces, carcass, and exhaled air for 72 hours
post administration. Virtually complete oral absorption occurred with a total
recovery of radioactivity of 93 to 98 percent. For this single dose, rats
excreted through the lungs 52 percent of the dose unchanged (~25 mg), while
the mice excreted only 11 percent (~0.56 mg). Hence, the rats metabolized an
average of 48 percent of their dose (23 mg) and the mice 89 percent (4.54 mg).
The ratio of the amounts metabolized by the rat versus the mouse (5.05) closely
approximates their relative surface areas, which as calculated from their
2/3
experimental body weights (rat b.w./mouse b.w.) is 4.46. Therefore, the
metabolic capacity of rats and mice for TCI appears to be proportional to
their surface areas. On a mg/kg basis the mice would appear to metabolize TCI
to an extent 1.9 times greater than rats. Furthermore, the high proportion of
the dose (52 percent) excreted unchanged via the lungs would suggest that a
single, orally administered 200 mg/kg dose exceeded the overall hepatic capacity
of the rat resulting in a significant first-pass effect.
Stott et al. (1982) exposed rats and mice to 10 and 600 ppm (54 and 3228
3 14
mg/m ) C-TCI for 6 hours. Their results are given in Tables 4-6 and 4-7.
For exposed mice, virtually 100 percent of the body net uptake was metabolized
with no evidence of metabolic saturation. The body burden (dose) from the
6-hr exposure was estimated as 78.5 umol of TCI/kg body weight (10 mg/kg) and
3138 umol of TCI/kg (412 mg/kg), respectively. In contrast, for rats 98
percent of the net uptake from 10 ppm exposure was metabolized but only 79
percent at 600 ppm (3228 mg/m ) exposure, suggesting an incremental approach
to saturation of metabolism in this species. For rats, the inhalation doses
were 35.8 umol/kg (4.7 mg/kg) and 1075 umol/kg (141 mg/kg), respectively.
These results indicate a saturation of metabolism in the rat. Support for
4-35
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this conclusion is found also in the observation that pulmonary elimination of
unchanged TCI was only 2 percent of the dose from 10 ppm (54 mg/m ) exposure
but 21 percent of the dose at 600 ppm (3228 mg/m ) (Table 4-7). 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
o
mg/m ) (Tables 4-6 and 4-7). However, on a relative body surface area basis,
the ratio for the amounts metabolized by the rat versus the mouse approximated
their relative body surface areas.
Multiple dose studies: Dose-dependent kinetics as opposed to first-order
kinetics requires multiple dose level studies to rigorously differentiate
first-order kinetics and to appropriately evaluate the associated kinetic
parameters. Two investigative groups, Prout and Provan (1984) and Buben and
0'Flaherty (1984), recently have used this approach to examine the kinetics of
TCI metabolism in rats and in mice.
Buben and 0'Flaherty administered doses of 0, 100, 200, 400, 800, 1600,
2400, and 3200 mg/kg of TCI in corn oil by intragastric intubation to male
Swiss-Cox mice in groups of 7 to 9 mice. The control groups consisted of 24
mice. These doses were given 5 days/week for 6 weeks. The subchronic dosing
ensured that steady-state conditions for metabolism of TCI and for metabolite(s)
excretion were achieved. Metabolism was measured by determining 24-hr urine
collections of TCA, TCE, and TCE-glucuronide, as determined by GC. Of the two
other metabolites assayed, ethylene glycol proved negative, and oxalic acid
was not increased over excretion by control animals. Metabolites in feces
represented less than 5 percent of the amount in urine. No significant day-to-
day variability in urinary metabolites excreted or week-to-week trends was
found.
The use of "total urinary metabolites" (TCA, TCE, and TCE-glucuronide) as
an index of metabolism is an approximation, as stressed by Buben and 0'Flaherty
themselves, because there is experimental evidence that HAEE and oxalic acid
may be significant urinary metabolites of mice, 0.7 and 4.1 percent, respec-
tively (Table 4-12), and COp in exhaled breath may amount to 6 to 9 percent of
the TCI dose (Tables 4-2, 4-6, and 4-12).
Thus, "total urinary metabolites" may underestimate actual metabolism by
at least 20 percent or more. However, urinary TCA, TCE, and TCE-glucuronide
excretion, as the principal metabolites, provides an appropriate reflection of
overall TCI metabolism.
4-36
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Figure 4-7 shows the relationship between dose and metabolism ("total
urinary metabolites") found by Buben and 0'Flaherty in mice. Up to a dose of
about 1600 mg/kg, a linear relationship between dose and amount metabolized
obtained with a constant 27.5 percent of the dose metabolized to the urinary
metabolites TCA, TCE, and TCE-glucuronide. This percentage of dose metabolized
is considerably lower than that observed above by Dekant et al. (1984) and
Stott et al. (1982) for mice. Above the 1600 mg/kg dose, the metabolism of
TCI shows a rapid decrease of the percent of dose metabolized and approached a
saturation of metabolism at about 2400 mg/kg. These observations would indicate
that TCI follows first-order metabolism (proportion to dose) in the mouse up
to a relatively high dose of 1600 mg/kg (~48 mg/mouse) and thereafter dose-
dependent and then zero-order kinetics follow at higher doses. Buben and
0'Flaherty did not fit their data to the Michaelis-Menten equation, although
it would have been instructive.
Prout et al. (1984) have carried out a similar investigation in rats
(Osborne-Mendel and Alderly Park Wistar) and in mice (B6C3F1 and Swiss Webster).
However, in this case the dosage series (10, 500, 1000, and 2000 mg/kg) was
given as single doses (i.e., not subchronically) in corn oil by stomach tube
14
as C-TCI. The metabolism can be expected therefore to reflect that of
single doses and not of steady-state metabolic conditions. But the use of
14
C-TCI allows a complete estimate of total metabolism. Thus, Prout et al.
14
(1984) measured C-radioactivity (expressed as percent of dose) in urine,
feces, and expired air over a collection period of 72 hours, with total
recovery of administered dose-radioactivity of 92 to 98 percent. Their results
are tabulated in Table 4-2 for Osborne-Mendel rats and for B6C3F1 mice;
entirely similar results were observed for Wistar rats and Swiss-Webster mice,
i.e., the disposition of TCI was independent of rat or mouse strain. The data
of Table 4-2 indicate that for both the rat and mouse, the percent of the dose
metabolized decreased with increase of dose size, and in tandem, the percent
of the dose excreted via the lungs as unchanged TCI increased with increase of
dose size. However, these results were more evident and occurred to a much
greater extent in the rat than in the mouse. Thus, a plot of dose against the
portion of the dose metabolized, Figure 4-9, indicates that for the rat the
metabolism of TCI approaches saturation at a dose of about 1000 mg/kg (~200
mg/animal), while for the mouse the metabolism of TCI is still linear up to a
dose of 2000 mg/kg (~60 mg/animal). These results with the mouse are in
4-37
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2000
1600
l 200
Q
iU
N
8
800
400
-O— B6C3F1 MICE
-D SWISS WEBSTER MICE
-A—-A.P. RATS
•O~OSBORNE-MENDEL RATS
500
1000
TCI DOSAGE, mg/kg
1500
2000
Figure 4-9. Relationship between administered single oral doses of c-TCI to rats and mice and
amount of dose metabolized in 24 hr, expressed as mg/kg b.w., as calculated from 14c- radio-
activity excreted in urine, feces, and expired air (other than unchanged 14C-TCI). Each data point
represents 4 rats or mice.
Source: Prout et al. (1984).
4-38
-------
accord with those of Buben and 0'Flaherty, who found that metabolism was not
saturated in mice up to doses of 2400 mg/kg (Figure 4-7). It can be concluded,
therefore, that metabolism of TCI is dose-dependent and saturable at doses
above 500 to 1000 mg/kg in the rat and 1600 to 2400 mg/kg in the mouse. Both
species, therefore, exhibit a very high hepatic capacity to metabolize TCI.
At lower doses in these species, metabolism is nearly linearly related to TCI
dose, i.e., "first-order" metabolism prevails. However, it is likely that in
both species TCI metabolism can be best described overall by Michaelis-Menten
kinetics, particularly at high dose levels.
4.4.3 Enzyme Pathways of Biotransformation
Current knowledge of the details of the enzymatic biotransformation of
TCI derives mainly from jm vitro studies, principally with liver cell fractions,
and from iji vivo studies with end metabolite identification in exhaled breath,
urine, feces, and bile. Figures 4-10, 4-11, and 4-12 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 trichloroethylene epoxide (Figure 4-10). The
formation of chloral/epoxide is the rate-limiting step in the metabolism of
TCI (Ikeda et al., 1980; Nomiyama and Nomiyama, 1979). Intramolecular rear-
rangement with migration of a chlorine atom is presumed to occur in the forma-
tion of chloral hydrate, in order to explain the genesis of TCE and TCA.
Conversion of chloral hydrate to TCE and TCA is shown in Figure 4-11.
4.4.3.1 Formation of ch1ora1--Byington and Leibman (1965) conclusively identi-
fied chloral hydrate as the product of TCI when incubated with rat liver
microsomes fortified with NADPH and Oy (^450 niono-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. Costa and
Ivanetich (1984) found that chloral hydrate was the major metabolite in isolated
TCI-treated hepatocytes from phenobarbital-pretreated or untreated rats.
Daniel (1963) provided evidence that chloride migration must occur in chloral
oc
formation since no dilution of Cl atoms with chlorine atoms from other body
4-39
-------
CI
H
/CI NADPH, 02
CI
\
H
CHLORIDE 1
^ C
MIGRATION ff
O
— CI
—
ci>
EPOXIDE
1
OH
H2O
OH ""
-i_ckCI
1 XQ
H _
CHLORAL
2HCI
o=c'
CI
o
o
H
H
H
+ CO
O
O
C C'
CK ^H
H20
H
H
CO2
FORMIC ACID AND
CARBON MONOXIDE
GLYOXYLIC ACID
Figure 4-10. Postulated scheme for the metabolism of trichloroethylene to chloral
(see Figure 4-11 for chloral metabolism) and to trichloroethylene epoxide and its
metabolites.
Source: Miller and Guengerich (1982).
4-40
-------
MITOCHONDRIA
CYTOSOL
NAD +
ALDEHYDE
DEHYDROGENASE
C CI3 COOH
[TCA]
CHLORAL
C CI3 CHO
CYTOSOL
NADH
ALCOHOL
DEHYDROGENASE
NADPH
ALDEHYDE
REDUCTASE
MICROSOMES
NADPH, 02
C CI3 CH2OH
[TCE]
GLUCURONYL
TRANSFERASE
C CI3 CH2O C6H9O6
[TCEGLUCURONIDE]
Figure 4-11. Metabolism of chloral hydrate.
Source: Modified from Ikeda et al. (1980).
4-41
-------
Cl Cl
\ / NADPH, 02
C = C
/ \ P-450
H Cl
Cl . i Cl Lewis H Cl
\/\/ . \
C —C
/ \
H Cl Cl-shift 0 C1N,
TCA
— *" c—c-ci
Catalyzed // \
TCE
Cl 0
\
CHLORAL
EPOXIDE
I
I
I H20
4-
OH OH rl n H20
Cl I \ ^- Cl
H"^^*^ LI
C-C-Cl > C12CHCOOH
\
H
DICHLOROACETIC
ACID
C — C
Cl
\
H
GLYOXALIC
ACID CHLORIDE
oxidation
hydrolysis
HO
\
OH
OXALIC
ACID
Figure 4-12.
phosphatidyl-
ethanolamine
alkylation
r — r
\
N - CH2 - CH2 - 0 - phosphatidyl
1 - phosphatidyl
I
reduction
HO — CH2— C '
NH — CH2 — CH2—OH
N-(HYDROXYACETYL)-AMINOETHANOL
Postulated scheme for the metabolism of TCI based on urinary
metabolite profiles of rats and mice. From Dekant et al.,
1984. Suggested pathways are indicated by dashed arrows.
4-42
-------
36
pools was observed in the conversion of Cl-TCI to its end products TCE and
TCA. Microsomal P.5g-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-trichloroethene oxide) has also been directly demonstrated
in liver microsomal preparations by trapping with p-nitrobenzyl pyridine
(Miller and Guengerich, 1982). In addition, a specific P.™ 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 with microsomes
from animals pretreated with phenobarbital but not 3-methylcholanthrene (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 conditions (ti ~ 1.3 minutes in water at pH 7.4, 37°C) Bonse et al.,
1975; Kline and Van Duuren, 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 (FeCl_, A1C1-, BF_), and Henschler and co-
workers proposed that the formation of chloral iji vivo could therefore be due
to catalytic action of the iron of P45Q in the trivalent form at the hydrophobic
microsomal site of formation of the oxide (Figure 4-2). Although Miller and
Guengerich (1982) found that excess ferric iron salts catalyzed the rearrange-
ment of TCI-oxide to chloral in nonaqueous solutions (CH^Clp or CH3CN), chloral
was not formed in aqueous media even when iron salts, ferriprotoporphyrin IX
or purified cytochrome P4c0» were present.
In studies with rat or mouse liver microsome fortified with NADPH and Op
(or a reconstituted P.™ 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.
4-43
-------
Addition of TCI-oxide to these systems did not produce chloral, but the oxide
disappeared with a tj -17 seconds or, in the presence of rat liver epoxide
hydrolase, 9 seconds. 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. Alternatively, these investigators postu-
lated an oxygenated TCI-P.-n transition state leading to two products: chloral
(with chloride migration) and a separate epoxide formation, as shown in Figure
4-11. The working scheme for the metabolism of TCI as presented in Figure 4-11
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 while rearrangement to chloral occurred in non-
aqueous media in the presence of excess ferric iron salt, the incubation of
TCI-oxide with purified P.50 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 P.5Q-mediated reaction
to chloral may be preferential. The possibility of TCI-epoxide being a secon-
dary, minor product in the metabolism of TCI may account for the lesser muta-
genic and oncogenic potential of TCI in comparison to other halogenated ethy-
lenes (Bolt et al., 1982; Dekant et a!., 1984).
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 oxida-
tion, presumably by cytochrome P.™ 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 chlorine bonded to the carbon, which is oxidatively attacked, is
released (dechlorination).
Cl+ 0"
nn n ' \ I n
/Cl C\ s \ 4 ^ 0
C = C ' -> C C -»• CKC — C '
A»T ^ ^ M n S U ^ >• U
LI ^ n \i i n n
This mechanism also results in "reactive" intermediates which can covalently
bind to cell constituents.
4-44
-------
4.4.3.2 Metabolism of chloral hydrate—The enzymatic pathways for the biotrans-
formation 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 4-12. 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 i_n 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 (K ,
-3
2.7 x 10 M, horse enzyme). The reaction is essentially unidirectional, since
conversion back to chloral hydrate has not been observed with this enzyme
(Sellers et al., 1972b; 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
-3
of 6.0 x 10 M. These investigators also found that rat liver microsomes in
the presence of NADPH and 02 oxidized TCE to chloral similarly to the oxidation
of methanol and ethanol to acetaldehyde (Liebler and DiCarli, 1969).
-4
Human red cells also reduce chloral hydrate to TCE (K , 4.0 x 10 M)
(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 defined as clearly. The most likely reaction for the oxidation of
chloral hydrate to TCA would involve the well-known enzyme acetaldehyde dehy-
drogenase. However, chloral hydrate has been reported not to be a substrate
for human acetaldehyde dehydrogenase (Kraemer and Deitrich, 1968; Blair and
Bodley, 1969; Sellers et al., 1972b). Cooper and Friedman (1958) obtained
4-45
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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 dehydrogenase 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 after 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 respectively in mitochondria and microsomes. Grunett (1973) found
the NAD-dependent mitochondrial enzyme to have a broad substrate specificity,
but chloral hydrate was not a substrate. However, Ikeda et al. (1980) found
that an aldehyde dehydrogenase prepared from rat liver mitochondria (NAD-
-3
dependent) converted chloral to TCA (K , 62.5 x 10 M). 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 contribut-
ing 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. Dal bey 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 dehydro-
genase. As noted above, human red blood cells actively reduce chloral to TCE.
4.4.3.3 Minor Metabolites and Pathways—For rats and mice exposed to TCI,
several metabolites (C02, oxalic acid, dichloroacetic acid, HAAE; Tables 4-11
and 4-12) other than TCA, TCE, and TCE-glucuronide have been identified in
4-46
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exhaled air, urine, feces, or bile. Some of these minor metabolites have also
been identified in human urine (dichloroacetic acid, HAAE). Together, these
metabolites represent less than 15 percent of the TCI dose in rodents (Table
4-12).
Dekant et al. (1984) have suggested that the appearance of small amounts
of dichloroacetic acid in the urine of rats and mice (Table 4-12), and possibly
of man (Table 4-11), stems from a limited rate of conversion of TCI-oxide to
chloral with escape of the epoxide from the hydrophobic environment of P^n,
and subsequent decomposition in hydrophilic conditions to dichloroacetic acid
and other compounds.
COp from TCI metabolism has been postulated by Miller and Guengerich
(1982) to originate from hydrolytic rearrangement or cleavage of the epoxide
to formyl chloride and glyoxalic acid chloride with hydrolysis to formic acid
and glyoxalic acid, respectively (Figure 4-11). These two metabolites, entering
the endogenous one- and two-carbon pools, are then metabolized further to CCL.
Green and Prout (1984) have shown that CCU may also be the product of decarbox-
ylation of TCA and dichloroacetic acid. These investigators have administered
14
C-TCI to rats and mice by gavage or intravenously and demonstrated that 7 to
14
15 percent of the dose was recovered in exhaled breath as CCL within 24
hours. Green and Prout suggest the formation of a coenzyme A ester of TCA
followed by degradation to oxalyl CoA and subsequent release of two moles of
co2.
Dekant et al. (1984) have recently identified two new metabolites of TCI,
oxalic acid and HAAE, in the urine of rats and mice. These investigators
suggest that oxalic acid may be formed as the end-product of enzymatic or
non-enzymatic cleavage of TCI-oxide as shown in Figure 4-13. Hydrolytic ring
opening produces the glycol which is expected to undergo spontaneous elimina-
tion of two molecules of HC1 to form glyoxalic acid chloride with subsequent
hydrolysis to glyoxalic acid, which then is oxidized further to oxalic acid.
The spontaneous formation of glyoxalic acid from TCI-oxide decomposition in
aqueous solution has been amply demonstrated (Table 4-12). HAAE also had not
been identified previously as a metabolite of TCI. Dekant et al. suggest that
TCI-oxide may interact with ethanolamine or phosphatidyl-ethanolamine in cell
membranes to produce HAAE as indicated in Figure 4-13. The minor metabolites
of TCI may occur as follows: if it is assumed that the conversion of TCI-oxide
to chloral is a rate-limiting step in the P.50-mediated metabolism of TCI,
4-47
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then at high substrate concentrations of TCI an accumulation of TCI-oxide may
occur and activate alternative pathways leading to the formation of metabolites
other than chloral.
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 TC! to chloral, as
outlined in Figures 4-11 and 4-13, suggest 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 P.™ systems to epoxide
intermediates which are subsequently "detoxified" by molecular rearrangement
(in the case of TCI to chloral). Further, reactivities, and hence, toxicities
of individual epoxide intermediates depend on the type of chlorine substitu-
tions, 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 carcino-
genic potential 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 premises of the P.J-Q 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 (dichloro-
acetyl chloride, formyl chloride) (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 P.50 oxidative products with chloral production four- to five-fold
4-48
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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 4-13 lists investigators who have demonstrated
covalent binding of TCI to microsomal protein nucleic acids and lipids i_n
vitro. In many of these experiments, j_n vitro covalent binding from TCI
microsomal metabolism was decreased by animal pretreatment with SKF-525A
(which inhibits P.5Q metabolism) or enhanced by phenobarbital and other in-
ducers 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 mercapto-
ethanol, indicating the importance of cellular sulfhydryl groups and glutathione
specifically for providing a "detoxification mechanism" for reactive inter-
mediates from TCI metabolism. These experiments provide ample evidence that
TCI is metabolized jji vitro to reactive products by P.™ 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). Mouse 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 months
old) have less capacity for microsomal metabolism, as reflected by covalent
binding of TCI, than either adult (11 months old) or young (4 months) rats.
These investigators have also compared the rat hepatic microsomal covalent
binding to added DNA for a series of aliphatic ha!ides, including TCI. The
degree to which TCI and other aliphatic halides form a DNA-adduct is summa-
rized in Table 4-14. TCI was bioactivated and covalently bound to DNA at a
level of 0.36 nmol/mg DNA, or fifth in the series of 10 compounds. Binding
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 5 hours in a closed system to initial concentrations of 100 and 1000 ppm
3 14
(538 and 5380 mg/m ) C-TCI and determined protein-bound metabolites in
4-49
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TABLE 4-13. IN VITRO COVALENT BINDING OF TCI
Investigator
System
Macromolecules
covalently bound
Additions
Van Duuren and Banerjee rat liver microsomes microsomal protein
(1976)
SKF-525A or 7,8 benzoflavone- * binding; GSH,
methylmercaptoimidazole, mercaptoethanol, urea-^
binding; epoxide hydratase inhibitor (trichloro-
propane epoxide) t binding; pretreatment with
phenobarbital- t binding
Bolt and Filser (1977) rat liver microsomes
Bolt et al. (1977)
Uehleke and Poplawski- rat and mice liver
Tabarelli (1977) microsomes
albumin, polylysine GSH, -J. binding
microsomal protein
lipid
Allemand et al. (1978) rat liver microsomes microsomal protein
Banerjee and Van Duuren Mice (B6C3F) and rat
(1978) (Osborne-Mendel)
microsomes
microsomal protein
Salmon sperm DNA
Laib et al. (1979)
rat liver microsomes yeast RNA micro-
somal protein
DiRenzo et al. (1982a) rat liver microsomes calf thymus DNA
rat liver microsomes microsomal protein
young (4 mo) vs adult
(11 mo) vs aged
(27 mo)
Mouse > rat; pretreatment with phenobarbital,
t binding
GSH or piperonyl butoxide - * 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
* ^450 system in aged; 4- binding; pretreatment
with phenobarbital abolishes difference
-------
TABLE 4-14. MICROSOMAL BIOACTIVATION AND COVALENT BINDING OF ALIPHATIC
HALIDES TO CALF THYMUS DNA
Binding to DNA
Aliphatic halides (nmol/mg/h ± SD)
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 atmos-
phere.
Source: DiRenzo et al. (1982b).
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 hours.
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 primary excretion. Lesser amounts were found
in lung, spleen, small intestine, and muscle. No major differences in percent-
age of the dose bound versus the different initial atmospheric concentrations
of TCI were observed. However, the extent of binding in liver and kidney for
TCI was less than for vinyl chloride and approximated that observed with
carbon tetrachloride.
Uehleke and Poplawski-Tabarelli (1977) determined in vivo binding for
14
mice (NMRI) following i.p. injections of C-TCI (0.2 uCi/10 umole in 5 (jl of
peanut oil/g b.w.). Binding to liver microsomes, mitochondria, and cytosol
protein was measured at 2, 6, 12, and 24 hours after injection. Peak binding
occurred after 6 hours (8, 4 and 1.8 nmol/mg protein) respectively, and there-
14
after declined slowly. When the same dose of C-TCI was given by gavage at
the 6-hours time, binding to microsomal protein was found to be only 5.4 nmol/
14
mg protein. C-TCI was also bound to hepatic lipids; similarly to protein
binding, 6 hours after i.p. dosage, the highest concentrations were 9.4 nmol/mg
4-51
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of endoplasmic fractions of livers and 5.6 nmol/mg in mitochondria! lipids of
the liver.
Allemand et al. (1978) investigated the metabolic activation of TCI and
14
irreversible binding in Sprague-Dawley rats after C-TCI (100 umol, 100 uCi)
was administered i.p. in 200 ul of methanol to nonpretreated and to phenobar-
bital-pretreated rats; the rats were sacrificed 4 hours later. Trace amounts
of irreversibly bound radioactivity were found on muscle proteins (3.1 mmol
equiv./g tissue), while 40 times more was found on liver proteins (117 nmol
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 hours after
administration, confirming 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 DNA 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
C-TCI in corn oil (each animal received 160 uCi/kg of radioactivity). After
6 hours, 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 DNA 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
DNA) and contamination with labeled protein could not be entirely ruled out.
Identification of DNA 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 hours later.
Hepatic DNA 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. These investigators concluded
that the ability of TCI to interact with DNA w 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 exposed to 10 or 600 ppm (54 or 3228 mg/m ) of C-TCI (11.3 mCi/nmol)
for 6 hours. The mice metabolized more inhaled TCI than rats at low and high
exposure levels (123 and 260 percent, respectively). The amount of hepatic
4-52
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and renal covalent binding was three-fold higher in mice than for the high
dose (Table 4-15) and four-fold greater for liver than kidney. Mice (4) were
14
also dosed with 1200 mg/kg of C-TCI with relatively high specific activity
(2.4 mCi/nmol) and a maximum estimate of a liver DMA alkylation level of
0.62 ± 0.42 alkylations/10 nucleotides was observed after isolation and
separation by HPLC (Table 4-16). However, no chemical 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 carcinogens such as DMN,
methylnitrosurea, etc. (footnote, Table 4-15). These results are in accord
with the conclusion of Parchman and Magee (1982) that the ability of TCI to
interact with DMA i_n 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 DMA
synthesis, and centrilobular hepatocellular swelling were 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
increased liver weight and elevated DMA 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 tumorigenie 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.
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, result-
ing 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
environmental chemicals (e.g., barbiturates, PCBs, etc.), may bring about an
increased rate of TCI metabolism. Numerous jji vivo and ijn vitro experimental
4-53
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TABLE 4-15. 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
Exposure Test
(ppm) Tissue animal
10 Liver Mouse
Rat
Kidney Mouse
Rat
600 Liver Mouse
Rat
Kidney Mouse
Rat
Binding (pmol-
eq(14C) TCI/ug
protein) ± SD
0.318
0.268
0.168
0.155
20.4
4.72
5.06
1.77
(0.004)
(0.013)
(0.014)
(0.007)
(2.49)
(0.415)
(0.667)
(0.200)
Ratio
(mouse/rat)
1.2
1.1
4.3
3.0
Source: Stott et al., 1982.
TABLE 4-16. 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
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).
"Maximum" alkylations possible due to the relatively large amount of dpm
associated with protein binding and metabolic incorporation into normal bases.
GCovalent 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 = (umol adduct/mol dN)
(nmol/kg dose).
None detected.
Source: Stott et al. (1982).
4-54
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studies with animals pretreated with microsomal inducers have demonstrated an
enhanced metabolism 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 ex-
posed to TCI who are also on a therapeutic regimen of disulfiram for alcoholism.
Disulfiram 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 4-12), can be considered detoxification mechanisms, disul-
firam 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 exposure (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 intolerance to alcohol
syndrome is ascribed to mutual inhibition of TCI and alcohol metabolism
producing increased plasma ethanol and TCE concentrations from competitive
inhibition of aldehyde dehydrogenase (Sellers et al., 1972a,b; Stewart et al.,
1974).
Muller et al. (1975) specifically investigated the interaction of TCI and
alcohol in human volunteers. After the volunteers inhaled TCI (50 ppm) for
6 hours 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 intolerance syndrome from combined exposure to TCI and
ethanol was due to increased accumulation of TCI in the CNS, resulting from
4-55
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depression of TCI oxidation. From experiments in rats, Nakanishi et al.
(1978) found that ethanol ingested after TCI exposure increased blood acetal-
dehyde 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 minutes prior to TCI exposure,
3
6000 ppm; 32280 mg/tn ) 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 versus -0.539 exposed, mg/dl/min).
Furthermore, rabbits treated with ethanol exhibited markedly increased suscep-
tibility to the development 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
3
(16 hours later) 400 ppm (2152 mg/m ) for 4-hr 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 in 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 hours after ethanol dosing. At this time, almost no blood ethanol was de-
tected and no increase occurred in microsomal protein and P.rQ liver contents.
Ethanol addition directly to the TCI microsomal incubations inhibited TCI
metabolism, thus demonstrating that ethanol was a competitor of TCI metabolism.
These investigators suggest that ethanol, when taken i_n 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 hours later, when ethanol blood level has dissi-
pated, the microsomal enzymes have an increased capacity to metabolize TCI.
4-56
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4.4.5.3 Warfarin. Sellers and Koch-Weser (1970) observed a potentiation of
the anticoagulant effect of Warfarin in patients after chloral hydrate inges-
tion. This increased potential for bleeding appeared to result from displace-
ment 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
P.™ system activity. This concept, according to these investigators, is not
in conflict 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
capacity to metabolize TCI and other chlorinated hydrocarbons is influenced by
chronological age. These investigators isolated hepatic microsomes from young
(4 months), adult (11 months) and aging (27 months) Fischer 344 rats. Bio-
transformation and subsequent covalent binding of labeled TCI and other com-
pounds 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 adults. Senescent rats were also
found to have significantly less hepatic P^Q (-35 percent), NADPH cytochrome
C reductase (-31 percent), and ethylmorphine N-demethylase activity (-43
percent). Pretreatment with phenobarbital, however, abolished these defi-
ciencies of aging rats. These investigators suggest a reduced metabolic
capacity as a function of age, which results in a decrease in bioactivation of
4-57
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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 unsym-
metric chlorinated ethylenes like TCI are more hepatotoxic than symmetric
ethylenes as perch!oroethylene 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
intracellular damage (Pessayre et al., 1979; Allemand et al., 1978; Reynolds
and Moslen, 1977). Figures 4-11 and 4-13 indicate that these reactive metabo-
lites may include TCI epoxide, dichloroacetyl chloride, and formyl chloride
(Section 4.4.4). Chloral, chloral hydrate, and TCE, as ordinarily used hyp-
notics, are not associated with significant hepatotoxicity (Goodman and Gilman,
1975).
Buben and 0'Flaherty (1984) have recently provided convincing evidence
that the hepatotoxicity of TCI is directly proportional to the extent to which
TCI is metabolized. These investigators administered to male Swiss-Cox mice
by gavage TCI in corn oil vehicle for 6 weeks (5 days/week), at dose levels of
100 to 3200 mg/kg. Metabolism was measured by the amount of urinary metabo-
lites (TCA, TCE, and TCE glucuronide) excreted per day (24-hr urine collec-
tions). Indices of hepatotoxicity measured in the dosage groups were increases
in liver weight, decreases in liver glucose-6-phosphatase (G6P) activity,
increases in liver triglycerides, and increases in SGPT activity. TCI signifi-
cantly affected two of the four hepatotoxicity parameters, liver weight, and
G6P activity, in a dose-related manner. Increases of liver triglycerides and
increases of serum SGPT occurred only at the highest dosage levels. However,
4-58
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dose-related percentage increases of liver weight and of percentage inhibition
of G6P activity exhibited biphasic curves (Figures 4-13 and 4-14) exactly
analogous to the dose-related metabolism curve (Figure 4-8). In each case,
the relationships between dose and toxic effect, and dose and metabolism were
linear up through 1600 mg of TCI/kg/day, but reaching a plateau at higher
doses. Furthermore, when these same hepatotoxicity data points were plotted
against the amount of metabolism (i.e., total urinary metabolites) at each
respective dose, linear relationships were observed throughout the entire dose
range (Figures 4-13 and 4-14). These observations indicate that the non-
linearity seen in the dose-toxic effect relationships can be fully explained
by the kinetics of TCI metabolism. Chemical and histopathologic examination
confirmed that the dose-related toxic responses, liver weight increase, and
liver GBP activity decrease were paralleled by cellular change. The increases
in liver size were attributable to hypertrophy of the liver cells, since a
dose-related decrease in the DNA concentration (mg/g liver) was found. Micro-
scopic examination revealed evidence of dose-related cellular damage with
cellular degeneration, swollen hepatocytes, karyorrhexis, and central lobular
necrosis. Hepatic cells with two or more nuclei or enlarged nuclei containing
increased amounts of chromatin were observed, suggesting that a regenerative
process was ongoing.
As TCI hepatotoxicity in mice parallels TCI metabolism, then covalent
binding of reactive metabolites to cellular macromolecules should also be
directly proportional to the extent to which TCI is metabolized. Presently,
there is less information about a possible dose-related binding of "reactive"
metabolites to macromolecules that results in an incremental increase in a
toxic liver response; nor have the critical target binding sites been identi-
fied. 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 in rats, as
measured by plasma SGOT, is potentiated by both ethanol and phenobarbital.
Ethanol acted by competitive substrate inhibition, and also by stimulation of
microsomal mixed-function oxidase system; phenobarbital potentiated toxicity
by induction of mixed function oxidase system and thereby increased TCI metab-
olism. Allemand et al. (1978) observed a similar potentiation of toxicity in
4-59
-------
80
<70
cc
H
>. 60
Q
O
m
ul 50
HI
o40
z
30
20
cc 10
r2 = 0.974
a = 0.036
b = 6.27
J L
J I I
400
1
800 1200 1600 2000
TCI DOSAGE, mg/kg
T
2400
2800
3200
r2 = 0.974
a = 0.105
b = 6.64
100
200
300 400 500 600
TOTAL URINARY METABOLITE, mg/kg
700
800
Figure 4-13. Top: Dose-effect relationship between chronic daily oral TCI dose and increase in liver
weight of mice after six weeks.
Bottom: Relationship between liver weight increase with chronic dosing at increasing levels (top) and
total urinary metabolite (TCA and TCE) excreted per day by mice at various dose levels.
Source: Buben and O'Flaherty (1984).
4-60
-------
35
30
25
+-«
c
8
I
zT 20
55
I 15
Q.
(O
O
10
400 800 1200 1600 2000
TCI DOSAGE, mg/kg
T
2400
2800
3200
r2 = 0.988
a = 0.051
b = 1.89
200
400
TOTAL URINARY METABOLITES, mg/kg
600
800
Figure 4-14. Top: Dose-effect relationship between chronic daily oral TCI dose and inhibition of
liver glucose-6-phosphatase activity (G6P) of mice after six weeks.
Bottom: Relationship between inhibition of liver G6P activity with chronic dosing at increas-
ing levels (top) and total urinary metabolite (TCA and TCE) excreted per day by mice at the
various dosages.
Source: Buben and O'Flaherty (1984).
4-61
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Reduced glutathione had a protection effect against TCI liver toxicity. TCI
acutely administered to rats initially decreased hepatic glutathione by 61
percent, but increased glutathione levels (106 percent) 16 hours later (Moslen
et a!., 1977b). Glutathione added to microsomal preparations decreased binding
14
of reactive products of C-TCI, suggesting a glutathione conjugation of
metabolites. Pessayre et al. (1979) found that single or acute TCI administra-
tion to rats decreased cytochrome PA,-n of phenobarbital-treated rats, but not
14
untreated animals, and increased covalent binding of C-TCI metabolites to
microsomal protein. Chronic administration to normal rats simultaneously
14
decreased P.™ cytochrome content and increased C-TCI metabolite binding,
but also induced other microsomal 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 toxi-
city. However, Green and Prout (1984), after daily gavage dosing of B6C3F1
mice for 180 days (1000 mg/kg), could find no evidence of an increase of the
amount of metabolism. The proportion of TCA/TCE in the urine increased and
thus suggests a stimulation of the TCA pathway at the expense of TCE (Section
4.2.3).
Poplawski-Tabarelli and Uehleke (1982) have investigated other mechanisms
that contribute to the toxic manifestations of halogenated aliphatic hydrocar-
bons, 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 forma-
tion with reduced P.5Q cytochrome; covalent interactions of metabolic interme-
diates with cytochrome P450; destruction of cytochromes by lipid peroxidation;
and production and interaction of CO with cytochromes, the overriding correla-
tion appeared with the simple physicochemical factors of high lipid solubility
and vapor pressure. The ability of chlorinated ethylene to inhibit mitochon-
drial 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 and ethylenes, including TCI, inhibited
oxygen consumption of isolated rat mitochondria; their inhibitory potency
increased in the order of the number of contained chlorines.
4-62
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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 NEURAL AND BEHAVIORAL EFFECTS
Ostlera (1953), in a historical discussion of TCI, cited descriptions of
its anesthetic, analgesic, and neurotoxic effects from reports as early as
1911. The symptoms described are remarkably similar to those found in more
modern literature. In 1931, Stuber reviewed a total of 283 cases of presumable
overexposure to TCI, including 26 fatalities. Principal findings indicated
involved neural and behavioral effects. No cases of injury to the liver were
reported. The above findings were further supported by a study of 384 cases
of industrial gassing poisonings by TCI and other halocarbons from 1961 to
1980 (McCarthy and Jones, 1983). TCI exposure was attributed to 288 reports.
The majority of symptoms were attributed to effects on the central nervous
system (CNS). However, gastrointestinal and respiratory symptoms also were
quite common. The observation that TCI caused paralysis of the trigeminal
nerve led to its early use in the treatment of trigeminal neuralgia. Boulton
and Sweet (1960) suggested that trigeminal palsies that occurred in patients
under TCI anesthesia may have resulted actually from inhalation of dichloro-
acetylene or phosgene, formed when exhaled TCI was passed through soda lime to
remove carbon dioxide (C0?). The formation of these impurities and their
subsequent toxicity resulted in a decline in the use of TCI as an anesthetic.
5.2.1 Human Studies
5.2.1.1 Short-Term Exposures.
5.2.1.1.1 Case reports. Numerous case histories have described the effects
of short-term exposure to high levels of TCI (Feldman et al., 1970; Longley
and Jones, 1963; Harenko, 1967; Masoero and Lavarmo, 1955; Vallee and Leclercq,
1935; Kleinfeld and Tabershaw, 1954; Tomasini and Sartorelli, 1971; Mitchell
5-1
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and Parsons-Smith, 1969; Maloof, 1949; Steinberg, 1981; Lawrence and Partyka,
1981). The earlier studies reported on the toxicity of TCI following anes-
thesia, in particular the occurrence of trigeminal palsies (Humphrey and
McClelland, 1944). Effects reported included dizziness, headache, nausea,
confusion, and, at very high levels, unconsciousness. Facial numbness and
blurred vision also were reported occasionally. Psychotic symptoms resulted
from ingestion of TCI and alcohol (Harenko, 1967).
5.2.1.1.2 Experimental exposures. Stewart et al. (1970) exposed six humans
3
to 200 ppm (1076 mg/m ) TCI for 7 hours. The subjects reported rapid adaptation
to the odor of TCI. Some complaints of dryness of the throat and mild eye
irritation occurred after the first 30 minutes of exposure.
Experimental exposures to 95 ppm (511 mg/m ) (Konietzko et al., 1975) or
up to 300 ppm (1644 mg/m ) for 2.5 hours (Ettema et al., 1975) had no effect
on such behavioral task performances as simple or choice reaction time, hand
steadiness, tapping tests, or pursuit tracking. While Ettema et al. (1975)
reported slight effects, repeated significance tests never produced a p value
below 0.10 and effects were not dose-related.
Vernon and Ferguson (1969) exposed eight males repeatedly to 0, 100, 300,
or 1000 ppm (0, 538, 1644, or 5380 mg/m ) at 2-hr intervals, with 3 days
separating sequential exposures. Statistical tests showed significant decre-
ments in the performance of groove-type hand steadiness, depth perception, and
3
pegboard tests at 1000 ppm (5380 mg/m ) only. It is noteworthy that plots of
trends of the scores demonstrated nonsignificant increases in errors at levels
3
lower than 1000 ppm (5380 mg/m ). Trend tests or studies with a larger group
of subjects perhaps would have been more informative. No effects were noted
on Muller-Lyer illusions, flicker fusion, and code substitution tests.
Ferguson and Vernon (1970) repeatedly exposed individuals to 0, 300, or
3
1000 ppm (0, 1644, or 5380 mg/m ) TCI for 2 hours. In one group of eight
males, individuals were given also either meprobamate (800 mg) or an antihis-
tamine, thonzylamine hydrochloride (50 mg). In a second group of six exposed
to TCI as above, an alcoholic drink sufficient to produce a blood alcohol
level of 20 to 30 mg/100 ml blood (2 mg/kg) was also given. By itself, TCI
exposure produced decrements in depth perception, hand steadiness, and pegboard
performance that were statistically significant only at 1000 ppm (5380 mg/m ).
The two drugs in addition to TCI exposure produced no effects beyond that
induced by TCI exposure alone. Alcohol, however, substantially augmented the
5-2
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effects of TCI such that effects were significant at 300 ppm (1644 mg/m ).
Flicker fusion and Muller-Lyer perception were unaffected by any of the condi-
tions.
Windemuller and Ettema (1978) exposed four groups of six males each for
2.5 hours to either (a) room air only, (b) 200 ppm TCI, (c) 1 ml/kg alcohol in
a mixed drink, or (d) both alcohol and TCI. Performance was measured in a
pursuit rotor task or by a binary choice reaction time test. Heart rate,
sinus arrhythmia, and respiration rate also were measured and interpreted as
correlates of "mental load." The only variable affected was sinus arrhythmia,
which was significantly reduced only by the combination of 200 ppm (1076
3
mg/m ) TCI and alcohol. According to the authors, behavioral performance was
at too high a level to show any decrement due to the experimental conditions.
A separate group of 15 subjects was studied in a design in which all partici-
pated under all conditions. The same results obtained.
In a demonstration project, Stopps and Mclaughlin (1967) showed that TCI
exposure involving one individual caused slight effects. During a 1.5-hour
exposure, TCI was administered sequentially in first an ascending and then
descending order (100, 200, 300, or 500 ppm; 538, 1076, 1644, or 2690 mg/m ).
Of a battery of tests, the Necker cube reversal illusion was considerably
affected. Because only one individual was exposed, such findings are difficult
to interpret. Kyi in et al. (1967) purported to show that optokinetic nystagmus
3
in 12 humans was affected by 1000 ppm (5380 mg/m ) TCI for 2 hours. However,
data were graphically displayed only and thus trends are difficult to see.
5.2.1.2 Long-Term Exposure
5.2.1.2.1 Case reports. Evaluation of case reports indicates that symptoms
involved in short-term exposure situations also are present in long-term
exposures but in more extreme and persistent forms (Harenko, 1967; James,
1962; Kleinfeld and Tabershaw, 1954; Mitchell, 1969; and Steinberg, 1981).
Dizziness, nausea, and headache persist even after cessation of daily exposure.
Longer exposures may produce ataxia, decreased appetite, sleep disturbances,
and perhaps even psychotic episodes. Trigeminal neuropathy was reported in
two cases (Mitchell, 1969; Feldman et al. , 1970). In some instances recovery
was reported, but in others, permanent damage seems to have occurred. Data
are lacking so that it is not possible to characterize the parameters of
exposure beyond which irreversible damage occurs. In some instances, death
occurred (Kleinfeld and Tabershaw, 1954; James, 1963). In four of six cases,
death occurred from 1 to 17 hours after cessation of exposure. In a case of
5-3
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TCI ingestion, death occurred several days after the incident. Kleinfeld and
Tabershaw (1954) conjectured that death was due to ventricular fibrillation.
It is possible that in cases where repeated TCI exposures were not sufficient
to produce anesthesia, a day-to-day accumulation took place in body tissues
(see Chapter 4).
5.2.1.2.2 Workplace exposure. Formal symptom surveys of workers exposed to
various levels of TCI on a daily basis revealed that many symptoms of short-
term exposures became chronic complaints. Grandjean et al. (1955) studied
73 workers, Bardodej and Vyskocic (1956) studied 75, and Lilis et al. (1969)
studied 70 workers. Complaints included vertigo, fatigue, headache, and
nausea. As in case reports, tremors, ataxia, and impaired vision also were
found. Reported alcohol intolerance in TCI-exposed workers could be due to
day-to-day accumulation of TCI in tissue (see Chapter 4) and the interaction
of alcohol with TCI. Bardodej and Vyskocic (1956) also reported frequent
cases of addictive behavior in which insomnia occurred unless TCI was sniffed.
In the study of Grandjean et al. (1955), complaint frequency was higher in the
85 ppm (457 mg/m ) exposure group than in the 14 and 34 ppm (75 and 183 mg/m )
exposure categories. Grandjean et al. (1955) also administered tests for
short-term memory and for measuring understanding of instructions along with
psychiatric interviews to 73 workers exposed to average levels of 14, 34, and
85 ppm (75, 183, and 457 mg/m3), as part of the study. In the 85 ppm (457
mg/m ) group there were higher frequencies of short-term memory loss, fewer
word associations, increased perseverance, and increased rates of misunder-
standing.
Workers have also been formally tested for trigeminal nerve function.
Barret et al. (1982) studied 11 unexposed and 20 TCI-exposed (undocumented
levels) workers. Compared to the unexposed group, 8 of the 20 exposed indivi-
duals had elevated thresholds for trigeminal nerve evoked potentials. Triebig
et al. (1982) studied trigeminal motor and sensory nerve conduction velocity
in 26 individuals occupationally exposed to an average of 40 ppm (215 mg/m )
TCI and in 24 workers having no TCI exposure. No differences between groups
were found, but variances were large and exposure levels small.
Konietzko et al. (1974) found EEC changes in workers exposed to TCI. The
EEC for six workers was collected during work on TCI-exposed days and on
nonexposure days. During exposure days (50 to 100 ppm TCI), there were more
bursts of alpha waves and the alpha waves had higher amplitude than on unexposed
days. No clinically abnormal EEC observations occurred during unexposed work
5-4
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or in a clinical setting. Increased alpha activity frequently occurs in
individuals who are in a relaxed state.
5.2.1.2.3 Experimental exposure. Stewart et al. (1970) exposed six individuals
to 200 ppm (1076 mg/m ) TCI for 7 hr/day, for 5 sequential days. Subjective
reports were collected in addition to blood and physiological measures. Odor
perception decreased over the course of repeated exposures as did complaints
of eye irritation, dryness of throat, and other mild symptoms. By the fourth
and fifth days, however, fatigue and sleepiness during exposure were reported
by five of the six individuals.
5.2.1.2.4 Summary of human neural and behavioral effects. The effects of
short-term TCI exposure range from mild eye irritation, nausea, vertigo,
headache, confusion, unconsciousness, and death, depending upon exposure
3
concentration. Mild irritation occurs at levels near 200 ppm (1076 mg/m ).
Hand steadiness, coordination, and possibly depth perception are affected at
1000 ppm (5380 mg/m ) and perhaps below (see Section 5.2.1). If combined with
alcohol ingestion, TCI can produce these effects at levels of 200 to 300 ppm
(1076 to 1614 mg/m3).
Extended exposure can increase the duration and intensity of nausea,
vertigo, and headache, but eye irritation and olfactory sensation are reduced.
Confusion, reduced cognitive performance, impaired short-term memory, sleep
disturbances, loss of appetite, addiction, alcohol intolerance, tremors,
ataxia, and trigeminal neuropathy also are reported. The threshold for such
complaints is difficult to estimate since such data are gathered from workplace
surveys with all of the attendant problems in quantification and control. It
appears that effects, however, are absent below 85 to 100 ppm (457 to 538
mg/m ).
5.2.2 Laboratory Animals
5.2.2.1 Short-Term Animal Exposures. Exposure to 30,000 ppm (161,000 mg/m )
TCI for about 20 minutes was lethal to 15 dogs (Baker, 1958). After about
5 minutes, dogs began to salivate heavily and to become ataxic. Between 5 and
10 minutes, they exhibited foreleg tonicity and became semicomatose. During
the last 10 minutes, dogs were unconscious and frequently exhibited convulsive
(particularly hindlimb) movements. Not surprisingly, no CMS pathology was
found during autopsy.
Grandjean (1963) exposed 29 rats to 400 ppm (2152 mg/m ) TCI and a group
of 11 to 800 ppm (4304 mg/m ) TCI, for 6 hours. Immediately upon termination
5-5
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of exposure, rats were tested in a swimming escape task. Rats were tested
with and without a 27 gram weight attached to their tails. In each test, rats
were given ten sequential trials. At 400 ppm (2152 mg/m ) no effects of TCI
were observed without the added load and only slight slowing effects were seen
with the added load. At 800 ppm (4304 mg/m ) effects were slight and inconsis-
tent without the added load; definite and consistent slowing of swimming time
occurred with the added load. During the sequential trials, animals exposed
3
to 800 ppm (4304 mg/m ) TCI under load showed the greatest effects on the
eighth, ninth, and especially the tenth trial. The author interpreted these
data to mean that TCI exposure produced greater fatigue since the effects were
dependent upon load and time.
In the above report, Grandjean (1963) also presented data on jiggle cage
activity levels from 12 rats per group exposed to 0, 400, 800, or 1200 ppm (0,
2152, 4304, or 6456 mg/m ) TCI for 5 hours. Activity was measured during
exposure and again for a period from 20 to 80 minutes after exposure. Activity
levels were significantly decreased during and after exposure to 1200 ppm
(6456 mg/m ). Means of the 400 and 800 ppm (2152 and 4304 mg/m ) groups also
were lower but not significantly so.
Grandjean (1960) exposed rats for 8 hours to either 200, 600, or 1600 ppm
o
(1076, 3228, or 8608 mg/m ) TCI which were then subsequently tested for explora-
tory behavior in a maze. Exploratory behavior was reported to have decreased
at 200 and 1600 ppm, but increased at 600 ppm (3228 mg/m ). Since no actual
data were presented, the information is difficult to interpret.
Baetjer et al. (1970) used hypothalamic electrical stimulation to reinforce
lever pressing behavior in rats. Groups ranging in size from 14 to 36 were
exposed to either 0, 2500, or 3500 ppm (0, 13, 450, or 18,830 mg/m ) TCI for
30 minutes on three sequential days. At each exposure level, one group was
placed on water deprivation for 3 days while the other group was not. Thus,
there were six groups of rats. TCI at both levels reduced the lever pressing
rate in a dose-dependent fashion. Especially at 2,500 ppm (13,450 mg/m ),
response rate reduction was less noticeable on day 2 than on day 1 and on day
3 than on day 2. Thus, it appears that short-term adaptation can occur.
Water-deprived rats were affected less and had delayed effects in comparison
to normals. The authors speculate that in brain cells of dehydrated rats,
either TCI levels accumulated more slowly or the brain cells were more resistant
to TCI effects. Another possible interpretation is that the interoceptive
stimuli from water deprivation plus the stimuli associated with TCI exposure
5-6
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simply increased the activity and, as a result, the lever pressing rate, with
respect to rats that were subjected to only one of the above. Hence, dehydra-
tion may have been unimportant except as another source of stimulation.
5.2.2.2 Repeated Exposures (4 days to 5 weeks). Savolainen et al. (1977)
recorded open-field activity in rats after the last of 4 days of exposure to
either 0 or 200 ppm (0 or 1076 mg/m ) TCI for 6 hr/day. During the first hour
after exposure, the activity level was higher in the exposed group, but by
17 hours postexposure the effect was no longer present.
Silverman and Williams (1975) exposed independent groups of 16 rats to
either 100, 200, 500, or 1000 ppm (538, 1076, 2690, or 5380 mg/m3) TCI for
6 hr/day, 5 days/week, for 5 weeks. For each of the abovementioned four
groups, a separate contemporaneous group of 16 unexposed controls was studied.
The activity of pairs of male rats was studied in their home cages for 5 minutes
after termination of exposure on days 1, 3, 10, 17, and 24. Where significant
TCI effects occurred, the effect was always a reduction of activity. At
3
1000 ppm (5380 mg/m ), the effect was evident on the first day. Other levels
of exposure showed a gradual decline in activity with the decline reaching a
3
criterion of significance in a dose-dependent fashion. The 100 ppm (538 mg/m )
group was not affected significantly at the end of 5 weeks and was further
exposed for a total of 8 weeks. This group was eventually affected in a
significant manner.
Goldberg et al. (1964) exposed groups of rats to 200, 560, 1568, and
4380 ppm (1076, 3013, 8436, and 23,564 mg/m ) TCI for 4 hr/day, 5 days/week,
for 2 weeks. Signaled electric shock escape-avoidance behavior was studied in
previously trained rats. The highest exposure level produced a significantly
greater rate of response inhibition than did any of the lower concentrations.
3
Concentrations from 200 to 1568 ppm (1076 to 8436 mg/m ) did not differ.
Another group of rats was trained on the same task during exposure. No signif-
icant differences were found although asymptotic performance was reduced in a
3
dose-related manner above 200 ppm (1076 mg/m ). The 200 ppm group seemed to
reach the asymptotic level faster than controls. It must be emphasized that
none of the acquisition curves were significantly different from each other
and the reported effects should, therefore, be viewed with caution.
Battig et al. (1960) exposed four groups of six rats each to either 0 or
3
600 ppm (0 or 3228 mg/m ) TCI for 3 to 4 hr/day. The daily exposure plan was
complex, with each group receiving TCI at least once during acquisition and
5-7
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extinction of a shuttle box escape-avoidance task. In no case was acquisition
affected by TCI exposure, but reaction times were increased and extinction was
slowed.
Three rats were trained in positively reinforced, rope-climbing behavior
under the control of a positive discriminative stimulus (Grandjean, 1960).
They were then tested immediately after exposure to either 0, 200, or 800 ppm
(0, 1076, or 4304 mg/m ) TCI. Apparently there were 7 sequential days at
3 3
200 ppm (1076 mg/m ) for 3 hr/day, followed by 5 days of 800 ppm (4304 mg/m )
for 3 hours. Controls were interspersed. It is not clear when the data
presented were collected; they are probably a summary over all days. There
was no effect on latency or failures to respond, but the number of responses
in the absence of a discriminitive stimulus was higher in the TCI-exposed
groups. There was no difference between the 200 and 800 ppm (1076 and 4304
3
mg/m ) groups. Little can be concluded from only three rats.
Goldberg et al. (1964) reported data on the performance of electric shock
3
escape-avoidance responses in 10 rats exposed to 125 ppm (672 mg/m ) TCI for
4 hr/day, 5 days/week, for a total of 14 days. Rats were tested during the
last hour of exposure. At the onset of exposure, rats immediately began to
receive more electric shocks (avoidance response failures) and continued to
perform more poorly than on pre-exposure days throughout the 14 days. Some
evidence of adaptation to TCI was observed. After the 14-day exposure period,
rats were tested for 7 more unexposed days during which performance returned
to and below pre-exposure levels. The evidence for adaptation is poor since
(a) no contemporaneous controls were studied and (b) the "adaptation" trend of
improvement continued for the 7 postexposure days.
5.2.2.3 Long-Term Exposures. Battig and Grandjean (1963) exposed groups of
10 rats to either 0 or 400 ppm (0 or 2152 mg/m3) TCI for 8 hr/day, 5 days/week,
for 44 weeks. Several behaviors were tested either immediately after daily
exposure or 16 hours after exposure. TCI increased times in a swimming escape-
avoidance test. The magnitude of the effect became greater as the number of
weeks of exposure increased. When tested 16 hours after exposure, no effect
was seen on the swimming test. Acquisition of shuttle box avoidance behavior
was not affected either immediately after exposure or 16 hours later. Hebb
maze performance was not affected when tested 16 hours after exposure. Tests
of exploratory behavior which were conducted immediately after exposure during
the last 2 weeks of the study showed an increased level of activity. This may
be due to the lack of adaptation to novel surroundings (Daschiell maze).
5-8
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Kjellstrand et al. (1980) studied the effects of continuous 320 ppm
(1721 mg/m ) exposure for 9 months on radial-arm maze performance in 24 gerbils.
An unexposed control group of 24 also was tested. Gerbils were removed from
exposure chambers for the test. An eight-arm maze test was started on day 59
and terminated on day 87. Performance was nearly perfect in both groups. At
day 237, a 16-arm maze test was begun with 16 gerbils per group. Again, no
significant differences were observed even though performance was not as good
as in the eight-arm maze. Eleven days after the termination of the 9-month
exposure, 32 gerbils were split into four groups: two groups of 8 previously
exposed to 9 months of TCI and two groups of 8 previously unexposed. All four
groups were exposed on alternate days to 2,300 ppm 1,1,1-trichloroethane for
6 hours and then tested in the 16-arm maze. Those gerbils that had previously
been exposed to TCI showed worse performance after 1,1,1-trichloroethane
exposure than after air exposure. Similar but somewhat less dramatic results
were obtained from tests 75 days after TCI exposure. The authors interpreted
these findings to mean that permanent damage to the CMS resulted during the
9 months of exposure to TCI. This interpretation is difficult to support
since exposure to 1,1,1-trichloroethane improved performance in gerbils pre-
viously exposed to TCI. It is more likely that the findings can be explained
in terms of state dependent learning.
Baker (1958) exposed dogs repeatedly to concentrations of TCI ranging
from 500 to 3000 ppm (2690 to 16,140 mg/m3) for 2 to 8 hours. He reported
anecdotally that animals exhibited such chronic symptoms as ataxia, excessive
salivation, head tremor, teeth gnashing, and extension of extremities. Upon
repeated exposure, dogs progressively became unconscious more quickly. Post-
mortem inspection of brain tissue in three dogs that had survived between 60
and 162 hours of TCI (unspecified concentration) revealed selective destruction
of cells in the Purkinje layer of the cerebellum and, to a lesser extent,
unselective cellular damage to the cerebral cortex. Changes to Purkinje cells
in rabbits injected intramuscularly with a total of 53 g of TCI were reported
by Bartonicek and Brun (1970).
Continuous exposure of gerbils to 0, 60, or 320 ppm (0, 323, or 1722
mg/m ) TCI for 3 months was followed by a recovery period of 4 months (Haglid
et al., 1981). Gerbils were sacrificed (6 to 7 per group) and the percentage
of S100 protein and DNA in various brain regions was determined. Although the
authors purport to find a consistent change in S100 protein percentages,
5-9
-------
inspection of the data reveals changes which are not dose-related and are
inconsistent across areas. DNA seems to be increased in the cerebellum,
sensory cortex, and motor cortex, but not in brainstem or hippocampus. However,
if one makes Bonferroni corrections to the significance tests, no significant
increases remain. This report should be viewed as presenting pilot data only.
5.2.2.4 Summary of Neural and Behavioral Effects in Laboratory Animals.
Effects of TCI upon behavior have been demonstrated at exposures as low as
400 ppm (2154 mg/m )for 6 hours or as low as 200 ppm (1076 mg/m ) for four,
6-hr days. If exposure is extended to 6 hr/day for up to 8 weeks, 100 ppm
(538 mg/m ) will produce behavioral effects. These effects include increased
fatigue, decreased activity level, and slowed extinction of learned behaviors.
These behavioral changes might well be regarded as analogous to the
effects of TCI on humans. Fatigue is a common complaint at low exposure
levels. Activity level reduction is another possible analog of fatigue.
Slowed extinction of a learned response might be related to short-term memory
loss. In general, the laboratory animal as an analog of human behavior is
speculative.
At very high repeated exposure levels, TCI apparently selectively destroys
the Purkinje cell layer in the cerebellum. Data regarding the neuropathological
effects of lower level exposures for long periods are inconclusive or absent.
Neuropathological investigations could provide a model of TCI-induced ataxia.
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 arrhyth-
mias, including bradycardia, atrial and ventricular premature contractions,
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
5-10
-------
attributed to increases of epinephrine release and circulating plasma levels.
These direct cardiac effects of TCI, with sensitization to catecholamines,
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 extra-
systoles, 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 etal., 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 extra-
systoles 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/m ) TCI, 4 hr/day, 6 days/
week, for 45 days. They found that severe blood dyscrasia was produced in
these animals. The characteristics of the hemochromocytometric tests and the
description of the bone marrow led the authors to conclude that chronic intoxi-
cation 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 i.p. with 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.
White and Carlson (1979) demonstrated that epinephrine-induced arrhythmias
in rabbits exposed to TCI could be decreased by phenobarbital treatment. This
increases metabolism of TCI. This would support the view that TCI, and not
its metabolites, is responsible for arrhythmias. Caffeine has been shown to
5-11
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potentiate the arrhythmogenicity of TCI in rabbits upon challenge with exogen-
ously 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 minutes
prior to exposure) and exposed for 1 hour to 6000 ppm (32,280 mg/m ) TCI under
dynamic airflow conditions. Epinephrine was infused until arrhythmias occurred
after 7.5, 15, 30, 45 and 60 minutes of exposure and 15 and 30 minutes post-
exposure. The authors reported a pronounced increase in 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/
m ) TCI. Rabbits were treated with saline (1 ml/kg) or ethanol (1 g/kg, i.v.
or p.o.) 30 minutes prior to exposure to 6000 ppm TCI for 1 hour. Epinephrine
was infused as described above. Rabbits treated with ethanol developed epine-
phrine-induced cardiac arrhythmias sooner and at lower doses of epinephrine
than control rabbits. 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.
Most recently, Carlson and White (1984) evaluated the effect of benzo(a)-
pyrene on the production of arrhythmias in epinephrine-induced, TCI-exposed
rabbits. Benzo(a)pyrene increased TCI metabolism but also led to increased
arrhythmias. Thus, it would appear that benzo(a)pyrene has intrinsic arrhy-
3
thmogenic action. Rabbits were exposed to 8100 ppm (43,578 mg/m ) TCI 48 and
72 hours subsequent to administration of 40 mg benzo(a)pyrene/kg.
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 hours prior to a 1-hr
exposure to 6000 ppm (32,280 mg/m ) TCI. Disulfiram or its metabolites, C$2
and diethyldethiocarbamate (1.35 mmoles/kg, i.p.), were administered at similar
3
time intervals also to rabbits exposed for 1 hour to 9000 ppm (48,420 mg/m )
TCI. Blood levels of TCI and its metabolites, trichloroethanol and trichloro-
acetic acid, were measured at periods ranging from 7.5 minutes of exposure to
30 minutes post-exposure. The metabolism of TCI was inhibited in all treatment
groups. When challenged with 0.5 to 3.0 ug/kg epinephrine, disulfiram pre-
vented the epinephrine-induced arrhythmias resulting from exposure to 6000 ppm
5-12
-------
(32,280 mg/m ) TCI. All treatment groups in the first 30 minutes of exposure
3
to 9000 ppm (48,420 mg/m ) 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 disulfiram- 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 in-
creased carbon dioxide tension (Dobkin and Byles, 1963). These effects were
noted in 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 Bui bring (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
3
35,000 ppm; 26,900 to 188,300 mg/m ) 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.
Coleridge et al. (1968) 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.
Lewis et al. (1984) attempted to determine if TCI alone (without previous
induction) can affect the activity of pulmonary mixed-function oxidase in mice
and if inhalation of this compound can injure the lungs directly. Animals
were exposed to 10,000 ppm TCI (53,800 mg/m ) for 5 days, 1 or 4 hr/day.
Pathologic changes such as thrombus formation and vacuolization of bronchiolar
epithelial cells were also noted in the animals with reduced microsomal NADPH
5-13
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cytochrome c reductase. The authors concluded that the decrease in pulmonary
enzyme activity may reflect direct damage to the lungs.
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 acti-
vity. 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
anesthesia. 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
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
5-14
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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/m ).
Phoon et al. (1984) reported recently on five individuals exposed to TCI
occupationally who had liver damage in conjunction with severe erythema (Stevens-
Johnson syndrome). It is unlikely that TCI was a causative factor in producing
the liver injury since other workers were unaffected and since the exposure
3
levels were estimated to be between 9 and 169 ppm (50 and 912 mg/m ).
In the case reports of occupational exposure to TCI described by Suciu
and Olinici (1983), factors other than TCI probably were more causally related
to the hepatorenal disturbances noted.
From the above reports, it is apparent that observations of liver or
renal dysfunction 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 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.
TCI has also been reported to have some abuse potential. Cases of delib-
erate 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 extrapolated to inhalation of the
same total dose over a long period of time, as in occupational exposure.
5.4.2 Animal Studies
Hepatotoxic potential has been evaluated in a number of animal species
under various exposure conditions and schedules. Findings are tabulated in
Table 5-1.
5.4.2.1 Inhalation Studies—In a series of experiments, Kjellstrand et al.
(1981; 1983a,b) evaluated the effects of TCI exposure on body and organ weights,
and plasma butyrylcholinesterase in Sprague-Dawley rats, various mouse strains,
and mongolian gerbils. Kjellstrand et al. (1981) found suggestive evidence
5-15
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that rats, NMRI mice, and gerbils (consisting of pooled males and females)
showed relative and absolute liver weight gain after continuous exposure to
3
150 ppm (807 mg/m ) for up to 30 days. The effect seemed to be more pro-
nounced in mice. During postexposure, relative liver weights decreased.
Study design limitations hampered evaluation of the statistical significance
of results; body weight gains were determined by linear regression.
In a follow-up experiment, Kjellstrand et al. (1983a) obtained more
convincing evidence that exposure to 150 ppm (807 mg/m ) for 30 days caused
liver weight gain in various mouse strains (wild, C57BL, DBA, B6CBA, A/sn, and
NZB). Plasma butyrylcholinesterase, a liver-produced enzyme of unknown func-
tion, also showed statistically significant increased activity in males of all
strains and in females of strains A/sn and NZB.
In a further extension of their studies, Kjellstrand et al. (1983b)
exposed male and female NMRI mice continuously (up to 120 days) to levels from
37 to 300 ppm (200 to 1614 mg/m3) and intermittently to 225 to 3600 ppm (1233
3
to 19,728 mg/m ). Regardless of the exposure schedule, enzyme activity was
highest in male mice under all exposure schedules. Peak activity was reached
about day 30. Activity in female mice was largely unaffected by TCI exposure.
Liver weights in both sexes increased under either continuous or intermittent
exposure. Peak increase in liver weight during 150 ppm exposure occurred at
about day 30.
Light microscopy revealed distinct changes in liver morphology at exposure
levels as low as 37 ppm (200 mg/m3). Rehabilitation during postexposure
revealed that the liver became histologically similar to air-exposed controls.
TCI exposure had little effect on body weight and spleen weight. While kidney
weight increased during exposure, weight gain decreased during postexposure
rehabilitation. It was concluded that TCI in this species induced a transient
abnormal state but no liver damage. It was noted that Shimada and Yoshida
(1980) proposed that elevations in butyrylcholinesterase activity may represent
fatty metamorphosis or toxic damage to the liver. The finding that butyrylcho-
linesterase activity is largely confined to male mice and that tumor induction
appears similarly sex-specific to males (NCI, 1976) suggests that further
investigation of the role of butyrylcholinesterase in metabolism warrants
attention.
Other inhalation studies (see Table 5-1) have been more limited in scope,
and findings were generally unremarkable (Gehring, 1958; Newell et al. , 1954.;
5-16
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TABLE 5-1. EFFECTS OF TCI EXPOSURE ON EXPERIMENTAL ANIMALS
Mode of Exposure
Exposure
Dose
Schedule
Effects
Reference
I
I—»
-^1
Inhalation
Mouse (NMRI)
Mongolian
gerbil
Rat (S-D)
Mouse (wild,
C57BL, DBA,
B6CBA, A/sn, NZB,
NMRI)
Mouse (NMRI)
150 ppn
150 ppm
150 ppm
150 ppm
37, 75, 150, and
300 ppm
Continuous
up to 30 days
for most groups
Continuous
for 30 days
Continuous
for 30 days
Continuous
for 30 days
Continuous
for 30 days
(also for 120 days
at 150 ppm)
Analysis of b.w. gain limited in mice
by poor study design. Relative liver
weight increased and then decreased
during rehabilitation.
No strong evidence for b.w. gain or
loss. Small but significant relative
liver weight Increases. Kidney weight
increases significant for males and
females.
Only increased b.w. for some female
rats. Small but significant in-
creases in relative liver weight.
All strains showed large increases
in liver weight. Small changes in
kidney and spleen weight. Plasma
butyrylcholinesterase activity in-
creased in males of all strains.
Males exposed to 300 ppm had 3.5
increase in butyrylcholinesterase;
females had slight changes at any
level. Relative liver weight was
linear function of concentration
ranged. Increase dropped when body
weight dropped. Body weight gain was
less than controls. Kidney weight in-
creased more in males (significant at
75 ppm). Spleen weight unchanged.
Butyrylcholinesterase activity peaked
at day 30; returned to normal during
postexposure. Liver weight increase
also peaked near day 30. Liver weight
returned to near normal during post-
exposure.
Kjell strand et al., 1981
Kjellstrand et al., 1983a
Kjellstrand et al., 1983b
-------
TABLE 5-1. (Continued)
Mode of Exposure
Exposure
Dose
Schedule
Effects
Reference
Mouse (NMRI)
225, 450, 900,
1800, and 3600
ppm
I
I—'
oo
Mouse
5500 ppm
Dog, rabbit, rat 500 to 1000 ppm
Rat
15,000 ppm
Rat, guinea pig, 35 and 700 ppm
dog, rabbit, monkey
30 days
(hours/day
x
days/week)
16x7
8x7
4x7
2x7
1x7
respectively
Acute
18 hr/day, 90 days
Intermittent
8 hr/day, 5 days/
week for 30 exposures
over a 6-week period.
After 30 days at 150 ppm, liver cells
larger and had fine vacuolization of
cytoplasm. Kuppfer cells increased in
cellular and nuclear size; connective
tissue infiltrated with inflammatory
cells. No bile stasis. During
postexposure, morphology similar to
controls.
Butyrylcholinesterase significantly
increased in males by average expo-
sure of 150 ppm irrespective of
pulse. Females unaffected. Liver,
body, and kidney weights increase un-
affected by pulse. Motor activity
increased at 3600 ppm but decreased
at 900 ppm. Liver morphological dif-
ferences more pronounced than with
continuous exposure. Mitoses more
frequent in groups exposed longer
intervals.
No increase in SGPT; histopathology
not performed.
No gross changes in liver, renal,
or blood functions.
No liver or kidney damage.
Histopathology not performed.
No signs of hepatotoxicity. No
mortality. 15 rats and guinea
pigs, 3 rabbits and monkeys, and
2 dogs exposed at each dose.
No changes in liver enzymes.
Kjellstrand et al., 1983b
Gehring, 1958
Nowill et al., 1954
Utesch et al., 1981
Pendergast et al., 1967
-------
TABLE 5-1. (Continued)
Mode of Exposure
Exposure
Dose
Schedule
Effects
Reference
en
t—*
10
Albino rat
Dog
Rat
Gavage
Male mice
Rat
Swiss mice
(mixed sexes)
Anesthetic
750 ppm
500 to 600 ppm
1% TCI
0, 250, 500,
1200, or 2400
mg/kg
0 and 1100 mg/kg
360 to 4300
mg/kg
2 hr
8 hr/day, 6 days/week
3 weeks
6 hr/day, 5 days/week
8 weeks
2 hr
5 days/week
3 weeks
Ix
No signs of parenchymal liver damage.
Distribution of acid phosphatase-
positive granules irregular and
diminished in number, 24 hr after
exposure.
Glycogen depletion and hydropic paren-
chymatous liver degeneration observed.
Test mixture may have contained con-
taminants.
Glutathione levels decreased during
TCI exposure of phenobarbital-treated
rats. Rebounded during postexposure.
Dose-related changes characteristic
of hepatocellular hypertrophy at all
dose levels. Slight changes at lowest
dose, increased liver weight at 500
'Ag/kg, severe centrilobular hepatocyte
swelling, giant cell inflammation,
and mineralized cells at highest dose.
No effect on kidney or body weight.
Slightly increased liver weight but
no histopathological changes. No
effect on kidney or body weight.
Fatty infiltration. Minimum lethal
dose: 2920 mg/kg. Minimum hepato-
toxic dose: 720 mg/kg.
Ramadan and Ramadan, 1969
Seifter, 1944
Moslen et al., 1977
Stott et al., 1982
Stott et al., 1982
Jones et al., 1958
-------
TABLE 5-1. (Continued)
Mode of Exposure
Rat
Weanling CD-I
ICR outbred albino
mice
(mixed sexes)
Exposure
Dose Schedule
85 ing/kg and ix
340 mg/kg
24 and 240 rag/kg 14 days
5 days
Effects
No effect on liver weight or trigly-
ceHdes. Effects only if pretreated
with phenobarbital.
Apparent dose-related increase in
liver weight.
Aniline hydroxylase significantly
Reference
Danni et al . , 1981
Tucker et al . , 1982
ro
o
Subcutaneous
Rat
Drinking Water
nnklng
Weanli
ing CD-I
ICR outbred albino
mice (mixed sexes)
730 mg/kg (day 0)
1460 mg/kg (day 1)
2910 mg/kg (day 3)
1460 mg/kg (days 4,
sacrifice (day 6)
500 mg/kg
0.1, 1.0, 2.5, and
5.0 mg/ml in IX
Emulphor
5)
2x
2x
2x
Ix
Ix
4 and 6 mo
elevated and aminopyrene N-demethylase
significantly decreased. Oral LD50 in
females: 1839 to 3779 mg/kg; in males:
2065 to 2771 mg/kg.
SCOT elevated within 12 to 16 hr.
Correlated with hepatocellular
damage seen in histopathology.
Decreased water consumption in females
at highest dose and in males at two
highest doses. Gross pathology un-
remarkable. Significant increase in
kidney weight (males) at highest dose
at 6 mo and in females at 4 and 6 mo.
Elevated protein and ketone levels in
urine in high-dose females (6 mo) and
two highest doses for males (6 mo).
Decreased RBC count in high-dose males
(4 and 6 mo), decreased leukocyte
counts, altered coagulation values in
males (4 and 6 mo), and shortened pro-
thrombin time (females, 6 mo).
Wirtschafter and
Cronyn, 1964
Tucker et al., 1982
-------
Utesch, 1981; Pendergast et al., 1967; Ramadan and Ramadan, 1969). In many of
these studies, histopathology was not performed.
5.4.2.2 Gavage—Stott et al. (1981) found dose-related changes characteristic
of hepatocellular hypertrophy at all dosage levels in mice. This study included
dosages comparable 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). In the Stott et al. study, male mice were given 0, 250,
500, 1200, or 2400 mg TCI/kg body weight/day, 5 days/week, for 3 weeks.
The rats received 0 or 1100 mg TCI/body weight/day. Rats did not show histo-
pathological changes but had slightly increased liver weights. The authors
suggest that the greater sensitivity of mice reflects their greater capacity
for metabolic activation. The species difference could very well be due to
metabolic saturation in the rat (chapter 4).
Relative liver weight and hepatic triglyceride content were used as
parameters in measuring liver injury in the study by Danni et al. (1981). No
injury in Wistar rats at dose levels of 85 mg/kg and 340 mg/kg was observed
unless the animals were first pretreated with phenobarbital. It was noted by
Moslen et al. (1977) and by Adams et al. (1951) that acute liver necrosis is
produced only when TCI inhalation is preceded by pretreatment with phenobar-
bital. In phenobarbital-treated rats, hepatic glutathione levels decreased
during TCI exposure but rebounded during the postexposure period (Moslen et
al., 1977).
In a 14-day gavage study with weanling CD-I and ICR outbred albino male
and female mice, the only effect noted was an apparent dose-related increase
in liver weight. Doses given were 24 and 240 mg/kg (Tucker et al., 1982).
5.4.2.3 Drinking Water—Tucker et al. (1982) evaluated the toxicity of TCI to
male and female mice administered TCI in a 1 percent solution of Emulphor.
Results of gross pathology on mice sacrificed at 4 and 6 months were unremarkable.
Dose levels were 0.1, 1.0, 2.5, and 5.0 mg TCI/ml. Decreased water consumption
was evident in females at the highest dose level and in males at the two
highest dose levels, compared to vehicle controls. Urinalysis and hematological
findings are indicated in Table 5-1.
5.5 IMMUNOLOGICAL AND HEMATOLOGICAL EFFECTS
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).
5-21
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Details of exposure and discussion of toxicological effects noted were reported
separately by Tucker et al. (1982) (see Section 5.4.2). 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, particularly
in the 4-mo 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 concentrations of
TCI. The proliferation capacity of the lymphocyte was not affected when
challenged with T-cell mitogens.
Hobara et al. (1984) investigated the acute effects of TCI on hematologic
parameters in anesthetized, mature, cross-bred dogs. A dose-related decrease
in leukocyte counts was observed over the TCI exposure range of 0 to 700 ppm
(0 to 3,766 mg/m ). Five dogs were exposed to each of the following treatments
for one hour: 200, 500, 700, 1000, 1500, and 2000 ppm (1026, 2690, 5380,
3
8070, and 10,760 mg/m ). In addition, five dogs were exposed to 700 ppm for
one hour. Leukocyte counts decreased at the start of exposure and at the end
of exposure reached minimum values. During postexposure, values gradually
recovered. No significant changes were observed in erythrocyte counts, hemato-
crit values, or thrombocyte counts. It was the authors' opinion that the
decrease in leukocytes reflects the sequestering of the leukocytes in tissues,
as a defensive mechanism.
Koizumi et al. (1984) found that 6-aminolevulinic acid dehydratase (ALA-D),
an enzyme important in heme synthesis in erythrocytes and liver, was inhibited
in rats exposed to 50, 400, or 800 ppm (269, 2152, or 4304 mg/m3) TCI continu-
ously for 48 or 240 hours. A dose-response relationship was observed. TCA
and TCE were not inhibitory j_n vitro. Inhibition of ALA-D (38 percent or 48
percent of the control in liver or in blood, respectively) observed after
5-22
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240 hours of exposure to 400 ppm (2152 mg/m ) was accompanied by the signifi-
cant elevation of 6-aminolevulinic acid synthetase (186 percent of control) in
liver and an increase in excretion of ALA in urine (142 percent of control).
No effects on weight, liver, or other hematological parameters were observed.
Fujita et al. (1984) also demonstrated an inhibition of ALA-D in rats
exposed to TCI for 240 hours. Exposure levels were 50, 377, and 754 ppm
(270, 2140, and 4128 mg/m ). TCI inhibited the holoenzyme only when incubated
with a mixed-function oxidase system, suggesting that metabolites play a role.
The apoenzyme (without the zinc cofactor) was inhibited in a dose-dependent
manner. After exposure to 377 ppm (2140 mg/m3), urinary ALA increased to 142
percent of control. In livers of exposed rats, cytochrome P-450 concentrations
and heme saturation of tryptophan pyrrolase were reduced. Both measures are
indices of the free heme pool. Lead levels in blood were normal, indicating
that lead was not responsible for the inhibition. Inhibition of ALA-D in the
liver was observed at the lowest dose and after 48 hours exposure to TCI.
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 (Maloof, 1949), generalized dermatitis resulting from contact
with the vapor (McBirney, 1954; Conde-Salazar et al., 1983), and possible
scleroderma (Reinl, 1957; Walder, 1983). 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. No
effects have been reported with exposure to TCI in dilute aqueous solutions.
Phoon et al. (1984) reported recently on severe erythema (Stevens-Johnson
syndrome) in five individuals occupationally exposed to TCI through vapor and
direct dermal contact. Duration of exposure ranged from 2 to 5 weeks and
exposure levels were estimated to be between 9 and 169 ppm (50 and 912 mg/m ).
The common occupational history and similarities in condition suggest that TCI
may have been a causative factor in eliciting a hypersensitive response.
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
5-23
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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
(MFC1) 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
3 3
ppm (37,122 mg/m ) for 2 hours and 10,400 ppm (55,952 mg/m ) for 2 hours.
This was confirmed 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-hr
exposure to 1 percent TCI in animals pretreated with other inducers of MFO,
including chlorinated 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 CS?, when given to the mice orally, 30 minutes before TCI, exerted a
protective effect, in that the increase in plasma glutamine-pyruvate trans-
aminase 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.
CS?, at levels of 3, 10, and 30 mg/kg, acted similarly to diethyldithiocarbamate
in suppressing the TCI-associated increase in plasma glutamate-pyruvate trans-
ami nase. The authors concluded that the protective mechanisms of diethyldithio-
carbamate and CS- may involve their interference with metabolic activation of
a variety of hepatotoxins.
Novakova et al. (1981) reported that an open-ended jjn 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.
5-24
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Pessayre et al. (1982) have obtained evidence that TCI is an effective
potentiator of hepatotoxic effects of CC1. jji 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 CC1., both solvents coadministered produced
extensive centrilobular necrosis. TCI given in vitro 5 hours prior to CC1.
_"_ *T
increased CCK-induced lipid peroxidation. Similarly, coadministration markedly
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 CC1. resembled those pro-
duced by a high dose of CC1., a finding consistent with the view that TCI
potentiated the hepatotoxicity of CCl^. It was further concluded that this
potentiation may be the result of increased CCl.-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/m ) TCI (3 rats in the highest exposure category
died). Animals ingesting ethanol prior to exposure to TCI showed a statisti-
cally significant (p <0.05) increase in SCOT, SGPT, and serum isocitric dehydro-
genase (SICD) at 5000 ppm (26,900 mg/m ). After exposure to 5000 ppm (26,900
mg/m ) for 4 hours, the SCOT 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/m ) or less for 4 hours
showed no marked alteration of serum enzyme levels. Neither ethanol-treated
nor nontreated rats showed any alterations of serum enzyme levels when exposed
3 3
for 8 hours to 100 ppm (538 mg/m ). All rats exposed to 5000 ppm (26,900 mg/m )
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.
5-25
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Muller et al. (1975) proposed that TCI and ethanol undergo similar meta-
bolic 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/m ) TCI for 1, 3, or 7.5 hours. 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,
administered to mice in an 18-hr pretreatment, potentiated the hepatotoxic
response to two doses of TCI, 11.1 mmol/kg and 16.7 mmol/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 SGOT. 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 Section 5.4.2), found that pretreatment of Wistar-Munich rats with ethanol
exacerbated the CNS-depressant effects of high doses of TCI. A group of rats
was given 5 ml/kg ethanol orally 1 hour before initiation of the TCI inhalation
regimen: 15 exposures cycles in a 5-hour period; each cycle consisted of 5
minutes of inhalation of 15,000 ppm (80,700 mg/m3) 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 SGOT 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 hours before the 5-hr intermittent TCI exposure regimen had no apparent
effect on the combined CNS-depressant action of ethanol and TCI. No histo-
pathological effects were observed in the livers or kidneys of these animals.
5-26
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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 (53,800 mg/m ) TCI continuously
for 2 hours. However, 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 SGOT 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 SGOT.
Pretreatment of male albino rats with either phenobarbital or 3-methy1
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 chol anthrene in corn oil, 40 mg/kg i.p. for 2 days. Animals were
exposed to TCI 24 hours after the last dose of phenobarbital or 48 hours after
the last dose of 3-methylcholanthrene in a dynamic inhalation chamber. Rats
were sacrificed 22 hours after exposure. Phenobarbital pretreatment increased
SGOT and SGPT three-fold above TCI controls. TCI exposure was 10,400 ppm
(55,952 mg/m ) for 2 hours. Similarly, 3-methylcholanthrene increased SGOT
and SGPT.
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 TL\r (50 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 excessive exposure to TCI is
central nervous system (CNS) dysfunction. Psychomotor function and subjective
complaints which have been studied in short-term controlled human studies, as
well as data from accidental exposure and epidemiological studies, indicate
that nervous system function probably is affected by TCI concentrations ranging
from 200 to 500 ppm (1076 to 2690 mg/m ). Dose-response relationships for
adverse health effects associated with TCI have not been established in man or
experimental animals.
5-27
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5-34
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6. TERATOGENICITY, EMBRYOTOXICITY, 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 Gil man,
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. Another study in the
avian embryo system (Bross et al., 1983) indicated that TCI is embryotoxic and
teratogenic in chick embryos. However, because administration 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 "fetotoxic" 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 jri 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
6-1
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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
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). Thirty to forty bred mice were exposed by
inhalation to TCI (99.24-percent pure) at an air concentration of 300 ppm
(1614 mg/m ) for 7 hours daily on days 6 through 15 of gestation. The results
from two separate trials of 12 and 18 pregnant animals were reported. The
first day of pregnancy was designated as the first day a vaginal plug was
observed. Concurrent pregnant control mice (30) were exposed to filtered air.
The mice were observed daily throughout pregnancy, and maternal body weights
6-2
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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)
cn
do Bell (1977)
Dorfmuel ler
et al. (1979)
Bellies 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-
hydri n
(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 9
CR - SO 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
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
Premating 3 wk + first 18-d
gestation, daily (rat)
+ first 21-d gestation, daily
(rabbit)
(maternal toxicity)
-------
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 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 the maternal animals 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/m ) TCI caused no
significant maternal, embryonal or fetal toxicity, and that TCI was not terato-
genic 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/m ) 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 sperma-
tozoa 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 examined 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
3
weight gain when dams were exposed to 300 ppm (1614 mg/m ) 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,
6-4
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litter size, the incidence of fetal resorptions, fetal sex ratios, or fetal
body measurements. 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
3
(1614 mg/m ) TCI causes little or no significant maternal, embryonal or fetal
toxicity in Sprague-Dawley rats.
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/m ) was administered to 17
dams by inhalation exposure, 6 h per day, from day 6 through day 15 of gesta-
tion. Control pregnant animals (21) were killed on day 20 of gestation.
Caesarean sections were performed and the number of fetuses, implantation
sites, resorption sites, and corpora lutea were counted. 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/m ) TCI exposure produced little or 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).
3
Exposure to TCI, 500 ppm (2690 mg/m ) 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
determined. The maternal livers, kidneys and lungs were weighed. The numbers
6-5
-------
TABLE 6-2. EXPERIMENTAL DESIGN OF BELILES et al. STUDY (1980)
ON SPRAGUE-DAWLEY RATS
Group
1
2
3
4
5
6
No.
21
16
20
16
20
32
TCI
ppm
0 (control)
0 (control)
500
500
500
500
Days of Exposure
(Controls exposed to air only)
Pre-gestational Gestational
None
(5 day/wk) 3 wk
None
(5 day/wk) 3 wk
None
(5 day/wk) 3 wk
0-18
0-18
0-18
0-18
6-18
6-18
of implantation sites., live and dead fetuses, and resorption sites were re-
corded. 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 (2690 mg/m3).
Dorfmueller et al. (1979) conducted a study of the teratogenic and feto-
toxic effects of TCI in Long-Evans rats exposed to a inhalation concentration
of 1800 ppm (9684 mg/m ) 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 hr/day, 5 days/wk for 2 weeks,
and the gestational exposure was 6 hr/day each day for days 0 to 20. Day 1 of
pregnancy was considered to be the day on which sperm from mating were observed.
On day 21 of gestation, 15 out of 30 dams/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
6-6
-------
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
5 day/wk; 2 wk
5 day/wk; 2 wk
none
none
Exposure
Gestational
1-20
none
1-20
none
fetuses counted. Each fetus was weighed, the sex was recorded, and then exam-
ined for external anomalies. Four fetuses from each litter were examined for
soft tissue changes, and four fetuses were evaluated for skeletal abnormalities.
Livers were removed from the remaining fetuses for measurement of mixed-function
oxidase activity.
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
3
(9684 mg/m ) 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
interactions were found in numbers of corpora lutea or implantation sites/
litter, fetal body weights, resorbed fetuses/litter, or sex ratios. No signi-
ficant 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
6-7
-------
in the general process of skeletal ossification which are not necessarily
3
irreversible. Thus, prenatal exposure to 1800 ppm (9684 mg/m ) 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, however, a small but statistically significant depression in post-
natal 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 treatment-related maternal toxicity, embryotoxicity, terato-
genicity, or significant postnatal behavioral deficits from exposure,
pre-gestational and/or gestational, to 1800 ppm (9684 mg/m ) 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/ TCI
group ppm
21
18
23
19
23
25
0 (control)
0 (control)
500
500
500
500
Days of Exposure
Pre-gestational Gestational
None
5 day/wk;
None
5 day/wk;
None
5 day/wk;
3 wk
3 wk
3 wk
0-21
0-21
0-21
0-21
7-21
7-21
Exposure to TCI, 500 ppm (2690 mg/m ) in air, was 7 hr/day, 5 day/wk during
a 3-week 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 lutea 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
6-8
-------
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.
The number of deaths in the TCI-treated groups was not significantly
greater than among controls. Deaths occurring during the experiment account
for the unequal 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/m ) 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
treatment group 3 and the malformation was not observed in the litters of
3
rabbits that were exposed to 500 ppm (2690 mg/m ) 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. It is not known if the increase in external
hydrocephalus observed in this study is a significant observation of a rare
biological anomaly. Additional studies would be needed to strengthen this
conclusion.
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. Skeletal examination revealed no unusual features, except for an
6-9
-------
increased incidence of changes related to retarded bone ossification, which
were not considered malformations 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/m ) TCI revealed little or no evidence
of maternal toxicity or embryotoxicity; the 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 occurrence. A definite conclusion concerning
this effect cannot be determined without additional studies that would strengthen
this observation.
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
anesthetics in use, and, therefore, it is impossible to make a specific associa-
tion of TCI with the miscarriages. Cote (1975) has pointed out weaknesses in
this study due to the lack of direct physician examination of the children and
a lack of stringent categorization of the types of birth defects. It was
suggested by Cote that additional long-term studies be conducted to resolve
these issues.
6.3 SUMMARY
Inhalation exposure studies in the mouse and rat by different investigators
indicate that TCI does not produce significant signs of developmental toxicity
except at levels which affect maternal well-being. One investigator, Beliles
et al. (1980), reported the occurrence of an unusual observation in rabbits,
hydrocephalus, in a few fetuses from one exposure group. However, this observa-
tion was not dose-related and the significance of this finding could not be
assessed from this study.
6-10
-------
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
conceptus is uniquely susceptible to the effects of TCI. It should be noted,
however, that the potential of TCI to produce external hydrocephalus in rabbits
has been discussed by Bellies et al. (1980) and could not be resolved by that
study. In addition, TCI has been implicated in producing adverse male repro-
ductive effects (see Chapter 7).
6-11
-------
6.4 REFERENCES
Beliles, R. P., D. J. Brusick, and F. J. Mecler. 1980. Teratogenic-Mutagenic
Risk of Workplace Contaminants: Trichloroethylene, Perch!oroethylene and
Carbon Disulfide. U.S. Department of Health, Education and Welfare (Contract
No. 210-77-0047).
Bell, Z. 1977. 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.
Bernstine, J. B. and A. E. Meyer. 1953. Passage of metabolites of chloral
hydrate into amniotic fluid. Proc. Soc. Exp. Biol. Med. 84:456-457.
Bernstine, J. B. , A. E. Meyer and R. L. Bernstine. 1956. Maternal blood and
breast milk estimation following the administration of chloral hydrate
during the puerperium. J. Obst. Gyn. 63:228-231.
Bernstine, J. B. , A. E. Meyer and H. B. Hayman. 1954. Maternal and foetal
blood estimation following the administration of chloral hydrate during
labour. J. Obst. Gyn. 61:683-685.
Bross, G. , D. D. Franceisco, and M. E. Desmond. 1983. The effects of low
dosages of trichloroethylene on chick development. Toxicol. 28:283-294.
Corbett, T. H. 1972. Anesthetics as a cause of abortion. Fert. Steril.
23:866-869.
Corbett, T. H. , R. G. Cornell, J. L. Endres, and K. Leiding. 1974. Birth
defects among children of nurse anesthetists. Anesthesiology 41:341-344.
Corbett, T. , G. Hamilton, M. Yoon, and J. Endres. 1973. Occupational exposure
of operating room personnel to trichloroethylene. Can. Anesth. Soc. J.
20:657-678.
Cote, C. J. 1975. Birth defects among infants of nurse anesthetists. Anesthe-
siology 42:514-515.
Dorfmueller, M. A., S. P. Henne, R. G. York, R. L. Bornschein, and J. M.
Manson. 1979. Evaluation of teratogenicity and behavioral toxicity with
inhalation exposure of maternal rats to trichloroethylene. Toxicol.
14:153-166.
Elovaara, E., K. Hemminki, and H. Vainio. 1979. Effects of methylene chloride,
trichloroethane, trichloroethylene, tetrachloroethylene and toluene on
the development of chick embryos. Toxicology 12:111-119.
Fink, R. 1968. Toxicity of Anesthetics. Williams andWilkins Co., Baltimore.
MD, pp. 270.
Goodman, L. S., and Gilman, A. G. 1980. The Pharmacological Basis of Therapeutics.
Ed. A. G. Gilman, L. S. Goodman and A. Gilman, 6th ed. New York: McMillan
Pub. Co., pp. 361-363.
6-12
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PDR, Physician's Desk Reference. 1981. Oradell, N.Y.: Medical Economics Co.,
p. 1258.
Schwetz, B. A., B. K. J. Leong, and P. J. Gehring. 1975. The effect of
maternally inhaled trichloroethy1ene, perchloroethy1ene, methyl chloroform,
and methylene chloride on embryonal and fetal development in mice and
rats. Toxicol. Appl. Pharmacol. 32:84-96.
U.S. Environmental Protection Agency. 1980. 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.
6-13
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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 indicative of DMA damage (gene conversion,
mitotic recombination, sister chromatid exchange, 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 that concerned testing of TCI for its
mutagenic potential in bacteria. Seven used Salmonella typhimurium (Henschler
et a!., 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 (NCI) 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; diisobuty-
lene, 0.02; ethylacetate, 0.052; pentanol-2, 0.015; butanol, 0.051. These
impurities were tested for mutagenicity with and without S9 mix from PCB-induced
Wistar rat livers. In Salmonella typhimurium, two impurities, epichlorohydrin
and 1,2-epoxybutane, were found to be mutagenic in tests conducted without
activation. Increases of twenty-five and eight times 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
7-1
-------
tetrachloride, chloroform, and 1,1,1—trichloroethane, both with and without
activation, but no data were presented. Similarly, no increased mutation fre-
quency was obtained with "purified" TCI (source and purity not given). Roughly
140 TA100 revertants were observed at each point tested over the entire dose
-3
range (from 1.12 x 10 M to 1.12 M), both with and without metabolic activation;
the spontaneous revertant count was 138 ± 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. The basis for reaching these conclusions is
inadequate for a number of reasons including flaws in the study design, such
as those mentioned above.
Margard (1978) also addressed the hypothesis that the mutagenic activity
of technical-grade samples of TCI is due to mutagenic stabilizers rather than
TCI 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 deter-
mining 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 sample contained 0.08
percent (w/w) epichlorohydrin, 0.02 percent N-methylpyrrole, 0.13 percent
N-propyl alcohol, 0.05 percent nitromethane, 0.10 percent 1,1,1-trichloroethane,
and 0.45 percent 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
7-2
-------
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
percent 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 precau-
tions were. The toxicity data suggest that the cells were exposed to the test
material, but it is not possible to determine toxicity adequately in a plate
incorporation test. To do this, a liquid suspension assay must be done.
Appropriate positive controls were reported and demonstrated that the 59 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 hydro-
carbons may prevent them from being adequately tested in standard plate incor-
poration 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 percent in the atmos-
phere. The purity of the sample tested was not reported, but it would have
been high, with an approximate minimum purity of 99.5 percent and stabilization
by 0.1 percent 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-hr 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 nonvolatile compound 1,1,1-trichloroacetic acid (>99.0 percent
pure, obtained from Matheson, Coleman, and Bell, personal communication with
Matheson, Coleman, and Bell), chloral hydrate (recrystallized from chloroform
7-3
-------
TABLE 7-1. MUTAGENICITY OF TECHNICAL-GRADE TCI IN SALMONELLA/MAMMALIAN
MICROSOME ASSAY
Activation
Toxicity
Tester
Strain
TA1535
TA100
Cone.
(ml/plate)
1.0
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
Rel.
Via.*
0.14
0.72
0.94
--
0.19
0.73
0.90
—
MUTAGENICITY OF
TA1535
TA100
0.1
0.5
0.1
0.05
0.01
0
1.0
0.5
0.1
0.05
0.01
0
97
172
22
246
175
175
225
317
0.39
0.70
0.90
--
0.55
0.55
0.71
"" ™"
Mutagenicity
No.
Rev.t
58
40
17
24
184
263
123
167
PURIFIED-GRADE
17
19
21
22
70
142
157
154
Nonactivation
Toxicity
Plate
Count
22
122
149
157
66
167
232
234
TCI
89
121
156
175
166
162
354
305
Rel.
Via.*
0.14
0.78
0.95
--
0.28
0.71
1.0
—
0.51
0.69
0.89
--
0.54
0.53
0.82
"
Mutagenicity
No.
Rev.t
58
42
21
23
173
181
136
160
15
20
20
19
114
158
145
158
*Relative viability of tests compared to the control plates.
tNumber of revertants observed (average).
Source: Adapted from Margard, 1978.
7-4
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six times, to achieve a purity of >99.0 percent), 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 are 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 as statistically significant by
the t test (P <0.01), the increases merely suggest, at best, 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 airtight exposure chambers and metabolic activation.
In tests employing Salmonella tester strain TA100, both with and without
an exogenous S9 metabolic activation, Bartsch et al. (1979) assessed the muta-
genic 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 percent pure and contained no detectable amounts of epichlorohydrin
or 1,2-epoxybutane at levels >I 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 10-1 desiccators for various lengths of time
at 37°C, in the dark. Upon completion of the exposure period the compound was
removed, and the plates were then covered, inverted, 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:
7-5
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Concentration in Air
(5% v/v)
Time
(hr)
Revertants/plate
+S9 -S9
Trichloroethylene
Vinyl
0
8
20
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 percent TCI vapor in air for 2 hours, a nearly twofold increase over the
spontaneous mutation frequency was observed (see table).
Concentration Air
(% v/v)
0
5*
5*
Time
(hr)
2
2t
Revertants/pl ate
+S9
75
100
135
-S9
70
80
70
*0.1 mM EDTA added to soft agar.
tPreincubated with S9, TA100, and 0.1 mM EDTA for 4 hours, then exposed
to TCI vapors.
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 percent pure, contained no detectable epoxides, and was stabilized
with 0.1 percent (w/w) thymol (personal communication with M. Kelly, V.A.
7-6
-------
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 percent. 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 percent (± 10 percent 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 percent above background (149 ± 12, n=15) at concentrations
of 1 percent (196 ± 13, n=15), and 3 percent (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.
Simmon et al. (1977) also reported that reagent-grade TCI, with no detec-
table epoxides, gave less than a twofold increase in revertant counts in
Salmonella typhimurium strain TA100. The sample was 99.85 percent pure, and
contaminants included 0.09 percent chloroform, 0.02 percent tetrachloroethy1ene,
0.01 percent pentachlorobutadiene, and 0.03 percent 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 spon-
taneous 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
2.0 ml TCI/91 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 2.0 ml
7-7
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TCI/91 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 analyt-
ical 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 approxi-
mate composition of the TCI product was probably: TCI, 99.9 percent; 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, 1,2-dichloro-
ethane, 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 £._ 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 percent.
A weak twofold mutagenic effect of TCE, expressed as a percentage of the
spontaneous mutation rate, was observed for arg , 232 ± 36, but the signifi-
cance of this response cannot be ascertained because no increase was observed
for the other markers; gal , 123 ± 23; MTR, 114 ± 18; nad , 100. Descriptions
of the methodology and the observed results were inadequate. For instance,
information on 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 signifi-
cance of the results cannot be assessed adequately.
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
7-8
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that TCI itself is mutagenic are at best suggestive; 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 (
-------
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 lower, by 44 percent and 35 percent, respectively, than 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
-4
tested by itself was, however, positive (3.71 ± 1.05 x 10 mutants at 50
as was the response of the positive control, dimethylnitrosamine
-4
(16.73 ± 3.24 x 10 mutants at 100 mg/kg), indicating that, for these chemicals,
the assay was working properly. No data were presented 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 condi-
tions employed.
Subsequent testing by the same laboratory (Rossi et al., 1983) was con-
ducted to investigate further the mutagenic potential of pure and technical
samples of TCI and two stabilizers contained in the technical -grade sample,
epichlorohydrin and 1,2-epoxybutane. Schi zosaccharomyces pombe strain PI was
used in i_n 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 j_n vitro studies in which 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) were found. By contrast, epichlorohydrin
and 1,2-epoxybutane caused dose-related increases in mutant frequency up to
about a tenfold 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-I 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 2 g/kg. These data suggest that the intraperitoneal
host-mediated assay in mice with Schi zosaccharomyces pombe may not be appro-
priate for testing halocarbons and their stabilizers.
7-10
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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)
Treatment
Dose
(mg/kg)
Mutants
M.F. x 10 (M ± S.E.)
Tri chloroethy1ene,
technical grade
Methylmethanesulfonate
None (control)
500
1,000
2,000
50
0.94 ± 0.08*
1.33 ± 0.28
1.32 ± 0.29
1.61 ± 0.45
4.16 ± 0.54
Tri chl oroethy 1 ene,
purified grade
Dimethylnitrosamine
Epichlorohydrin
None (control)
1,000
2,000
100
None (control)
2
10
50
1.45 ± 0.06
1.59 ± 0.22
1.36 ± 0.21
16.73 ± 3.24
1.66 ± 0.03
1.50 ± 1.20
2.05 ± 0.51
3.71 ± 1.05
*Lower than the other negative control values.
Source: Loprieno et al., 1979.
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: TCI, 99.9
percent; water, evaporation residue, chloroform, 1,2-dichloroethane, carbon
tetrachloride, 1,1,2-trichlorethane, other impurities, all £20 ppm; diisopro-
pylamine, 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 TCI 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-hr incubation period,
the animals were sacrificed to remove yeast cells from the peritoneum for
plating and counting. Plates were incubated at 32°C for 4 to 5 days before
~4
mutant colony counting. Mean numbers (± S.D.) x 10 of mutant colonies were
as follows: control, 1.80 ± 0.48; low dose, 1.18 ± 1.18; mid-dose, 1.03 ±
7-11
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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 of the test mice.
The mutagenic properties of technical-grade TCI in the XV 185-14C haploid
strain of Saccharomyces cerevisiae were assessed i_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 incu-
bated in liquid YPG medium at 30°C until a stationary growth phase was achieved.
o
Cells were harvested, washed, and adjusted to a population of 1 x 10 cells/ml.
In order to estimate its mutagenic effect in the absence of metabolic activa-
tion, cells treated with 1.11, 5.56, 111.1, or 222.2 mM of TCI during incuba-
tion 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 exces-
sive 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 percent cell death). The revertants/10 survivors
for lys 1-1, his 1-7, and horn 3-10, corresponding to 1-hr 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 conduc-
ted with metabolic activation is uncertain, because the increases were noted
only when there was greater than 99 percent killing. The positive control in
the tests with metabolic activation (EMS) was inappropriate because it is a
direct-acting mutagen. TCI without metabolic activation was ineffective as a
mutagen at all doses tested, but increased numbers of revertants were noted
7-12
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TABLE 7-3. MUTAGENICITY OF TECHNICAL-GRADE TCI AT THE HIS 1-7
LOCUS OF SACCHAROMYCES CEREVISIAE
Dose
(mM)
0
1.11
5.56
111.1
222.2
Positive control
-S9
(6-hr
incubation)
Revertant
14.3
16.7
22
0
0
83*
Percent
Survival
100
90
72
<0.13
<0.013
61
+S9
(4-hr
incubation)
Revertants
19.2
15.9
16.8
22,000
>29,000
6300t
Percent
Survival
99
72
65
<0.1
<0.1
0.23
2-acetylaminofluorene at 20 ug/ml.
'''EMS at 8 pi/ml-
Source: Adapted from Shahin and Von Borstel, 1977.
with activation. With respect to the 48-hr treatment without activation, the
maximum number of revertants per 10 survivors for the 3 loci individually was
10 to 15 for untreated controls (98 percent survival), 12.8 to 21.4 for 1.11
mM (73 percent survival), 11.4 to 17.1 for 5.56 mM (64 percent survival), and
0 for 11.1 mM and 222.2 mM (<0.1 percent survival) of TCI. The lack of rever-
tants with the two highest concentrations appears to correspond to failure of
the cells to survive after plating. The largest number of revertants per 10
survivors produced by 2-acetylaminofluorene (222.2 mM) was 102 (lys 1-1) after
a 6-hr treatment, and 673 (his 1-7) and 488 (horn 3-10) after a 24-hr treatment.
It may be that there is a "window" of effective concentrations in which 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; it was in this dose range that the
weak positive responses discussed below were obtained by Bronzetti et al.
(1978) and Call en et al. (1980) in strain D7. 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
7-13
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the response cannot be ascertained or ruled out (personal communication with
R.C. Von Borstel, University of Alberta). These results do not allow a conclu-
sive evaluation to be made.
An increase in point mutations and gene conversion by a certified ACS
reagent grade of TCI in Saccharomyces cerevisiae strains D4 and D7 was observed
by Bronzetti et al. (1978) in cell suspension and in host-mediated assays. 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 percent and stabilization with either 25 ppm diisopropylamine or 20 ppm
triethyl amine. In the cell-suspension experiment, strain 07 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, ade recombinants, and trp convertants) and on
complete media (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 DMA 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 introduc-
tion 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 ce.ll viability in the cell suspension system but did
not induce a mutagenic response. Survival was reduced to approximately 87
percent with the 10-mM dose and to 65 percent with the 40-mM dose, compared to
100 percent for the controls. TCI did produce concentration-dependent increases
in point mutation frequencies with metabolic activation at doses of 10, 20,
30, and 40 mM. Positive responses were also observed in the host-mediated
7-14
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TABLE 7-4. MUTAGENICITY OF REAGENT-GRADE TCI AT THE ILV LOCUS IN
SACCHAROMYCES CEREVISIAE
Suspension Tests Strain D7
Concentration
(mM)
Revertants/106
(Mean x ± S.E.)
Percent 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 ± 0.16
1.77 ± 0.12
2.24 ± 0.17
3.23 ± 0.32
Mice
No. of
Treatment Mice
Control TCE 3
4
Control TCE 2
3
Control TCE 2
4
100
87.5 ±1.0
64.1 ±2.1
100
81.6 ± 2.9
67.1 ± 2.8
55.5 ± 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 ± 0.19
0.21 ± 0.02
0.79 ± 0.37
Source: Adapted from Bronzetti et al., 1978.
7-15
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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, in which 0.79 ± 0.37 x 10
revertants were found in the treated animals versus 0.21 ± 0.02 x 10 in the
negative controls.
Like Bronzetti et al. (1978), Call en et al. (1980) also employed a TCI
sample obtained from the Fisher Scientific Company. They used Saccharomyces
cerevisiae strains D4 and D7 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 survivors, respectively) without an
exogenously supplied metabolic activation system (67 percent cell survival).
A severely toxic effect was observed at 22 mM. A positive response without
activation was observed by Call en et al. (1980) but not by Bronzetti et al.
(1978). The reason for this is not known, but it may be due to the growth
stage of the yeast cells when tested. Call en et al. (1980) found TCI to be
mutagenic to cells from log phase cultures but not to cells from stationary
cultures of 07. 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 Saccharo-
myces can metabolize TCI. They analyzed whole-cell suspensions of strain D5
in the log phase of growth in a spectrophotometer, after exposure to unspecified
amounts of TCI. Peaks with maximums of 450 and 505 mM were observed. The
peak at 450 nm appeared to reach a maximum after roughly 5 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 TCI enters exposed yeast cells and suggest TCI is
metabolized by a yeast cytochrome P.5Q-independent monooxygenase system.
7-16
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TABLE 7-5. MUTAGENICITY OF TCI AT THE ILV LOCUS IN
SACCHAROMYCES CEREVISIAE D7
Survival
Concentration Total
(mM) Colonies
0
15
22
1291
802
3
Percent
199
67
0.3
ilv 1
Total
Revertants
43
59
Revertants
106 Survivors
3.6
7.4
Source: Adapted from Callen et al., 1980.
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 to 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
Schi zosaccharomyces pombe host-mediated assays are not considered to diminish
the positive results in Saccharomyces because of the inherent insensitivity of
the S_. pombe test (fprward 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 interspecific 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 inflores-
cences with 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 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
7-17
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value cannot be verified. The mutation frequencies (± S.E./100 cells) for
6-hr 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.
7.1.2.2 Animals.
7.1.2.2.1 Insects (Drosophila). Abrahamson and Valencia (1980) fed 1000 ppm
(5380 mg/m ) TCE in 0.2 percent 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, screening the F_ descen-
dents 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 3 females each and then transferred without
anesthetization to fresh females at 2- to 3-day intervals, until a total of 4
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 were not
ascertained (personal communications with L.Valcovic, Food and Drug Administra-
tion, sponsor of the "study; and Allied Chemical Co.).
TCI was not mutagenic in the above test. The frequency of lethals (number
of lethals/number of chromosomes tested) was 0.12 percent (19/9248) in the
feeding experiment, 0.17 percent (4/2344) in the injection experiment, 0.31
percent (31/10,009) in concurrent controls, and 0.24 percent (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 (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. Threehundred one-day-old B Y y ac , (K 1+2) /X.Y ,
INEN2,f males were placed in each of three screen cages and subsequently
7-18
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3
exposed to 0, 100, and 500 ppm (0, 538, and 2690 mg/m ) TCI vapor from North
Strong (estimated to be 99.9 percent pure by infrared spectroscopy) for 7
hours. An unspecified number of males serving as positive controls were fed
0.025 M EMS in 1 percent sucrose for 8 hours. Two hundred males per treatment
8 8
group were individually mated to sequential groups of 3 In(l)sc , sc 1
Ml Ml
B/In(l)dl-49 B , y ac v B 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 F..
Ml
generation. F-. males of the genotype y ac v B /In(l)EN2,f were mated to
S L
virgin Y /X.Y , In(l)dl-49, v f B females, and the occurrence of recessive
lethal mutation on the In(l)EN2, f chromosome was detected by the lack of
males with forked bristles in the F2 generation. The frequencies of X or Y
chromosome loss were 0.07 percent and 0.17 percent for the low and high doses,
respectively, compared to 0.06 percent for the concurrent negative control.
These values are not significantly different; however, the 0.17 percent was
close to a significant value. 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 percent and 0.05 percent for the low
and high doses, respectively, and losses of the short arm of the Y chromosome,
0.02 percent for both dose levels, were not significantly different from the
0.01 percent 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 percent and 0.16 percent
for the low and high doses, respectively, compared to 0 percent for the concur-
rent negative control and 0.11 percent of the historical negative control.
The frequency of sex-linked recessive lethal mutations for the positive control
(EMS) was 21.56 percent 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, c
7-19
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p/cc 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/+, c p/+ +, 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
percent 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 percent chloride (C12), and 0.0001 percent 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 endpoint, twice weekly between 2 and 5 weeks of
age.
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 percent), 2 low-dose pups (1.3 percent), and 2 high-dose pups (3.92
percent). Known mutagens, including ethyl methanesulfonate, were positive (26
percent 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 mice, commercial samples of TCI are judged to be capable of causing gene
mutations after metabolic activation. Because of the requirement of activation,
it is not likely that epoxide stabilizers present in the commercial samples
account for the effect. However, because high doses of commercial-grade TCI
were required to elicit a positive effect, the material must be considered to
be only weakly mutagenic. The data for purified samples of TCI do not conclu-
sively 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 (Bellies et al., 1980). The rest of the studies are presented
next.
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7.2.1 Experimental Animals
7.2.1.1 Bone Marrow. Bellies 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 (0, 538, or
2690 mg/m ) TCI by inhalation, both acutely and subchronically. The positive
control was TEM at 0.3 mg/kg given i.p. in 0.85 percent saline. Acute exposure
consisted of a single 7-hr session in the exposure chamber. Subchronic exposures
consisted of 5 daily exposures of 7 hours. Bone marrow cells were then collec-
ted; slides were prepared and coded, and 50 cells/animal were scored. There
was no increase in the number of aberrations in either study. The percentage
of cells with aberrations were 0.4 to 2.4 in the negative control group compared
to 0 to 2.7 in the TCI-treated group. The positive control TEM was clastogenic,
causing 7.2 to 8.5 percent 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
and 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 hour (American Industrial Hygiene
Association, 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 (0, 269, 1086, and 2421 mg/m ) TCI in air to cause dominant lethal mutations
in NMRI-Han/BGA mice. The sample contained 99.5 percent TCI stabilized with
100 mg/1 triethanolamine. Analysis of a liquid sample also identified 5 ppm
1,2-dichloroethylene, 9 ppm cis-l,2-dichloroethylene, tetrachloride, 10 ppm
1,1,2-trichloroethane, and 815 ppm tetrachloroethylene. Fifty males per dose
were exposed for 24 hr in a 1000-1 volume glass chamber equipped with a ventila-
tor. The temperature was kept between 22-24°C and the humidity between 70-80
percent. For rapid equilibration the calculated volume of liquid TCI was
7-21
-------
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) 1/hr. After the
24-hr 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 cervical 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 (2421 mg/m3) 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 micro-
nucleus test. CD-I mice were divided into 9 groups, 6 of which were given up
to 3000 mg/kg TCI (analytical grade 99.5 percent 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 and stained, and
polychromatic 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 percent gum arabic
solution) than in the untreated control (1.0 ±0.5 versus 0.5 ± 0.4 percent).
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).
7-22
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TABLE 7-6. MUTAGENICITY OF TCI IN THE DOMINANT LETHAL ASSAY, USING NMRI MICE
1
ro
GO
Dose
in ppm
(rate)
(200 1/h)
0
50
202
(300 1/h)
0
450
*Dominant
F. = 1-
Female
Total
Mated % w/Implants Implants
498
482
493
503
488
lethal factors (FL).
(live implants per
89.6
90.1
91
93.4
92.4
female
5578
5360
5703
6142
5927
of test group)
Total
Dead
Implants
443
479
531
502
533
x 100
Total
Live
Implants
5135
4881
5172
5640
5394
Live % Dominant
Implants/ Lethal
Female Factors*
11.5
11.2 2.6
11.5 0
12.0
11.97 0.25
Source: Adapted from Slacik-Erben et al., 1980.
-------
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 to what other chemical substances they may have been exposed. 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 percent (X ± SD) hypodiploid cells, which
was significantly elevated compared to the control group, 6.5 ± 3.2 percent
hypodiploid 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 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 indivi-
duals possessing >13 percent hypodiploid cells were abnormal. Workers were
divided according to whether or not they possessed >13 percent hypodiploid
cells, and these groups of workers were subsequently further characterized
(Table 7-7). Nine individuals had greater than 13 percent hypodiploid cells.
These individuals worked under higher daily peak concentrations of TCI (as
measured on three occasions over a 2-week period for 8 hr each time) than did
their fellow workers. The average daily concentrations for these 2 groups of
workers were 75 ± 14 ppm versus 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 techni-
cal changes had been made in the plants. This report may provide suggestive
evidence that the exposures resulting from the manufacture of technical-grade
7-24
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TABLE 7-7. NUMERICAL CHROMOSOME ABERRATIONS IN TCI WORKERS
Parameter
>13% Hypodiploid Cells
(n* = 9)
Mean Value Standard Error
(X)
>13% Hypodipoloid Cells
(n* - 18)
Mean Value Standard Error
(X) (SJ
Daily average
concentration
(ppm)
Average maximum
concentration
(ppm)
75.0
205.8
17.4
96.3
61.4
115.7
*n = Number of individuals studied.
Source: Adpated from Konietzko et al., 1978.
23.1
57.6
Exposure 5.9
(years)
Total dose
(ppm x day) 0.55 x 106
Age 38
(years)
Alcohol 18.9
(g/day)
Nicotine
(cigarettes/day) 12.8
4.8 5.9 5.1
0.72 x 106 0.31 x 106 0.39 x 106
9.4 40.4 9.7
29.3 33.6 37.5
12.7 10.1 10.2
7-25
-------
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 to
exposure to TCI renders this study inconclusive.
7.3 OTHER STUDIES INDICATIVE OF MUTAGENIC ACTIVITY
Additional studies have been conducted on the genotoxicity of TCI. These
studies do not measure mutagenic events per se, in that they do not demonstrate
the induction of heritable genetic alterations, but positive results in these
test systems show that DNA 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 end point 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
convertants at 40 mM compared to 16.3 ± 0.42 x 10 convertants for the negative
control). No increase was noted in the test conducted without activation.
Toxicity was observed in both tests with a 40 to 50 percent 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.
7-26
-------
Call en 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 percent at 15 mM and by >99
percent at 22 mM TCI. A fivefold increase in trp convertants was detected at
15 mM compared to the negative controls (76 x 10 versus 14 x 10 , respec-
tively). 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 vs. 3 x
-3
10 , respectively). These end points 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 DMA 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 percent of the atmosphere in the presence of 19 percent
oxygen, 5 percent carbon dioxide, and 75.83 percent nitrogen for 1 hour with S9
mix prepared from Aroclor 1254-treated male rats. After exposure, the concen-
tration of TCI gas in the flask was measured by gas chromatography and found
to be 59 percent 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. Onehundred 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 toxi-
city in the treated cells, further suggesting that they did not receive an
adequate dose. In the second place, no concurrent positive controls were
employed to ensure that the test system was working properly. These defici-
encies reduce the weight of the negative response reported by White et al.
(1979).
7-27
-------
Gu et al. (1981) collected blood from six individuals occupationally
exposed to TCI, cultured their peripheral lymphocytes, and scored the chromo-
somes 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, i_n 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 versus 7.9 ± 0.2). There was no information
about the levels of TCI to which the subjects were exposed 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, respec-
tively, for 68 to 72 hours, and increases in SCEs were observed, compared to
the untreated cultures. The results are tabulated as follows:
1.
2.
3.
4.
5.
Group
Control
Trichloroethanol (1)
Trichloroethanol (2)
Chloral hydrate
TCI in vivo
No. of
Karyo types
212
24
72
52
194
X ± SD
7.
9.
9.
10.
9.
9 ±
8 ±
2 ±
7 ±
1 ±
3.
3.
3.
4.
4.
0
2
3
0
9
Comparison
Between
1
1
1
1
&
&
&
&
2
3
4
5
P Value
from
t Test*
<0.05
<0.01
<0.001
<0.01
*Calculated by Gu et al.
The results obtained by Gu et al. (1981) are suggestive of a positive re-
sponse. 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 suggestive responses obtained
by Gu et al. (1981) in in vitro studies with trichloroethanol (>99.5 percent
7-28
-------
pure, Fluka; Groups 2 and 3) 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 DNA 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; 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 pi/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 between 1.25 to 10 pg/ml and 2.5 to 20
jjg/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 genotoxic potential, because
the positive control (BaP) was only effective at one dose (2.5 (jg/ml); higher
doses resulted in a negative response, although this may have been due to cell
killing.
Perocco and Prodi (1981) collected blood samples from healthy humans,
separated the lymphocytes, and cultured 5 x 10 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
MerckSchuchardt, Darmstadt, FRG, 97-99 percent pure). The tests were conducted
both in the presence and in the absence of PCB-induced rat liver 59 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 [ H] deoxythymidylic acid (TdR) after 4 hours of culture (2661 ± 57
dpm in untreated cells, compared to 1261 ± 36 dpm in cells treated with 5
pi/ml TCI). Subsequently, 2.5, 5, and 10 pi/ml (0.028, 0.056, and 0.11
jjmole/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
7-29
-------
TABLE 7-8. UNSCHEDULED DMA SYNTHESIS IN WI-38 CELLS
I
co
o
Test
Nonactivation
Solvent control, DMSO
MNNG
TCI
Activation
Solvent control, DMSO
BaPb
TCI
Compound
Concentration
(Ml/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
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
Source: Adapted from Beliles et al., 1980.
-------
3
estimated by measuring dpm from incorporated [ H]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 ± 34 at 5
Ml/ml, compared to control value of 612 ± 26). The increase was not statisti-
cally 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). 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," con-
tained 99.9 percent TCI. No information was provided about the remaining com-
ponents. 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
3
( 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 incuba-
tion period, the slides were developed and examined microscopically to count
developed silver grains in the radiotrack emulsion over the cell nuclei.
7-31
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Williams and Shimada (1983) used 2-acetylaminofluorene and vinyl chloride
as positive controls for the conventional and modified exposure assays, respec-
tively. 2-acetylaminofluorene was highly active (170 grains/nucleus at 10 M)
and by comparison, vinyl chloride was weakly active at 2.5 percent (5 grains/
nucleus) and 5 percent (11 grains/nucleus) in increasing unscheduled DNA syn-
thesis compared to the negative controls (3 grains/nucleus).
In the modified HPC DNA repair assay, cells were exposed to dosages up to
2.5 percent TCI in the air for 3 or 18 hours. No increase in the grain count
was observed (the number of grains/nucleus ranged from 0.2 ± 0.2 to 3.2 ± 2.5
in treated cells, compared to 2.75 ± 2.76 in the untreated controls). Similar-
ly, negative results were reported in the conventional assay, in which the
cells were exposed to concentrations as high as 1 percent 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 end point measured may not actually reflect repair synthesis
of chromosomal DNA in the nucleus (Lonati-Galligani et al., 1983). Enhanced
3 3
incorporation of H-TdR into mitochondrial DNA or suppressed HTdR incorpora-
tion into mitochondrial 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 than the highest dose tested for TCI.
7-32
-------
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
_c _p
reported to be nontoxic between 10 M and 10 M. A negative response was
obtained in a test conducted in rat cells. Positive and negative controls
were employed for both sets of experiments and responded appropriately, but
the aforementioned concerns 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
is weakly active in damaging DMA.
7.4 EVIDENCE THAT TCI REACHES THE GONADS
Although not bearing on the ability of TCI to cause mutations, two studies
have been conducted to assess its ability to induce sperm morphological abnormal-
ities in mice (Land et al., 1979; 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)F1 male mice (13 weeks old) to air (negative
control), 0.02 percent and 0.2 percent anesthetic-grade TCI vapor 4 hr/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 percent Eosin Y. Slides were prepared, mounted, coded, and
evaluated by scoring 1000 sperm/slide at 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.
7-33
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Agent
Air
TCI
Concentration %
0.02
0.2
Number of
Animals Studied
15
5
5
% Abnormal Sperm
± S.E.
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 hr/day for 5 days to 0, 100, or 500 ppm
(0,538, or 2690 mg/m ) TCI. After dosing, groups of 4 animals were killed at
the end of 1 week, 4 weeks, and 10 weeks. Sperm were collected from the
caudae epididymides and suspended in 0.85 percent 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 (2690 mg/m ) group compared to the negative controls
for weeks 1 and 4 (i.e., 18.9 percent versus 6.8 percent and 23.5 percent
versus 8.1 percent, respectively). A significant increase was also noted in
the 100-ppm exposure group, compared to the negative control for week 4 (14.8
percent versus 8.1 percent).
7.5 MUTAGENICITY OF METABOLITES
Trichloroethylene is metabolized to trichloroethanol, trichloroethanol
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 which have not
been identified but which are thought to occur (e.g., TCI oxide). Some muta-
genicity studies bearing on a few of TCI's metabolites are discussed below.
See the chapter on metabolism for a more detailed discussion.
7-34
-------
Kline et al. (1982) tested the mutagenic potential of TCI oxide in
Salmonella strain TA1535 and E. coli strain WP2 uvrA. They observed no in-
creases in revertant colonies at doses ranging from 0.25 to 5 mM. Preincubation
tests were conducted, and toxicity was observed at the highest doses, demon-
strating 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 1,3-dichloropropene oxide). The
genotoxicity of these compounds was also assessed by differential growth
inhibition of the DNA polymerase-deficient E. 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 polA cells (up to 40
percent) 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 TA100), 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 tri-
chloroethanol (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,
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 weakly 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 of SCEs in cultured human
lymphocytes, compared to untreated control cells (i.e., 10.7 ± 4.0 versus 7.9
± 3.0). Again, the response was merely suggestive of an effect.
7-35
-------
7.6 BINDING TO DNA
Stott et al. (1982) studied the pharmacokinetics and macromolecular
Interactions of TCI in B6C3F1 male mice and male Osborne-Mendel rats. As part
of their study, mice were given 1200 mg/kg [ C] 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
14
C content was determined by liquid scintillation counting. Analysis of the
data indicated that the ability of TCI to alkylate DNA was low. A maximum
estimate of the average DNA alkylation level was 0.62 ± 0.42 alkylations/10
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 iji 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 end point.
Other tests provide evidence that commercially available TCI damages DNA.
Suggestive and weak-positive responses have been observed in yeast (gene
conversion 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 sugges-
tive positive effects have been shown. TCI or a metabolite(s) may be capable
of binding to DNA, but only minimally.
7-36
-------
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
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 observation that TCI causes adverse effects in the
testes of mice suggests TCI may cause adverse testicular effects in man, too,
provided that the pharmacokinetics of TCI in humans also results in its distri-
bution 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.
7-37
-------
TABLE 7-9. SUMMARY OF TESTS FOR MUTAGENICITY OF TCI
CO
CO
Test
Category
I . Gene
Mutations
Organism Type of Test
Salmonella Reverse mutations
typhimurium in vitro
Plate.
incorporation
tests
Vapor
exposure
Escherichia Forward and
coli reverse mutations
Schi zosaccharomyces Forward mutations
pombe (Host-mediated
assays)
Purity
of TCI Results
Technical
grade
Technical +
grade
Purified
Anesthetic
grade
Purified *
Purified3 *
Reagent *
grade
Analytical *
grade
Technical *
grade
Comments
No control to test
effectiveness of
S9 mix. No
precautions to
prevent evaporation.
Twofold increase
1.8-fold increase
1.3-fold increase
1.7-fold increase
'Positive for reverse
mutations only at
arg locus (twofold
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)
i
CO
Test Purity
Category Organism Type of Test of TCI Results
Purified
Purified
Purified3
Saccharomyces Reverse mutations Technical x
cerevisiae (in vitro) arade
ACS reagent +
grade
Technical +
grade
Tradescantia Forward mutations Unknown *
Drosophila Sex- linked Technical
melanogaster recessive lethals grade
Technical
grade
Mouse Spot test Technical +
grade
Comments
Epichlorohydrin
and epoxybutane
were also negative.
High toxicity
Fourfold increase
both host-mediated
assay and liquid
suspension test.
Twofold increase
Sixfold increase
Reference
Loprieno
et al . , 1979
Rossi et al. ,
1983
Mondino,
1979
Shah in and
Von Borstel ,
1977
Bronzetti
et al . , 1978
Callen
et al . , 1980
Schairer
et al . , 1978
Abrahamson
and Valencia,
1980
Beliles
et al . , 1980
Fahrig
et al . , 1977
+ = Positive
- = Negative
* = Suggestive
a = No detectable epoxides
x = Inconclusive
-------
TABLE 7-9. (continued)
-J
o
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 Purified
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.
Twofold increase
with metabolic
activation.
Reference
Bellies
et al . , 1980
Bellies
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 Type of Test
Mitotic
recombination
Mouse Sister chromatic!
exchange
Human Sister chromatid
exchange
Unscheduled DNA
synthesis
Rat HPC DNA
repair
assay
Purity
of TCI
Technical3
grade
Technical3
grade
Anesthetic
grade
Occupational
exposure
Technical
grade
Technical
grade
Stabilized
Unstabilized
Technical
grade
Results Comments
+ Fivefold increase
+ Fourfold increase
- No positive
controls. No
evidence of
toxicity.
* Increases
correlated with
presence of TCI
metabolites tri-
chloroethanol and
trichloroacetic
acid in the blood
* 1.5 to 1.8-fold
increases
X
Vinyl chloride
only weakly
active.
-
-
Reference
Callen
et al . , 1980
Callen
et al . , 1980
White
et al . , 1979
Gu et al. ,
1981
Bellies
et al . , 1980
Perocco and
Prodi, 1981
Williams and
Shimada,
1983
Wi 1 1 i ams and
Shimada,
1983
Williams,
1983
+ = Positive
- = Negative
* = Suggestive
a = No detectable epoxides
x = Inconclusive
-------
TABLE 7-9. (continued)
i
-E»
ro
Test
Category
IV. Evidence
TCI Reaches
the Gonads
V. Mutagenicity
A. TCI-oxide
Organism
Mouse
Mouse
of Metabolites
Salmonella
typhimurium
Escherichia
coli
Purity
Type of Test of TCI
HPC DNA Technical
repai r grade
assay
Morphological Anesthetic
sperm grade
abnormalities
Technical
grade
Reverse mutations
Reverse mutations
Differential
killing of repair
deficient bacteria
Results Comments Reference
+ 8- to 20-fold Williams,
increases 1983
* 1.8- fold increase Land
et al . , 1979
+ Threefold increase Beliles
et al . , 1980
Kline
et al . , 1982
Kline
et al . , 1982
+ 40% decreases in Kline
survival of Pol- etval., 1982
vs. Pol + cells
B. Tri chl oroethanol
Salmonella
typhimurium
Human
lymphocytes
Reverse mutations
Sister chromatid
exchange
Waskell,
1978
* Gu et al . ,
1981
+ = Positive
- = Negative
* = Suggestive
a = No detectable epoxides
x = Inconclusive
-------
TABLE 7-9. (continued)
Test
Category Organi sm
C. Trichloroacetic Acid
Salmonella
typhimurium
-J D. Chloral Hydrate
00 Salmonella
typhimurium
Human
lymphocytes
Purity
Type of Test of TCI Results
Reverse mutations
Reverse mutations *
Sister chromatid *
exchange
Comments Reference
Waskell,
1978
1.6-fold increase Waskell ,
1978
Gu et al . ,
1981
+ = Positive
- = Negative
* = Suggestive
a = No detectable epoxides
x = Inconclusive
-------
7.8 REFERENCES
Abrahamson, S. and R. Valencia. 1980. Evaluation of substances of interest
for genetic damage using Drosophila 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 dichloro-
butenes produced by rodent or human liver tissues. Arch. Toxicol. 44:
249-277.
Beliles, R.P., D.J. Brusick, and F.J. Mecler. 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 trichloro-
ethylene in yeast. J. Environ. Pathol. Toxicol. 1: 411-418.
Callen, D.F., C.R. Wolf, and R.M. Philpot. 1980. Cytochrome P-450 mediated
genetic activity and cytotoxicity of seven halogenated aliphatic hydrocar-
bons 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 recombina-
tion 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. Henschler. 1975. Mutageni-
city iji 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. Toxicol. Eur. Res. 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 Duur&n. 1982. Mutageni-
city of chloroalkene epoxides in bacterial systems. Mutat. Res. 101: 115-
125.
7-44
-------
Konietzko, H. W. , W. Haberlandt, H. Heilbronner, G. Reill, and H. Weichardt.
1978. Cytogenetlsche Untersuchungen an Trichlorathylen-Arbeitern. Arch.
Toxicol. 40: 201-206.
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 autoradiographic method for detecting DMA 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. Iji vivo mutagenicity studies with trichloroethylene and
other solvents (preliminary results). Unpublished.
Margard, W. 1978. Summary report on i_n vivo bioassay of chlorinated hydrocar-
bon 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.
Mondino, A. 1979. Examination of the live mutant activity of the trichloro-
ethylene compound with "Schizosaccharomyces pombe." Unpublished.
National Cancer Insititute (NCI). 1976. Carcinogenesis bioassay of trichloro-
ethylene. CAS no. 79-01-6. NCI-CG-TR-2.
Perocco, P. and G. Prodi. 1981. DNA damage by haloalkenes in human lymphocytes
cultured i_n vitro. Cancer Lett. 13: 213-218.
Rossi, A. M. , L. Migliore, R. Barale, and N. Loprieno. 1983. In vivo and i_n
vitro mutagenicity studies of a possible carcinogen, trichloroethylene,
and its stabilizers, epichlorohydrin and 1,2-epoxybutane. Teratog.
Careinog. Mutagen. 3: 75-87.
Russell, L.B., and B.E. Matter. 1980. Whole-mammal mutagenicity tests. Evalua-
tion of five methods. Mutat. Res. 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 jji situ exposures. Preliminary results from the
Tradescantia plant test sytem. EPA Pub. 600/9-78-027. Pp. 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.
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.
7-45
-------
Slacik-Erben, R., R. Roll, G. Franke, and H. Uehleke. 1980. Trichloroethylene
vapours do not produce dominant lethal mutations in male mice. Arch.
Toxicol. 45: 37-44.
Stott, W.T., J.F. Quast, and P.G. Watanabe. 1982. The pharmacokinetics and
macromolecular interactions of trichloroethylene in mice and rats. Toxicol
Appl. Pharmacol. 62: 137-151.
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, G.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.
7-46
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8. CARCINOGENICITY
8.1 DESCRIPTION AND ANALYSIS OF 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 inhala-
tion, gavage, subcutaneous injection, and topical application. The results are
summarized in Table 8-1. Of the studies done, the evidence for the carcinoge-
nicity of TCI consists of statistically significant increases of hepatocellular
carcinomas 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, significant 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 pre-
liminary 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 CARCINOGENICITY BIOASSAYS IN ANIMALS
TCI chemical
Study purity
NTP, 1982 Purified
NCI, 1976 Technical grade
Bell et al., Technical grade
(MCA) 1978
Maltoni, 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, Han:Wist
Males
Females
Hamsters, Syrian
Males
Females
Dose levels,
route
1000 mg/kg/day
gavage, 103 wk
500, 1000 mg/kg/day
gavage, 103 wk
1119, 2339 mg/kg/day
869, 1739 mg/kg/day
gavage, 78 wk
549, 1097 mg/kg
gavage, 88 wk
100, 300, 600 ppm
inhalation, 24 mo
100, 300, 600 ppm
inhalation, 24 mo
250, 50 mg/kg
gavage, 52 wk
100, 500 ppm
inhalation, 78 wk
100, 500 ppm
inhalation, 78 wk
100, 500 ppm
inhalation, 78 wk
Results
Treatment -related
hepatocellular carcinomas
in males and females
Renal adenocarcinomas
in treated males
Treatment-related
hepatocellular carcinomas
in males and females
Negative
Increased incidence of
hepatocellular carcinomas
in males and females
with dose
Negative
Negative
Increased incidence of
malignant lymphomas
in females
Negative
Negative
[continued on the following page)
8-2
-------
TABLE 8-1. (continued)
Study
TCI chemical
purity
Species
Dose levels,
route
Results
Van Duuren et al.,
1979, 1983
NTP, 1982
Maltoni, 1979,
1985
Henschler et al.,
1984
Purified
Purified TCI
epoxide
Purified
Purified
Purified and
stabilized
Swiss mice
ICR/Ha
Female
Female
Female
Female
Male
Female
Female
Female
Rats, Osborne-
Mendel
Marshall 540,
August 28807,
AC I
Mice, B6C3F1
Swiss albino
Rats, Sprague-
Dawley
Swiss mice
ICR/Ha
1 mg, 3x/wk, 581 d
topical
1 mg, 3x/wk, 14 d
2.5 ug phorbol
myristate acetate,
topical 452 d
0.5 mg sc/wk, 622 d
0.5 mg, once wk
gavage, 622 d
1 mg TCI epoxide,
3x/wk, 2.5 pg
phorbol myristate
acetate, topical,
452 d
2.5 mg TCI epoxide
3x/wk, topical for
526 d
0.5 mg TCI epoxide,
once wk, sc, 547 d
Gavage, 104 wk
Inhalation, 78 wk
Inhalation, 104 wk
Gavage, 78 wk
Negative
Negative
Negative
Negative
Negative
Negative
Negative
In preparation
In press
Negative
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°C to 24°C and 40% to 60%, respectively; room air was exchanged
10 to 15 times 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 < 0.05);
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
-------
500
0
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MALE RATS
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10
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500-
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Till
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TIME ON STUDY (WEEKS)
80
90
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-»—
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TIME ON STUDY (WEEKS)
8-6
-------
PROBABILITY OF SURVIVAL
PROBABILITY OF SURVIVAL
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TABLE 8-2. ANALYSIS OF PRIMARY TUMORS IN MALE RATS3
KIDNEY: TUBULAR-CELL ADENOCARCINOMA
Tumor rates
vehicle-
control
Low
dose
High
dose
Overall13
Adjusted0
Terminal
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
Terminal
Stati sti ca I 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
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.
^The configuration of tumor incidences precludes the use of this statistic.
SOURCE: NTP, 1982.
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 adenocarcinomas
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 adenocarcinomas 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 di-
lated, 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 de-
creases 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 carcinogenicity bioassays is the highest
dose that can be administered during the chronic study which will not be expec-
ted to alter the animals' survival rate from effects other than carcinogenicity,
Thus, TCI doses beyond that maximally tolerated, particularly in male rats, ap-
pear to have been used in this study. The toxicity of TCI in the kidney tubule
8-10
-------
region, as cytomegaly, 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 adeno-
carcinomas in high-dose males compared to vehicle-control males 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 F344/N
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 could 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, >L 99.0% (w/w);
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-
8-11
-------
ment 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 carcinogenicity study
were estimated from survival, body weight, and necropsy results from a preli-
minary 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 con-
sumption, it would appear that decreases in food consumption and body weights
ultimately were a result of toxicity from treatment with TCI. For example,
food consumption 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
Dosage
group
Low-dose
males and
females
High-dose
males and
females
Dose (mg
TCI/kg
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
period
(weeks)
7C
9c
14C
48d
32
7C
9c
14c
48d
32
Time-weighted
av. dose& (mg
TCI/kg body wt)
549
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 = I (dose in mg/kg x no. of days at that dose)/
I (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.
dDosing 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.)
SOURCE: NTP, 1982.
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
-------
TABLE 8-4. MEAN BODY WEIGHTS, FOOD CONSUMPTION, AND SURVIVAL - TRICHLOROETHYLENE
CHRONIC STUDY - MALE RATS
Vehicle-control
Time
interval
(weeks)
0
00
£ 14
26
54
78
110
Body
Mean
(g)
193
504
570
616
559
382
weight3
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
weighed
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
Calculated using individual animal weight.
bAverage weight per animal per week.
SOURCE: NCI, 1976.
-------
TABLE 8-5. MEAN BODY WEIGHTS, FOOD CONSUMPTION, AND SURVIVAL - TRICHLOROETHYLENE
CHRONIC STUDY - FEMALE RATS
Vehicle-control
00
1
1— >
in
Time
interval
(weeks)
0
14
26
54
78
110
Body
Mean
(g)
146
302
351
388
373
326
weight3
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
weighed
20
20
19
17
16
8
Body
Mean
(g)
144
271
293
315
317
311
Low
weight
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
animals
weighed
50
44
34
28
20
13
Body
Mean
(g)
144
262
286
307
317
311
High
wei ght
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.
bAverage weight per animal per week.
SOURCE: NCI, 1976.
-------
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)—Maltoni (1979) summarized the results of a study done
to estimate the carcinogenic potential of TCI in Sprague-Dawley rats. Thirty
males and 30 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-dichloro-
ethylene, chloroform, carbon tetrachloride, and 1,1,2-trichloroethane. No de-
tectable epoxides were found in this test material, and 10 ppm diisopropylamine
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, al-
though 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 poten-
tial 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 mg/kg/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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01
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TIME ON STUDY (WEEKS)
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10 20 30 40 50 60 70
TIME ON STUDY (WEEKS)
80 90 100 110
Figure 8-5. Growth curves for mice administered
trichloroethylene in corn oil by gavage.
SOURCE: NTP, 1982. 8-21
-------
PROBABILITY OF SURVIVAL
PROBABILITY OF SURVIVAL
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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 _£ 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 inci-
dence 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 treat-
ed 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 mg/kg/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
LIVER: ADENOMA
Tumor rates
Overallb
Adjustedc
Termi nal^
Statistical tests6
Life table
Incidental tumor test
Cochran-Armitage Trend,
Fisher Exact Tests
Vehicle-
control
3/48 (6%)
9.1%
3/33 (9%)
Dosed
8/50 (16%)
27 .5%
1/16 (6%)
p = 0.022
p = 0.191
p = 0.113
LIVER: CARCINOMA
Tumor rates
Overall0
Adjustedc
Terminald
Statistical tests6
Vehicle-
control
8/48 (17%)
22.1%
6/33 (18%)
Dosed
30/50 (60%)
92.5%
14/16 (87.5%)
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
Overall
Adjusted0
Terminal^
Statistical tests6
Vehicle-
control
11/48 (23%)
30.7%
9/33 (27%)
Dosed
38/50 (76%)
97.1%
15/16 (94%)
Life table
Incidental tumor test
Cochran-Armitage Trend,
Fisher "xact 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. The incidental tumor test regards these
lesions as non-fatal. The Fisher Exact Test compares the overall incidence
rates directly.
SOURCE: NTP, 1982.
8-24
-------
TABLE 8-7. ANALYSIS Oc PRIMARY TUMORS IN FEMALE MICE3
LIVER: ADENOMA
Tumor rates
Vehicle-
control
Dosed
Overall*3
Adjusted0
Terminal
2/48 (4%)
6.2%
2/32 (6%)
Statistical tests6
Life table
Incidental tumor test
Cochran-Armitage Trend,
Fisher Exact Tests
8/49 (16%)
30.2%
6/23 (26%)
p = 0.016
p * 0.023
p = 0.049
LIVER: CARCINOMA
Tumor rates
Overall6
Adjusted0
Terminal^
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
Overallb
Adjusted0.
Terminal^
Statistical tests6
Vehicle-
control
4/48 (8%)
12.5%
4/32 (13%)
Dosed
19/49 (39%)
63.8%
13/23 (57%)
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.
°Kaplan-Meier estimated lifetime tumor incidence after adjusting for intercurrent
mortality,
''Observed tumor incidence at terminal kill.
6Beneath 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 Fisher Exact Test compares the overall incidence
rates directly.
SOURCE: NTP, 1982.
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 hepatocellular 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 hepatocellular 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
8-26
-------
8-week subchronic test. In the carcinogenicity study, time-weighted average doses
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
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 oil
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
5
11
17
83
Treatment
period
(weeks)
6C
6C
66C
12
6C
6C
66C
12
12C
66C
12
6^
6C
66C
12
Time-weighted
av. dose" (mg
TCI/ kg body wt)
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.
bTime-weighted average dose = I (dose in mg/kg x no. of days at that dose)/
I (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.
SOURCE: NCI, 1976.
8-27
-------
Mice were permitted to survive until terminal sacrifice at 12 weeks fol-
lowing 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 slight-
ly higher consumption in treated males as compared to matched controls. There-
fore, 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
-------
TABLE 8-9. MEAN BODY WEIGHTS, FOOD CONSUMPTION, AND SURVIVAL - TRICHLOROETHYLENE
CHRONIC STUDY - MALE MICE
Vehicle-controls
Time
interval
(weeks)
!° o
o
D
14
26
54
78
90
Body
Mean
(g)
17
28
31
32
34
34
weight3
Std. dev.
(g)
0.5
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
wei ght
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
animals
wei ghed
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.
SOURCE: NCI, 1976.
-------
TABLE 8-10. MEAN BODY WEIGHTS, FOOD CONSUMPTION, AND SURVIVAL - TRICHLOROETHYLENE
CHRONIC STUDY - FEMALE MICE
Vehicle-controls
00
1
CO
0
Time
interval
(weeks)
0
14
26
54
78
90
Body
Mean
(g)
14
23
25
28
29
28
weight3
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
(9)
14
23
26
27
29
30
Low dose
weight
Std. dev.
(9)
0.6
0.3
0.7
0.5
0.7
0.6
Food
(g)
0
23
23
21
25
27
No. of Body
animals Mean
weighed (g)
50 14
49 24
49 25
45 26
41 28
40 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.
SOURCE: NCI, 1976.
-------
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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) diffe-
rences 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 adenocarcinoma, or sheets of highly ana-
plastic 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
CO
Dose 0 mg/kg
Number with
tumors/number
of animals 1/20
Incidence 0.05
p-value
Number with
metastases 0/20
Males
1169 mg/kg
26/50
0.52
a
1.39 x 10-4
4/50
2339 mg/kg 0 mg/kg
31/48 0/20
0.646 0
a
3.44 x 10~6
3/48 0/20
Females
869 mg/kg 1739 mg/kg
4/50 11/47
0.08 0.234
a
1.75 x 10-1 1.35 x 10-2
0/50 0/47
Statistically significant (p < 0.05) increase compared to matched vehicle-controls (0 mg/kg) by Fisher Exact
Test.
SOURCE: NCI, 1976.
-------
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 compounds were responsible for the observed results is considered low.
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
*l,l,2,2-tetrachloroethane, 3-chloropropene, chloropicrin, 1,1-dichloroethane,
chloroform, sulfolene, iodoform, ethylene dichloride, methyl chloroform, 1,1,2-
trichloroethane, tetrachloroethylene, hexachloroethane, carbon disulfide,
trichlorofluoromethane, 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 applica-
tion, and initiation-promotion experiments with TCI and an initiation-promotion
experiment 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 experi-
ments were below those which could have been tolerated by the animals. TCI
doses used in the skin treatment experiments were selected to permit a compa-
rison with TCI oxide, with respect to carcinogen!city, 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 yg phorbol myristate acetate
(PMA) to the skin three times weekly until termination of the study at 452 days.
In addition, a different group of 30 females was similarly treated with an ap-
plication of 1.0 mg TCI epoxide, a metabolite of TCI, followed by applications
of 2.5 ng PMA; 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; 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 immedi-
ately 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
8-36
-------
routinely done on skin, liver, stomach, and kidney in the skin application
experiments, injection site tissue and liver in 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 un-
treated controls, respectively. Lung tumors were diagnosed as benign papillo-
mas, 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 applica-
tions 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 higher TCI doses could have more strongly challenged the mice for
carcinogenicity in the skin treatment experiments, but Van Duuren et al. (1983)
noted that low activity or lack of activity of chloroolefins tested for carci-
nogenicity in skin treatment experiments in their laboratory was expected be-
cause of evidence for the relatively low level of epoxidizing enzymes in mouse
8-37
-------
TABLE 8-12. MOUSE SKIN BIOASSAYS OF TRICHLOROETHYLENE AND TRICHLOROETHYLENE OXIDE
(30 FEMALE HA:ICR SWISS MICE PER GROUP)
Initiation-promotion3
Repeated application'3
Compound
Dose, mg/ Days to Mice with Dose, mg/ Days to Mice with No. of mice
application/ first papillomas/ application/ first papillomas/ with
mouse tumor total papillomas mouse tumor total papillomas distant tumors^
Trichloroethylene 1.0
Trichloroethylene
oxide 1.0
7,12-Dimethyl-
264 4/9 (l)c
371
3/3
1.0
NT
9 lung
1 stomach
benz[a]anthracenee 0.02
00
1
co
00 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.
dLung tumors were benign papillomas, and stomach tumors were papillomas of the forestomach.
ePositive control agent.
fStatistically significant by chi-square test compared to controls.
SOURCE: Van Duuren et al., 1979.
-------
TABLE 8-13. SUBCUTANEOUS INJECTION OF TRICHLOROETHYLENE
(30 FEMALE HA:ICR SWISS MICE PER GROUP)
Compound and
dose3
Trichloroethylene, 0.5
B-propiolactone,d 0.3
•J0 Trioctanoin, 0.05 mL
CO
No treatment (100 mice)
Days on
test
622
378
631
649
No. of mice
with local
sarcomas'3
0
24
0
0
P value0
NS
<0.0005
_
-
aCompound subcutaneously injected in left flank once weekly in 0.05 ml trioctanoin. Dose is mg per
injection per mouse.
bHistopathologically characterized as fibrosarcomas.
CNS = Not significant. Statistical significance calculated by chi-square test compared to controls.
dPositive control agent.
SOURCE: Van Duuren et al., 1979.
-------
TABLE 8-14. CARCINOGENICITY OF TRICHLOROETHYLENE BY INTRAGASTRIC FEEDING IN
MALE AND FEMALE HA:ICR SWISS MICE (30 MICE PER GROUP)
oo
I
Compound and
dosea
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
tumors'5
1
2
24(23)
25(23)
0
0
5
8(1)
P value0
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.
SOURCE: Van Duuren et al., 1979.
-------
skin relative to other organs such as liver. 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, both from
the mice dosed orally as well as from the mice dosed by subcutaneous injection,
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. (1980) 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/L air), or 500 (2.8 mg/L 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 inhala-
tion chambers were monitored with direct ultraviolet spectrophotometry, and the
flow rate of the TCI air mixture was adjusted to yield a threefold turnover of
8-41
-------
the total volume each hour. A description of the analytical method was provi-
ded by D. Henschler, University of Wurzburg, as follows: Exposure levels were
automatically controlled to 100 or 500 ppm, as appropriate, and were continu-
ously 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 equip-
ment, and the registered band indicated +_ 7% of the median at 100 ppm.
No effect of TCI on body weight gain was observed. A statistically signifi-
cant (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.
8.1.3.2 Bell et al. (1978; audit of Industrial Bio-Test Laboratories, Inc.
study)--A carcinogenicity study of TCI vapor in Charles River rats was con-
ducted at Industrial Bio-Test Laboratories, Inc. (IBT) from 1975 to 1977, and
audit findings by the Manufacturing Chemists Association (MCA) have been repor-
ted (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/L 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 hema-
tology and clinical chemistry were not given pathologic examinations.
8-42
-------
TABLE 8-15. ESTIMATION OF PROBABILITY OF SURVIVAL OF RATS (%)a
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.
SOURCE: Henschler et al., 1980.
According to the audit report by the MCA, there were noticeable deficien-
cies 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 measure-
ments 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
8-43
-------
00
I
I I I I
0 20 40 60 80 100 120 140 160 180200 220240 260280 300320 340360 380400 420440 460 480 500520 540560 580600 620640 660680 700
PPM MAX. VALUE
2040 ppm
Figure 8-9. Trichloroethylene histogram of chamber concentration measurements
T-l (100 ppm).
SOURCE: Bell et al., 1978.
-------
oo
I
en
0 20 40 60 80 100 120140 160 180 200220240 260280 300320 340360 380 400420440 460480 500520 540560 580600 620 640660 680700 +
PPM MAX. VALUE
1676 opm
Figure 8-10. Trichloroethylene histogram of chamber concentration measurements
T-l (300 ppra).
SOURCE: Bell et al., 1978.
-------
917-8
FREQUENCY
GO
O
O
CO
rt>
t-t-
00
c
(T)
to
r+
O
O)
O
3-
CU
3
CT
ro
-s
O
O
3
O
(D
CU
fD
CU
fD
-------
study, and additional information from the Chemical Manufacturers Association
has been requested regarding exposure levels obtained during the final 18
months of this study.
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 carci-
nogenicity study on TCI in Syrian hamsters. The TCI sample and the experimen-
tal conditions were those used in the carcinogenicity study of TCI in Han:Wist
rats by Henschler et al. (1980) discussed herein. Thirty male and 30 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 18 months. The initial age of the animals was not given. Terminal sacri-
fice was at 30 months. All animals were subjected to necropsy and histopatho-
logic 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 similar-
ity 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.
8-47
-------
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
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 18
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 (%)a
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.
SOURCE: Henschler et al., 1980.
8-48
-------
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
significant (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.
TABLE 8-17. ESTIMATION OF PROBABILITY OF SURVIVAL OF MICE (%)a
Group
Control
Week
1
50
75
100
115
126
156
M
100.0
93.3
83.3
66.7
33.3
23.3
-
F
100.0
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
82.8
37.9
10.3
3.4
-
aBy the Kaplan and Meier (195.8) method.
SOURCE: Henschler et al.t 1980.
8-49
-------
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
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 immunosuppressive 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
8-50
-------
20
15
10'
LL)
03
D CONTROLS
A 100ppm
O 500 ppm
O
Y
\ i
s
s
t
36 40 50 60 70 80 90 100 110 120 130
WEEKS
Figure 8-12. Incidence of malignant lymphomas in female mice.
SOURCE: Henschler et al., 1980.
8-51
-------
that of Henschler et al. (1980) in which a coexistence of persistent antigenic
stimulation and chronic immunosuppression is pathogenetically related to malig-
nancy 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 experi-
ments 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 carcinogenicity 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 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 un-
treated control group, consisted of 140 mice of each sex. Except for 40 mice/
sex/group assigned to interim sacrifice for evaluation of clinical chemistry
and hematology, a pathologic examination was scheduled for each animal. Sur-
viving animals were sacrificed 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 carcinogenicity 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
8-52
-------
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 reexami nation 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
pathology was done by the Fisher Exact Test and the chi-square analysis (Table
8-18). Significant (p < 0.05) increases in the incidences of hepatocellular
adenoma, hepatocellular carcinoma, and hepatocellular adenoma/hepatocellular
carcinoma combined in treated males compared to untreated males and the inci-
dence of hepatocellular adenoma/hepatocellular carcinoma combined in treated
females compared to untreated females were calculated as shown in Table 8-18.
With respect to the treatment groups which were on study concurrently, statis-
tically significant (p < 0.05) increases in both the number of hepatocellular
carcinomas alone and hepatocellular adenomas/hepatocellular 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 inci-
dence 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 hepa-
tocellular 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. A recently completed carcinogenicity study by Dr. C. Maltoni and
8-53
-------
TABLE 8-18.
STATISTICAL ANALYSIS OF THE INCIDENCE OF PRIMARY HEPATOCELLULAR NEOPLASMS
IN CONTROL AND TRICHLOROETHYLENE TREATED B6C3F1 MICE
CO
I
Hepatocellular adenoma
Incidence
Percent
Chi square
Fisher Exact Test
Hepatocellular carcinoma
Incidence
Percent
Chi square
Fisher Exact Test
Hepatocellular adenoma/
hepatocellular carcinoma
combi ned
Incidence
Percent
Chi square
Fisher Exact Test
Hepatocellular adenoma
Incidence
Percent
Chi square
Fisher Exact Test
Hepatocellular carcinoma
Incidence
Percent
Chi square
Fisher Exact Test
Hepatocellular adenoma/
hepatocellular carcinoma
combined
Incidence
Percent
Chi square
Fisher Exact Test
Untreated
controls
2/99
2.02%
18/99
18.18%
20/99
20.20%
Untreated
controls
2/99
2.02%
6/99
6.06%
8/99
8.08%
T-ia
7/95
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%
0.572
0.2265
4/100
4.00%
0.116
0.8382
9/100
9.00%
0.000
0.5083
Males
T-2b
7/100
7.00%
1.820
0.0873
31/100
31.00%
3.741
0.0262*
38/100
38.00%
6.794*
0.004407*
Females
T-2
1/94
1.06%
0.002
0.8672
9/94
9.57%
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
10/97
10.31%
4.504* 0.032 0.214 0.329
0.0150* 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* 0.0138*
T-3 T-l vs. T-2 T-l vs. T-3 T-2 vs. T-3
4/99
4.04%
0.172 1.364 0.000 0.719
0.3413 0.9824 0.7461 0.2007
13/99
13.13%
2.096 1.599 4.205* 0.303
0.0730 0.1026 0.0188* 0.2916
17/99
17.17%
2.930 0.020 2.250 1.211
0.0427* 0.4427 0.0662 0.1353
*Significant at 95% level.
a T-l: 100 ppm
b T-2: 300 ppm
c T-3: 600
SOURCE: Bell et a!., 1978.
-------
associates can provide further evaluation of the carcinogenic potential of TCI
vapor in B6C3F1 mice.
8.1.6 Other Carcinogenicity Evaluations of Trichloroethylene--Although 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 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
8-55
-------
TABLE 8-19. SUMMARY OF NEGATIVE CARCINOGENIC ITY DATA FOR TRICHLOROETHYLENE
Species
Dogs
Rats
Guinea pigs
Monkeys
Rabbits
Cats
00
en
en Mice
Rats
Guinea pigs
Monkeys
Rabbits
Dogs
Rats
Number
16
12
11
2
4
8
28
15
15
3
3
2
37 newborn and 2
adults per group
Exposure3
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/wk, unspecified treatment
period
Inhalation
3,825 mg/m3, 8 hr/day, 5 days/wk,
6 months
189 mg/m3 continuous for 90 days
Inhalation
2,000 ppm, 8 hr/day, 5 days/wk, _
10 weeks followed by 2 weeks
without treatment
Results Reference
No tumors; no deaths Seifter, 1944
Three rats died; Adams et al.,
no tumors 1951
No tumors; no deaths Monsinger and
Fiorentini, 1955
No tumors; no deaths Rudali, 1967
No tumors; no deaths 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).
SOURCE: Waters et al., 1977.
-------
repeated skin application and subcutaneous injection in chronic studies with
female ICR/Ha mice. The epoxides were cjj^-1-chl oropropene oxide (cis-CPO);
trans-1-chloropropene oxide (trans-CPO); cis-1,3-dichloropropene oxide (cis-
DCPO); trans-l,3-dichloropropene oxide (trans-DCPO), trichloroethylene oxide
(TCIO); and tetrachloroethylene oxide (PCEO). The epoxides were prepared by
a modification of the method of Kline et al. (1978) to obtain pure samples.
However, cj_s_-DCPO and trans-DCPO contained 10 to 15% rn-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, inclu-
ding histopathologic examination of treatment sites and liver, in preliminary
toxicity tests of 6 to 8 weeks duration.
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 ace-
tone 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 cha-
racterized 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 histopatho-
logically examined. Routine sections examined microscopically included skin,
8-57
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TABLE 8-20. CHRONIC SKIN APPLICATION IN MICE
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
fi rst tumor
Median survival
time/test duration
in days
No. of mice with
papilloma/total
papillomas
No. of mice with
squamous cell
carcinoma
P value3
00
en
OO
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-Di chl orobenzene ,
3.5 mg
Dichloroacetyl chloride
250 yg
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
10C
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.
bThree were keratoacanthomas.
C0ne animal developed a fibrosarcoma and a squamous cell carcinoma.
dOne was a histiocytoma, and one was a keratoacanthoma.
eOne was a fibrosarcoma in the treatment area.
fpCEO was applied at 5 yL (7.5 mg) by Eppendorf pipet followed immediately by 0.1 mL acetone by micropipet.
90ne was a keratoacanthoma.
nTwo were fibrosarcomas in the treatment area.
SOURCE: Van Duuren et al., 1983.
-------
TABLE 8-21. CHRONIC SUBCUTANEOUS INJECTION IN MICE
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 0.05 ml tricaprylin
Compound
Median survival
time/test duration
in days
No. of animals
with local tumors
P value3
ci§_-CPO, 1 mg
trans-CPO. 1 mg
cjs_-DCPO, 500 yg
482/551
418/556
500/557
1 fibrosarcoma 0.052
1 carcinoma13
1 leukemia
4 fibrosarcomas 0.003
1 carcinoma
3 fibrosarcomas 0.003
1 anaplastic sarcoma
1 carcinoma and
mammary tumor
trans-DCPO, 500 yg
TCIO, 500 yg
PCEO, 500 yg
m-Dichlorobenzene,
165 yg
Dichloroacetyl chloride,
50 yg
dJ^-Diepoxybutane, 330
yg (positive control)
Tricaprylin, 0.05 ml
No treatment
(100 animals)
498/557
547/558
491/558
525/560
>560/560
475/553
>524/561
551/580
5 fibrosarcomas0
1 fibrosarcoma
1 anaplastic sarcoma
1 leukemia
0
1 fibrosarcoma
9 fibrosarcomase
2 carcinomas
1 mammary tumor
0
1 fibrosarcoma
0.003
NS^
NS
NS
<0.0005
aAll mice with local tumors included in calculation of p values: X^ 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.
SOURCE: Van Duuren et al., 1983.
8-59
-------
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 d^l-
diepoxybutane. The above-mentioned impurities jn-dichlorobenzene and dichloro-
acetylchloride 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 sig-
nificant (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) inci-
dences of malignant distant tumors were observed.
As discussed by Van Duuren et al. (1983), chloroepoxides have been con-
sidered as activated carcinogenic metabolites of chloroalkenes. The negative
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. (1979) 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 (cis 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 initi-
ating activity in an initiation-promotion study. As indicated by the authors,
the epoxides tested would be direct-acting agents which would not have to rely
8-60
-------
on metabolic activation at the sites of carcinogenic action in their study.
Under the conditions 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 triethyl amine 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, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 100 U penicil-
lin, and 100 yg streptomycin per ml. Liquid TCI was diluted 1:1000 in growth
medium before exposure of cells to desired TCI doses.
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 yM) of TCI were selected
for the transformation assay.
In the transformation assay, quadruplicate cultures of F1706 cells were
treated at 50% confluency with the two selected concentrations of TCI. Cells
8-61
-------
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 pM. 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 subdivision, 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 subcul-
tures after treatment. The ability of transformed cells to induce local fibro-
sarcomas 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 abil-
ity of transformed cells to grow in semi-solid agar and to induce local fibro-
sarcomas after subcutaneous injection into rats (Table 8-22). Transformation
of cells exposed to the positive control agent 3-methylcholanthrene was
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 acti-
vation system and by testing the ability of the F1706 cells to produce TCI
8-62
-------
TABLE 8-22. IN VITRO TRANSFORMATION OF FISCHER RAT EMBRYO CELL CULTURES BY TRICHLOROETHYLENE
00
Compound
Trichloroethylene
3-Methy 1 chol anth rene
Acetone
Medium
Dose
(uM)
1.1 x 10*
1.1 x 103
3.7 x 10'2
1:1,000
Morphological
transformation3
+5 (44)
+5 (44)
+4 (37)
(NA)
(NA)
Growth in
agar at P + 4&
9
8
124
0
0
Tumor incidence0
No. of animals with tumors/
No. of animals inoculated
13/13 (48)
12/12 (55)
12/12 (27)
0/13 (82)
NO
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).
bNumbers 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 C0£ 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.
SOURCE: Adapted from Price et al., 1978.
-------
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. On the other hand, the cell transfor-
mation test is simply an in vitro screening system, designed to provide at
best only suggestive evidence for carcinogenicity. Additionally, cells were
exposed to liquid TCI diluted in growth medium; therefore, since TCI is a
volatile substance, exposure of cells to TCI as a vapor could have indicated
how different exposure conditions possibly could have influenced its ability to
transform F1706 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 semisolid agar as an
endpoint, as part of a larger study (Purchase et al., 1978) done to screen che-
micals 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
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-
8-64
-------
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 (106) were incubated with test chemical and S-9 mix
at 37°C for 4 hours. To evaluate compound vapors, cells (2.5 x 10^) 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 treat-
ment 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 LCso
as a positive result. The spontaneous transformation frequency of BHK cells
(72 experiments) in this study was 50 +_ 16 per 106 survivors. Suitability of
the soft agar medium for colony growth was checked by assays with polyomatrans-
formed 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
8-65
-------
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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 transformation, expo-
sure of cells to TCI as a vapor could have provided a comparison of the trans-
formation potential of TCI as a vapor and TCI in liquid solution, as was done
for VC. Thus, the negative results reported in this study may, in fact, be due
to its incompleteness.
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 car-
cinogenic 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 limi-
ted intrinsic metabolic capability.
8.1.8.2 Trichloroethylene Oxide
8.1.8.2.1 DiPaolo and Doniger (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); cis-l,3-dich1oropropene oxide (c-DCPO); trans-1,3-dichloropropene
8-67
-------
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-chlo-
roperbenzoic acid. Each epoxide was provided pure, as determined by nuclear
magnetic resonance analysis, by Drs. Kline and Van Duuren of New York Univer-
sity.
Cells for culture were obtained from Syrian hamster embryos removed from
their mothers on the twelfth and thirteenth days of gestation. Primary cul-
tures 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 atmos-
phere 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
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.
8-68
-------
Results of the cell transformation assay with TCIO show a weak transfor-
mation effect, with high concentrations (>1 mM) capable of producing cytotox-
icity 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 cor-
related 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-DCPO
(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 transformation, 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:NMRI mice with inhalation treatment, and male
Fischer 344 rats with gavage treatment. Thus, there are at least four studies
which indicate that TCI-exposed rodents show elevated cancer incidence. The
NCI/NTP studies show an unequivocal increase in mouse liver tumors. The other
two studies just cited provide additional support to the overall body of evi-
dence, particularly since they were carried out in different animal species or
8-69
-------
TABLE 8-23. TRANSFORMATION AND SURVIVAL OF SYRIAN HAMSTER EMBRYO
CELLS TREATED WITH DIVERSE CHLOROALKENE OXIDESa
Compound Dose, mM
Acetone
c-CPO 0.11
0.27
0.55
1.1
t-CPO 0.22
0.55
1.1
2.2
c-DCPO 0.005
0.01
0.02
t-DCPO 0.01
0.025
0.05
TCEO 1.1
2.5
5.0
PCEO 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
0.26
0.83
0.26
2.2
2.2
1.1
0.26
0.26
1.1
-
0.83
0.56
1.4
aPlastic dishes (60 mm) were seeded with 300 cells from a secondary culture with
irradiated hamster cells (6 x 104) 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 sol vent-treated
cultures.
SOURCE: DiPaolo and Doniger, 1982.
8-70
-------
Compound
Structure
Half-Life (mm)
c/s-1 -Chloropropene oxide
-------
strains. 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 treatment; male and female
Charles River rats, male and female Han:Wist rats, male and female Syrian
hamsters, and male HanrNMRI 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 subcu-
taneous 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; however, when vinyl chloride was
tested under the same conditions as TCI, no transformation occurred with either
compound (see complete section 8.1 for details).
8.2 DESCRIPTION AND ANALYSIS OF EPIDEMIOLOGIC STUDIES
There are currently six epidemiologic studies which relate to the issue of
TCI exposure and cancer.
8.2.1 Axelson et al. (1978)
Axelson 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 inclu-
ded 3,643 of the 7,688 total person-years of observation in the study. Since
8-72
-------
the 1950s the TCI manufacturer had provided, free of charge, two urinary ana-
lyses 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/L) and high (ave-
rage reading of TCA in the urine of more than 100 mg/L). A level of 100 mg/L
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 expec-
ted 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 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
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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 19753
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
Exposure categorization is based on trichloroacetic acid (TCA) in the urine,
with low exposure referring to those not exceeding a concentration of 100 mg/L
TCA in urine on average.
SOURCE: Adapted from Axel son et al., 1978.
study cohort. Secondly, the definition of high and low exposure for the sub-
cohort 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 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
8-74
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less than 10 years of observation. Thus, less than 259 people had been fol-
lowed for as long as 10 years, which itself may not be a long enough observa-
tion period in which to observe an increase in cancer mortality, considering
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 carcino-
gen, 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;
however, there are severe limitations in interpreting the data. It should also
be noted that the primary author has reported in personal communications (Axel-
son 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 Tola et al. (1980)
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
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 mg TCA/L
urine. The list of names of persons whose urinary TCA levels had been measured
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was supplemented 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 Popula-
tion Data Register of the Central Statistical Office in Finland, and cancer
death information 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 yg/L urine, which, according to Axelson
et al. (1978), corresponds to an ambient exposure level of 30 ppm TCI. Further-
more, 78% of the workers had urinary TCA levels below 50 mg/L urine. An addi-
tional 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 com-
parison 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/L urine and one worker with clinical poisoning had
cancer; however, the difference of three cases from the expected number was not
statistically 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
8-76
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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. The actual durations of exposure for the
cohort were unknown.
TABLE 8-25. COMPARISON OF WORKERS EXPOSED TO TRICHLOROETHYLENE WITH THE TOTAL
FINNISH POPULATION FOR MORTALITY FROM ALL CAUSES AND FOR CANCER MORTALITY
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
SOURCE: Adapted from Tola et al., 1980.
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8.2.3 Malek et al. (1979)
Malek et al. (1979) studied the incidence of cancer among a group of 57
dry-cleaners in Prague, Czechoslovakia, who used TCI as a cleaning solvent.
Exposure to TCI was verified by analysis of the urine for trichloroacetic acid.
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 be 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 Novotna et al. (1979)
Novotna et al. (1979) identified 63 histologically confirmed cases of
liver cancer in the city of Prague, Czechoslovakia, for the years 1972 and 1974,
and reviewed their employment histories for evidence of exposure to TCI. The
study used no controls. On the basis of employment information dating back to
January 1, 1957, 56 of the cases could be identified as never having been occu-
pationally exposed to TCI. For seven cases there was no occuptional history.
The fact that none of the liver cancer cases had any occupational exposure
to TCI is not particularly surprising considering that in the City of Prague,
which has a population of approximately one million, there were, at the time
of the study, only 544 workers exposed to TCI at 63 work places. The authors
indicated, however, that at one time there had been 1,000 work places where
there was TCI exposure, but they did not indicate when this was.
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8.2.5 Harden et al. (1981)
Hardell et al. (1981) studied the chemical exposure histories of 169 men
who had malignant lymphomas (Hodgkins and non-Hodgkins disease) and 338 con-
trols matched by age, sex, and place of residence. The men were aged 25 to 85,
and were identified from the records of the Department of Oncology, University
Hospital, Umea, Sweden for the period 1974-1978. Controls werre extracted from
the National Population Register. The study was specifically undertaken to
examine the association between malignant lymphoma and exposure to phenoxy
acids or chlorophenols, but exposure to other chemicals, including TCI, was
also evaluated. Exposures were ascertained by means of a self-administered
questionnaire which included questions on employment, leisure time activities,
exposures to various chemicals, and drug and smoking habits.
For the analysis of solvent exposures, including TCI, the authors elimi-
nated the matching. Seven cases and three controls were reported as having had
"high-grade" exposure to TCI (exposed continuously for one week or more or ex-
posed repeatedly to brief exposures for at least one month). When compared
to 60 cases and 222 controls with no exposure to solvents, chlorophenols, or
phenoxy acids, the unadjusted odds ratio, calculated by the Carcinogen Assess-
ment Group, of being a case and having an exposure to TCI is 7.88 (95% CL,
2.30, 27.04). Without adjusting for age, this result is somewhat question-
able, however. Also, the ascertainment of chemical exposure by self-admini-
stered questionnaire is of doubtful value because of the varying recall abil-
ities of individuals. Also, it is possible that the liver cancer cases may
have been exposed to TCI prior to January 1, 1957, the earliest date for which
the authors had employment data. Fifteen to 17 years is not particularly long
when considering the latency period for a carcinogen. Thus, the limitations
of this study preclude its usefulness in evaluating the carcinogenicity of TCI,
8-79
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although further investigation of the results is suggested.
8.2.6 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.
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
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that the list of 95 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 not a good basis for a statistical comparison.
Lastly, for those workers with TCI exposure, it is not known how long they had
been exposed, or how recent their exposures were.
8.3 RISK ESTIMATES FROM ANIMAL DATA
The evidence for the carcinogenicity of TCI reviewed in this document con-
sists primarily of positive mouse studies (NCI, 1976; NTP, 1982; Henschler et
al., 1980) and one marginally positive rat study (NTP, 1982). These positive
animal studies reported an incidence of excess tumors of several types. Addi-
tional supporting evidence for the carcinogenicity of TCI includes the fact
that TCI has been reported to be weakly mutagenic in numerous test systems and
to have a low order of DNA binding. A metabolic intermediate, trichloroethy-
lene oxide, has been reported as positive in cell transformation studies; there
are no known differences among species with regard to metabolic pathways or
metabolic profiles. The NCI and NTP studies, which show an unequivocal increase
in liver tumors in B6C3F1 mice, serve as the basis for cancer risk quantifica-
tion in this document.
The evidence for the carcinogenicity of TCI has an uncertain classifica-
tion depending upon the differing scientific interpretations of the relevance
of mouse liver tumors (hepatocellular carcinomas) to human cancer risk. How-
ever, the more conservative public health view would regard TCI as a probable
human carcinogen. It is important to note that the quantitative estimations of
the impact of TCI as a carcinogen are made independently of the overall weight
of evidence for the carcinogenicity of TCI. The calculations are made only
under the presumption that TCI is a human carcinogen.
8-81
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8.3.1 Selection of Animal Data Sets
For some chemicals, several studies in different animal species, strains,
and sexes, each run at several doses and different routes of exposure, may be
available. A choice can be made, when appropriate, as to which of these data
sets to use in the mathematical extrapolation model. It may also be appropri-
ate to correct for differences in metabolism between species and for different
routes of administration.
For TCI, the data from the NTP (1982) and NCI (1976) oral gavage lifetime
studies in male and female mice, showing a significant dose-related excess of
tumors (hepatocellular carcinomas), have been used. Tumors were found at both
the "high" dose (approximately the maximum tolerable dose) and the "low" dose
(one-half the maximum tolerable dose). Table 8-26 gives the time-weighted
average dosage and tumor incidence in male and female mice for the NCI and NTP
studies. TCI in corn oil vehicle was given once a day by gavage (vehicle only
was given to controls); in the NTP study, 0.5 ml of oil was used to dose B6C3F1
mice by gastric intubation 5 days/week, starting at 8 weeks of age, continuing
for 103 weeks, and sacrificing at 112 and 115 weeks. In the NCI study, about
0.3 mL of corn oil was used, varying with body weight, to dose B6C3F1 mice by
gavage 5 days/week, starting at 5 weeks of age, continuing for 78 weeks, and
sacrificing after 90 weeks (see Section 8.2.1 for details of these studies).
The time-weighted average gavage doses for both studies are given in Table 8-26.
A dose-response relationship for both sexes is apparent for each of the
two doses used in the NCI study. It should be noted that the chronic daily
gavage doses used in the NTP and NCI studies are in the range reported for
acute oral toxicity of TCI, LDjo to 1059, in other strains of mice (see Chap-
ter 5). However, animal survival in the NCI lifetime study (1.5 year) was 44%
for high-dosed males, 72% for low-dosed males, and 84% to 89% for females.
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TABLE 8-26. INCIDENCE OF HEPATOCELLULAR CARCINOMAS IN MALE AND FEMALE MICE (B6C3F1) IN THE NTP (1982)
AND NCI (1976) GAVA6E STUDIES
oo
00
CO
Average Time-weighted
terminal average gavage dose3
Study
NTP
NCI
aSum (dose
Sex weight (g) mg/kg/day
M 40 0
1,000
F 35 0
1,000
M 33 0
1,169
2,339
F 26 0
869
1,739
in mg/kg x no. of days at that dose)
mg/animal/day
0
40
35
0
38.6
77.2
22.6
45.2
Incidence
8/48
30/50
2/48
13/49
1/20
26/50
31/48
0/20
4/50
11/47
(17%)
(60%)
(4%)
(27%)
(5%)
(52%)
(65%)
(0%)
(8%)
(23%)
Sum (no. of days receiving any dose)
-------
Similarly, in the 2-year NTP study, 32% of the males and 46% of the females
survived chronic administration of the single daily dose given, an amount which
approximated the low dose of the NCI study. Presumably, TCI is subjected to
elimination processes during the 24-hour interval between doses and on week-
ends, and hence the chronic LD may be much higher than the acute LD, parti-
cularly when the half-life is very short. Prout et al. (1984) estimated the
T 1/2 of TCI in B6C3F1 mice as only 1.75 hour, while Green and Prout (1984)
demonstrated that chronic daily oral dosing of TCI (1,000 mg/kg/day) in mice
(180 days) produces a consistent daily amount of urinary metabolites, indica-
ting that accumulation of TCI with chronic daily dosing does not occur. Prout
et al. (1984) and Green and Prout (1984) studied the metabolism and kinetics of
TCI in B6C3F1 mice given TCI by gavage in corn oil at doses of 10 to 2,000
mg/kg, i.e., including the conditions of the NTP and NCI bioassays.
8.3.2 Interspecies Dose Conversion
8.3.2.1 General Considerations—Carcinogenesis is a complex process, not en-
tirely understood, and no observational evidence exists to validate satisfac-
torily the extrapolated predictions of carcinogenesis from animal models to
humans. Thus, there is no scientific basis per se for choosing one extrapola-
tion method over another in extrapolation of the carcinogenic response. How-
ever, in extrapolating the dose-carcinogenic response relationships of labora-
tory animals to humans, the doses used in the bioassays must be adjusted in
some way to allow for such differences as size and metabolic rates. Therefore,
other biological information, particularly interspecies data, is examined for
TCI, since physiologic, biochemical, and toxicologic responses other than
cancer may provide information as to what factors might be considered and what
method is generally appropriate for the extrapolation from laboratory animals
8-84
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to humans for this chemical.
The major components requiring consideration in determining an appropriate
extrapolation base for scaling carcinogenicity data from laboratory animals to
humans are: 1) toxicologic data, 2) metabolism and kinetics, and 3) covalent
binding. The biological basis for extrapolation of dose-carcinogenic response
relationships has been outlined and discussed previously by Davidson (1984) and
Parker and Davidson (1984).
8.3.2.1.1 Toxicologic Data. The acute lethal concentrations or LDsgs for
inhalation and oral exposures to TCI are remarkably similar across species
(mice, rats, dogs, and humans), presumably due to an equally effective con-
centration leading to central nervous system depression, resulting in death
(Smith, 1966). Klassen and Plaa (1966, 1967) and Tucker et al . (1982) have
investigated other toxicologic end points (liver and kidney enzyme activity
changes and other modalities of liver and kidney damage), but these observa-
tions have not been extended across more than one to two species (see Chapter
5). Hepatotoxicity of TCI has been shown to be directly related to the extent
of the metabolism of the parent compound (Buben and 0' Flaherty, 1985).
8.3.2.1.2 Metabolism and Kinetics. The carcinogenic potential of TCI is
generally considered to reside in cellular "reactive" intermediate metabolites
of TCI (e.g., TCI oxide, chloral, dichloroacetyl chloride, formyl chloride)
rather than in the intact molecule itself. These metabolites have been pos-
tulated to be responsible, for example, for the hepatotoxicity of TCI (as
expressed by cellular morphological and functional changes) and its cellular
genotoxicity (as expressed by DNA binding), although the primary mechanism
(genotoxic or nongenotoxic) for TCI's carcinogenic potential in mice has not
been satisfactorily established. The metabolism of TCI has been relatively
8-85
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extensively studied in three species: mice, rats, and humans. Limited phar-
macokinetic data are also available. (See Chapter 4 for a detailed review of
the metabolism and pharmacokinetics of TCI.) The pertinent metabolism and
pharmacokinetic information which might be applied to extrapolation of TCI
dose-carcinogenic response relationships from laboratory animals to humans
is discussed in the sections that follow.
In mice, rats, and humans, the principal end metabolites of TCI are common:
trichloroacetic acid (TCA), trichloroethanol (TCE) and TCE-glucuronide, and the
precursor metabolite, chloral. In addition, TCI oxide has been demonstrated to
be formed by both mouse and rat hepatic microsomes in vitro (Miller and Guenge-
rich, 1982). Minor metabolites, including carbon dioxide, dichloroacetic acid,
oxalic acid, and N-(hydroxyacetyl)-aminoethanol (HAEE), have also been identi-
fied for each of these species (see Chapter 4). Green and Prout (1984) and
Dekant et al. (1984) made comparative studies of the biotransformation of TCI
in rats and mice and failed to detect any major species or strain difference in
metabolism. Thus, there is persuasive experimental evidence that the metabolic
pathways for TCI are qualitatively similar in mice, rats and humans.
The relative magnitude of metabolism of TCI across species, as an index of
cellular damage from "reactive" metabolites, provides information for determi-
ning a "mouse to man" extrapolation scaling factor.
Four types of studies have been done to investigate dose-metabolism rela-
tionships: 1) single-dose studies across species, using 14C-TCI and urinary
radioactivity and/or other methods as measures of metabolism, 2) kinetic models
based on inhalation kinetics, 3) comparative balance studies between rat and
mouse with l^C-TCI at one or two dose levels, and 4) multiple dose level stu-
dies in rats and/or mice. Comparative balance studies, which determine the
metabolic fate and disposition of TCI (amount metabolized, amount excreted
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unchanged, etc.), have been carried out after oral dosing and after inhalation
exposures in both rats and mice.
8.3.2.1.2.1 Oral Studies. The balance studies in which TCI dosing was by
the oral route are the most pertinent to the conditions of the NTP and NCI bio-
assays. Prout et al. (1984) administered 14C-TCI in corn oil in the form of
single intragastric doses of 10, 500, 1,000, and 2,000 mg/kg to male Osborne-
Mendel and Wistar-derived rats, and to male B6C3F1 and Swiss mice. Exhaled
breath, urine, feces, and carcass were analyzed for 14C-TCI radioactivity
(and unchanged TCI) for up to 72 hours after dosing, and expressed as a per-
cent of dose administered. The results of these studies, as calculated and
re-expressed from the data of Prout et al., are given in Table 8-27. Regarding
these data, several general observations can be made comparing the metabolism
of TCI in the rat and mouse.
As indicated in Table 8-27, the recovery, from 91% to 98%, of 14C-radio-
activity in this experiment indicates that virtually complete gastrointestinal
absorption of TCI occurred for all doses in both rats and mice. Peak blood
levels occurred at about 1 hour in mice and at 3.5 hours in rats. Some 80% to
90% of the TCI was excreted, either in the form of metabolites or unchanged in
exhaled breath, within the first 24 hours.
There appears to have been no rat strain difference or mouse strain dif-
ference in the disposition of TCI.
For mice, oral doses of less than 1,000 mg/kg were virtually completely
metabolized. Significant amounts of unchanged TCI appeared in exhaled air at
doses of 1,000 mg/kg (18%) and 2,000 mg/kg (14%), indicating saturation of
metabolism beginning to occur at doses approaching 1,000 mg/kg. For rats,
oral doses of less than 500 mg/kg were virtually completely metabolized, with
8-87
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TABLE 8-27. DISPOSITION OF 14C-TCI 72 HOURS AFTER SINGLE ORAL DOSES TO MALE OSBORNE-MENDEL AND
WISTAR-DERIVED RATS AND TO MALE B6C3F1 AND SWISS MICE
Mice (mean of 4)
Dose
(mg/kg)
no
i
00
00 10
500
1000
2000
Dose/
animal3
(mg)
0.30
15.0
30.0
60.0
Metabolized
(mg equivalent) -
0.28
13.73
23.28
46.90
Exhaled
(mg equivalent)
0.01
0.90
5.25
8.16
Dose
(mg/kg)
10
500
1000
2000
Dose/
animal a
(mg)
2.0
100.0
200.0
400.0
Rats (mean
Metabolized
(mg equivalent)
1.93
55.40
79.00
80.40
of 4)
Exhaled
(mg equivalent)
0.03
42.70
112.40
311.20
aBased on experimental weight of animals: rats, average 200 g; mice, 30 g.
SOURCE: Calculated from the data of Prout et al., 1984.
-------
saturation occurring at about this dosage level. These observations of satura-
tion of metabolism are in accord with those of Filser and Bolt (1979) and of
Andersen et al. (1980) who also observed saturation of metabolism in the rat
during inhalation exposure.
For oral doses of 10 to 2,000 mg/kg (covering the range used in the NTP
and NCI mouse bioassays, Table 8-26), the ratio of the amount metabolized in
the rat versus the amount rfietabolized in the mouse was as follows:
Dose Ratio: Rat/mouse
(mg/kg) (mg metabolized/animal)
10 (1.93/0.28) = 6.89
500 (55.40/13.73) = 4.03
1000 (79.00/23.28) = 3.39
2000 (80.4/46.90) = 1.71
At the highest dose, the ratio of the amount metabolized in the rat ver-
sus the mouse is smaller than it is at lower doses, probably due to saturation
of metabolism in the rat. At the lowest dose, closer to 100% of the assimila-
ted dose is metabolized in both rats and mice. These data indicate that, for
the dose range used in the cancer bioassays, the metabolism and, hence, the
fractional TCI doses metabolized for rats and mice are more consistent with
a V|2/3 base (surface area) than a V|l*0 base (mg/kg).
Green and Prout (1984) orally dosed rats and mice subchronically for 180
days with TCI in corn oil at a level of 1,000 mg/kg/day. The relative propor-
tions of urinary metabolites (TCA and TCE-glucuronide) were unaffected by
strain differences. The amount of the daily dose metabolized stayed constant
(Table 8-28), although the ratio of TCA to TCE-glucuronide increased. These
observations indicate that 1) even at a high dosage level, TCI does not accu-
mulate significantly with daily dosing, i.e., the total body half-life is suf-
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TABLE 8-28. METABOLISM OF TCI IN B6C3F1 MICE:
EFFECT OF CHRONIC DOSING (1,000 mg/kg/daya)
Day
1
10
180
1
10
180
Metabolized
(mg equivalent)
Chronic
18.27
22.50
15.75
Single-dose controls
18.27
19.35
16.86
Expired unchanged
(mg equivalent)
5.28
4.08
7.11
5.28
4.62
3.75
aBased on experimental weight of mice averaging 30 g, the daily dose per mouse
equals 30 mg in 0.5 corn oil.
SOURCE: Recalculated from the data of Green and Prout, 1984, based on an
after-dosing collection period of 24 hours.
8-90
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ficiently short (1 to 2 hours) so that each daily dose is cleared (metabolized,
exhaled, etc.) within the 24-hour dosage interval; and 2) the increased ratio
o'f TCA to TCE-glucuronide suggests a possible adaptation of metabolic pathways,
which may occur simply because TCA has a longer half-time in the body than TCE.
Prout et al. (1984) estimated the T 1/2 for TCI in mice at 1.75 hours; there-
fore, 98% elimination occurs in 10.5 hours (T 1/2 x 6), and for some 14 hours
of each dose interval (24 hours), exposure to the highly reactive intermedi-
ates formed from TCI is practically nil. For the rat or human with longer
half-times of elimination, proportionally less time free from exposure to the
active chemical species is available (T 1/2 = 4 to 5 hours for humans).
Dekant et al. (1984), using balance studies, compared the metabolism of
TCI in female rats (Wistar) and female mice (NMRI). 14C-TCI was administered as
a single dose by gavage in corn oil at 200 mg/kg. Radioactivity was determined
in urine, feces, carcass, and exhaled air for 72 hours post-administration.
Total recovery of radioactivity was 93% to 98%, and a virtually complete oral
absorption is indicated by the results shown in Table 8-29. For this single
dose, the rats excreted 52% of the TCI dose through the lungs unchanged, while
the mice excreted 11% of the dose unchanged. The ratio of the amount of TCI
metabolized by the rat versus that metabolized by the mouse was as follows:
Dose Ratio: Rat/mouse
(mg/kg) (mg metabolized/animal)
200 (23.04/4.56) = 5.05
The comparative surface area ratio of the two species, as calculated from
their experimental body weights, is (240/24.4)°-67 = 4.46. At this dose,
the amounts of TCI metabolized by the rat and the mouse are closely propor-
tional to their relative surface areas.
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TABLE 8-29. DISPOSITION OF 14C-TCI RADIOACTIVITY FOR 72 HOURS
AFTER SINGLE ORAL DOSE (200 mg/kg) TO RATS AND MICE (NMRI)
Mice (av. of 3)
Absolute dose
5.1 mg/animal
mg equivalent
per animal
Rats (av. of 2)a
Absolute dose
48 mg/animal
mg equivalent
per animal
Expired TCI
Unchanged
Metabolized
0.56 (11.0%)
24.96 (52.0%)
COo
Urine
Feces
Carcass
Washes
Total
0.31
3.89
0.25
0.10
0.01
4.56 (89.4%)
5.12
0.91
19.78
0.86
1.39
0.10
23.04
48.0
(48.0%)
aBased on experimental weight of animals: female rats, 240 g; female mice,
25.5 g.
SOURCE: Recalculated from the data of Dekant et al., 1984.
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Buben and 0'Flaherty (1985) recently examined the extent of TCI metabo-
lism in male mice during subchronic administration by gavage in corn oil vehi-
cle given for 6 weeks (5 days/week; conditions similar to those of the TCI car-
cinogenicity bioassays). While the strain was Swiss-Cox and not B6C3F1, which
was used in the NTP and NCI bioassays, the results in this study are probably
relevant and generally applicable. A dose-amount metabolized curve was esta-
blished with doses of 0, 100, 200, 400, 800, 1,600, 2,400, and 3,200 mg/kg/day.
Metabolism was measured by urinary metabolites excreted per day (24-hour urine
collections). TCA (15% to 30%), TCE, and TCE-glucuronide (70% to 85%), as
determined by gas chromatography, were found to be the principal metabolites;
oxalic acid was not found to be a significant metabolite. Metabolites in feces
represented less than 5% of the amount in urine. No significant day-to-day
variability nor week-to-week trends were found in the urinary metabolites
excreted. Presumably, because of the relatively short half-life of TCI (1.75
hours), urinary metabolites did not accumulate over several days with chronic
dosage.
Figure 4-8 (in Chapter 4) shows the biphasic-appearing relationship ob-
served between metabolism (amount of urinary metabolites) and TCI dose. The
initial portion of the curve, from 0 to 1,600 mg/kg TCI, is linear; the second
portion shows relatively abrupt plateauing, with an evident saturation of meta-
bolism. In the linear portion of the curve, from 0 to 1,600 mg/kg dose, 27.5%
of the dose was converted into urinary metabolites; above 1,600 mg/kg, the
fraction of each dose metabolized decreased. For comparison, Green and Prout
(1984) orally dosed mice daily for 6 months with TCI in corn oil at a level of
1,000 mg/kg/day, and found that 525-mg equivalents of TCI/kg body weight were
metabolized per day (calculated from data in Table 8-28). Buben and 0'Flaher-
ty (Figure 4-8) observed, for this dose of TCI, a metabolism of 318 mg urinary
8-93
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metabolites/kg body weight. The data of Green and Prout were obtained with
l^C-TCI and provide a measure of total metabolism. It is likely that Buben
and 0'Flaherty's measurements of the urinary metabolites underestimated the
total metabolism, since the measurements did not include TCI metabolism to
carbon dioxide, metabolites in feces and bound to cellular macromolecules, and
other metabolites in urine in addition to TCA, TCE, and TCE-glucuronide. The
molecular weight of TCI is 131.4, that of TCA is 163.4, and that of TCE is
149.4. If an adjustment is made on the basis of 1 ymol TCI being metabolized
to 1 pmol of TCA or TCE, and the mg equivalent represented by urinary metabo-
lites is 272 mg/kg body weight. In comparison to the findings of Green and
Prout, this represents an underestimation of about 50%. Such a comparison is
in accord with the observations of Green and Prout that about 53% to 75% of the
orally administered TCI at 1,000 mg/kg/day was metabolized (Table 8-28). Buben
and 0'Flaherty, on the other hand, observed a TCI metabolism fraction of 27.8%.
An important observation can be made on the basis of the data of Buben and
O'Flaherty (Figure 4-8) and Prout et al. (Table 8-27), seen in relation to the
NTP and NCI bioassay data. The NTP average gavage assay dose (1,000 mg/kg/day)
is within the linear portion of the dose-amount metabolized curve of Buben and
O'Flaherty (Figure 4-8). However, the saturation of metabolism is approached
in the "high" doses (1,739 and 2,339 mg/kg/day) of the NCI bioassay and a lower
percentage of the administered dose is metabolized at these doses. Assuming
that Swiss-Cox mice accurately represent the metabolism characteristics of
B6C3F1 mice, the metabolism (or fractional dose metabolized) contributing to
a carcinogenic response can be estimated from Figure 4-8 in the following
manner:
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Urinary
metabolites Percent increase
Gavage dose from Figure 4-8 of metabolites
(mg/kg/day) (mg metabolites/kg/day) with dose
NTP Males: 1000 318
Females: 1000 318
NCI Males: 1169 372
2339 625 68
Females: 869 276
1739 540 96
Thus, with a twofold increase of TCI dosage to male mice of the NCI study,
metabolism increased by only 68%. This observation is consistent with the
failure to observe a greater dose-related tumor increase in male mice in the
NCI bioassay (Table 8-26).
Buben and 0'Flaherty demonstrated that certain indices of TCI hepatotoxi-
city (increased liver weight and decreased liver glucose-6-phosphatase activity)
correlated well with the amount of TCI metabolized by mice. The degree of
hepatotoxicity (as measured by each of these toxicity parameters) when plotted
against urinary metabolite excretion was linear, suggesting that the hepatotox-
icity of TCI in mice is directly related to the metabolism of TCI.
8.3.2.1.2.2 Inhalation Studies. Stott et al. (1982) exposed rats and
mice to 10 and 600 ppm of ^C-TCI for 6 hours and estimated the amount metabo-
lized by collecting the radioactivity excreted in urine, feces, expired air,
etc., and by determining TCI in expired air for 50 hours post-exposure. Tables
8-30 and 8-31 summarize their results. The following general observations can
be made on the basis of these data:
For mice exposed to TCI for 6 hours at 10 ppm and 600 ppm, virtually all
of the total TCI uptake was metabolized. At 600 ppm exposure for 6 hours, all
8-95
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TABLE 8-30. DISPOSITION OF C-TCI 50 HOURS AFTER INHALATION EXPOSURE
OF MALE B6C3F1 MICE
10 ppm for 6 hr
TCI expired
Total body dose
Total metabolized
mg-eq/kg
0.08 (0.8%)
10.31
10.24 (99.2%)
mg/animala
0.003
0.36
0.36
600 ppm for
6 hr
mg-eq/kg nig/animal
10.01 (2.4%)
412.30
402.32 (97.6%)
0.35
14.43
14.0
aBased on 35 g mouse; calculated from data.
SOURCE: Stott et al., 1982.
TABLE 8-31. DISPOSITION OF 14C-TCI 50 HOURS AFTER INHALATION EXPOSURE
OF MALE OSBORNE-MENDEL RATS
TCI expired
Total body dose
Total metabolized
10 ppm
mg-eq/kg
0.10 (2.1%)
4.70
4.60 (97.8%)
for 6 hr
mg/animala
0.03
1.18
1.15
600 ppm for 6 hr
mg-eq/kg mg/animal
29.83 (21.1%) 7.46
141.24 35.31
111.29 (78.9%) 27.82
aBased on 250 g rat; calculated from data.
SOURCE: Stott et al., 1982.
8-96
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of the assimilated dose metabolized was equivalent to that of an oral dose to
mice of 500 mg/kg, and was approximately two- to fourfold less than the body
metabolic load in the NTP and NCI mouse bioassays. For rats as well, exposure
to 10 ppm resulted in the metabolism of virtually all of the TCI pulmonary
uptake; however, at 600 ppm some of the total assimilated dose was exhaled
unchanged (21%), indicating nonlinear kinetics and suggesting the approach of
saturation of metabolism at this exposure concentration. At 600 ppm exposure
of rats for 6 hours, the total body metabolic load roughly approximated that
from an oral dose of 500 mg/kg when 42% of the dose was exhaled unchanged.
These observations of approaching saturation of metabolism in rats at 500 ppm
are in accord with those of Andersen et al. (1980), who estimated saturation
at about 1,000 ppm and a Vmax (maximum rate of the saturable process) of 28
mg/kg/hour, and are considerably higher than the observations of Filser and
Bolt (1979), who estimated saturation at about 65 ppm but a comparable Vmax of
28 mg/kg/hour. From the data of Stott et al. (1982), the Vmax can be estimated
as 19 mg/kg/hour.
The ratios of the amount of TCI metabolized by the rat versus the mouse at
10 and 600 ppm (Tables 8-30 and 8-31) are as follows:
Exposure
ppm, 6 hours Ratio; Rat/mouse
10 (1.15/0.36) = 3.19
600 (27.82/14.0) = 1.99
Therefore, on the basis of comparative surface area (250 g/35 g)0-67 = 3.7, the
ratio of the amounts metabolized by the rat versus the mouse approximates their
relative surface areas at the lower dose. At the higher dose, the ratio of
the amount metabolized in the rat versus the mouse is smaller, probably due to
8-97
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saturation of metabolism in the rat. These inhalation results are comparable
to those observed after oral administration of TCI.
8.3.2.1.3 Metabolic Profile. The experimental evidence is consistent with
the metabolic pathways for TCI being qualitatively similar in mice, rats, and
humans. In these species, the following principal end metabolites are common:
trichloroacetic acid (TCA), trichloroethanol (TCE) and TCE-glucuronide, and
their precursor metabolite, chloral. In recent studies, Green and Prout
(1984) and Dekant et al. (1984) made comparative studies of biotransformation
of TCI in rats and mice, and failed to detect any major species or strain
differences in metabolism. In addition, TCI oxide has been demonstrated to
be formed by both mouse and rat hepatic microsomes in vitro (Miller and Guen-
gerich, 1982).
Dekant et al. (1984) isolated and identified, by rigorous methodology, the
metabolites in exhaled air and urine of mice and rats given an oral dose of 200
mg/kg of l^C-TCI. The metabolite profiles for these two species are shown in
Table 4-11 (Chapter 4). Previously, TCE, its glucuronide, and TCA had been
identified as urinary metabolites of TCI for mice, rats, and humans, and carbon
dioxide had been identified as an end metabolite in the exhaled air of mice
and rats (see Chapter 4). Hathaway (1980) detected traces of dichloroacetic
acid in mouse urine, but only after very large (2 ml TCI/kg) oral doses. To
explain these results, Hathaway proposed a "spillover" model, in which an
excess of compound saturates "deactivation mechanisms" of the TCI oxide formed,
producing an intermediate that becomes available for reactions that include
spontaneous hydrolytic dechlorination to dichloroacetic acid (Kline and Van
Duuren, 1977). However, Figure 4-8 shows that, at the dose of 200 mg TCI/kg,
metabolism is far from saturated in mice (or rats). Dekant et al. (1984)
8-98
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identified dichloroacetic acid in the urine of both mice and rats at this dose.
In addition, these investigators identified two previously unrecognized metabo-
Tites, oxalic acid and N-(hydroxyacetyl)-aminoethanol (HAAE) in the urine of
both of these species. They also identified significant amounts of HAAE in the
urine of human volunteers exposed to 200 ppm TCI for 6 hours. There are no
reported human studies in which carbon dioxide and oxalic acid, both normal
endogenous compounds, have as yet been identified as specific metabolites of
TCI, since doing so would require administration of labeled TCI. Nonetheless,
the observations of Dekant et al., and of others, indicate that qualitatively,
the pathways of TCI metabolism are similar in mice, rats, and humans (see
Chapter 4 for a more detailed review).
Whether there is a quantitative difference in TCI metabolism in mice,
rats, and humans is a question that remains unresolved. Dekant et al. (1984,
Table 4-11) have noted that, in the case of the principal urinary metabolites,
the ratio of TCE to TCA is much greater in the mouse than in the rat. However,
they suggest that the smaller relative amounts of TCA in the urine of mice may
be due to an active TCA enterohepatic recirculation, with TCA excreted in the
feces in a conjugate form. In humans, the urinary ratio of TCE to TCA varies,
but is generally found to be about 2:1.
8.3.2.1.4 Covalent Binding. Banerjee and Van Duuren (1978) compared covalent
binding of ^C-TCI to mouse and rat microsomes and observed that mice micro-
somes are two- to fourfold more active in metabolizing TCI to reactive cova-
lent-binding metabolites than are rat microsomes. The covalent binding is
proportional to the metabolism of TCI in these species. Stott et al. (1982)
also investigated covalent binding in vivo after 6-hour exposures of mice and
rats to 10 and 600 ppm l^c-TCI. The time of maximum binding post-exposure was
8-99
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found to be 0 and 3 hours, respectively, for liver and kidney. These results
are given in Table 8-32. At low exposure concentrations of TCI (10 ppm), rat
and mouse covalent binding per yg of protein occurred to the same extent,
with the amount of liver binding being two- to threefold greater than kidney
binding. At high TCI exposures (600 ppm), which are metabolism-saturating
concentrations for the rat (Tables 8-30 and 8-31), the amount of covalent
binding was three- to fourfold greater per yg of protein in mice than in
rats, but was in proportion to TCI metabolism; therefore, the ratios of total
amounts of covalent binding in the rat versus the mouse approximates the rela-
tive metabolic rates in these species. Stott et al. also observed DNA aklyla-
tion in mice dosed with 1.2 g/kg high-specific l^C-TCI. Their results showed
that TCI or its metabolites are capable of binding to DNA, but that the binding
ability was low when compared to dimethylnitrosamine and aflatoxin. Similar
results have been reported by Parchman and Magee (1982), and Di Renzo et al.
(1982).
8.3.2.2 Calculation of Human Equivalent Doses from Animal Data—The pharmaco-
kinetic and metabolic data that most closely approximate the conditions of the
NTP and NCI carcinogenicity bioassays are found in the following studies: 1)
Prout et al. (1984), oral administration of single doses of TCI at dose levels
up to 2,000 mg/kg to rats and mice; 2) Green and Prout (1984), chronic (180
days) oral administration of TCI at a dose level of 1,000 mg/kg/day to rats and
mice; 3) Dekant et al. (1984), oral administration of a single dose of TCI at a
dose level of 200 mg/kg to rats and mice; and 4) Buben and O1Flaherty (1985),
chronic (6-week) oral administration of TCI at levels up to 2,000 mg/kg to
mice. The comparative studies of TCI metabolism in both mice and rats of Prout
et al. (1984), Green and Prout (1984), and Dekant et al. (1984), as well as
8-100
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TABLE 8-32. COVALENT BINDING TO LIVER AND KIDNEY MACROMOLECULES OF
14C-TCI IN MALE B6C3F1 MICE AND OSBORNE-MENDEL RATS
Exposure
ppm Tissue
10 liver
kidney
600 liver
kidney
Species
mouse
rat
mouse
rat
mouse
rat
mouse
rat
Binding
pmole-eq
per ng
protein
Av. of 4
0.318
0.268
0.168
0.153
20.4
4.72
5.6
1.77
SOURCE: Adapted from Stott et al., 1982.
8-101
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the inhalation studies of Stott et al. (1982), provide evidence that, for a
given dose, such as those used in the NCI and NTP bioassays, the magnitude of
TCI metabolism in these species closely approximates their relative surface
areas. For the risk calculation, it is assumed that the amount of TCI metabo-
lized per m2 surface area is equivalent among species.
The carcinogenicity data of the NTP and NCI bioassays show, in male and
female B6C3F1 mice, a significant dose-related excess incidence of hepatocell-
ular carcinomas at time-weighted average dosage levels of 1,000 mg/kg in the
NTP study, and "low" and "high" doses for female and male mice in the NCI study
of 869 and 1,739 mg/kg, and 1,169 and 2,339 mg/kg, respectively (Table 8-26).
The data of Buben and 0'Flaherty (1985, Figure 4-8) clearly indicate that, in
Swiss-Cox mice, the metabolism of TCI is linearly proportional to oral doses
up to at least 1,600 mg/kg. Hence, except for the "high" doses (1,730 and
2,339 mg/kg) given to female and male mice in the NCI study, the amount of
metabolites formed and contributing to a carcinogenic response is directly
proportional to the dose, which in turn, may be proportional to the tumori-
genic response. Therefore, the dose-metabolism curve of Buben and O'Flaherty
can be used in calculating TCI metabolism from the bioassay doses and in cor-
recting for the saturation metabolism associated with the "high" doses of the
NCI bioassay. However, since the Buben and O'Flaherty dose-metabolism curve
was based solely on urinary metabolites (and hence underestimates the total
amount of metabolism), a more definitive measure of metabolism, using l^C-TCI
as in the studies of Prout et al. (1984) and Green and Prout (1984) can be
used to estimate the total TCI metabolism associated with the bioassay doses.
Also, urinary metabolites are assumed to be linearly proportional to total
metabolism.
8-102
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8.3.2.2.1 NTP Study. In this study, the time-weighted average gavage dose
was 1,000 mg/kg/day, or, based on the average terminal weight for the male
and female mice, 40 mg/male mouse/day and 35 mg/female mouse/day (Table 8-26).
In other studies, with similar doses given either singly or chronically in
corn oil to B6C3F1 mice, virtually complete absorption occurred; a portion of
the dose was excreted unchanged in exhaled air (Prout et al., 1984; Green and
Prout, 1984; Dekant et al., 1984). On the basis of these data, the fractional
dose metabolized for oral doses of between 30 to 60 mg per animal averages 78%.
These doses are on the linear portion of the dose-metabolism curve for mice of
Buben and 0'Flaherty (1984, Figure 4-8). Hence, the daily body metabolic load
(78% of the administered dose) for the mice in the NTP study is 780 mg/kg.
The relationship between the experimental administered dose (mg/animal)
and the amount metabolized is estimated on the basis of data from Prout et al.
(1984) (see Table 8-27), using the "Michaelis-Menten" type of equation, M = a x
d/(b + d), where d is the experimental administered dose (mg/animal) and M is
the corresponding amount metabolized. The parameters a and b are determined by
least-square estimates and are equal to 594.1 mg and 702.97 mg, respectively,
with a coefficient of determination r^ = 0.99. This equation is used to con-
vert a bioassay dose in mice to the metabolized dose. Assuming a lifetime of
104 weeks, the daily lifetime average effective exposure (LAE) of the mice is
given by:
104 weeks 5 days
LAEf/i = 104 weeks x 7 days x metabolized dose, mg/animal/day
and the equivalent dose for humans,
= LAEf/| x scaling factor
8-103
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The scaling factor for mouse to humans is: (wH/wAf where WH and WA are,
respectively, the human and animal body weights;
male mouse (70/0.040)2/3 = 145.21; female mouse (70/0.035)2/3 = 158.74
The lifetime average human equivalent doses for the NTP mouse carcinogeni-
city bioassay of TCI, expressed in units of mg/day, mg/m2 surface area/day, and
mg/kg/day, are presented in Table 8-33.
However, a total daily dose administered as a single bolus, such as in a
bioassay, may not be biologically equivalent to the same total daily dose
resulting from a continuous 24-hour exposure (e.g., air pollutant) or from
several intermittent smaller doses (e.g., drinking water). As has been pointed
out by Beliles (1975), a 24-hour continuous exposure to a given concentration
of an atmospheric pollutant is not necessarily the simple equivalent of one-
fourth the 8-hour workplace exposure.
8.3.2.2.2 NCI Study. For this study, the time-weighted average gavage doses
("high" and "low" doses) in mg/kg/day given to male and female B6C3F1 mice are
given in Table 8-26, and the corresponding dose per single animal per day is
given as calculated from the average terminal weight for the male and female
mice of 33 g and 26 g, respectively. As noted previously, doses in corn oil to
B6C3F1 mice in this range have been observed experimentally, when given as a
single oral dose or chronically (daily for 180 days), to be completely absorbed
from the GI tract, although a portion of the dose effects a first-pass of the
liver and is excreted in exhaled air unchanged (Prout et al., 1984; Green and
Prout, 1984; Dekant et al., 1984). The data of these investigators indicate
that the percentage of TCI dose metabolized for oral doses of between 30 and 60
mg per animal averages 78% (Table 8-27). Furthermore, these doses are on the
8-104
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TABLE 8-33. DOSE CONVERSION FOR THE NTP BIOASSAY (1982) IN B6C3F1 MICE
00
I
Oral
experimental dose
(time-weighted)
Sex
Male
Female
mg/kg/day
0
1,000
0
1,000
mg/day
0
40
0
35
Animal
metabolized dose
mg/day
0
31.98
0
28.17
Human
equivalent dose3
(lifetime
average exposure)
mg/day
0
3,317.23
0
3,194.06
mg/m2/dayb
0
1,793.10
0
1,726.52
mg/kg/day
0
47.39
0
45.62
Tumor
incidence
8/48
30/50
2/49
13/49
human equivalent dose (mg/day) = animal
aAssuming the animals were exposed to TCI for their entire lifetimes,
metabolized dose (mg/day) x 5/7 x (WH/WA)2'3
where W/\ = 0.040 kg; terminal average weight for male mice
0.035 kg; terminal .average weight for female mice
WH = 70 kg; weight for standard man
The factor 5/7 reflects the fraction of days per week that the animals received treatment.
bSurface area for standard man = 1.85 m2 (Diem and Lentner, 1970).
-------
linear portion of the dose-metabolism curve for mice of Buben and 0'Flaherty
(1984). However, the higher doses of 1,739 and 2,339 are on the nonlinear,
"saturating" portion of the curve and, as estimated from this curve (Figure
4-8), further adjustments of 96% and 68%, respectively, are appropriate,
assuming the same curve for the B6C3F1 strain mouse. The daily amounts of dose
metabolized for the mice in the NCI study are estimated below in the same
manner as was done for the NTP study (see Table 8-34), using the data from the
Prout et al. (1984) study, and by also including the data from Buben and 0'Fla-
herty, which affects the higher dose only, for comparison. The data from the
Buben and 0'Flaherty study are especially important to consider because this
study reflects a chronic dosing schedule, making it more relevant to the condi-
tions of the NTP and NCI carcinogenicity studies. The daily lifetime average
exposure (LAE) of the mice is given by
78 weeks 5 days
LAEM = go weeks x 7 days x amount metabolized, mg/animal/day
and the equivalent dose for humans,
LAE^ = U\EM x scaling factor
The scaling factors for mouse to man (WH/WA)2/3 are:
male mouse (70/0.033)2/3 = 165.09
female mouse (70/0.026)2/3 = 193.53
The lifetime average human equivalent doses for the NCI mouse carcinogeni-
city bioassay of TCI, expressed in units of mg/day, mg2 surface area/day, and
mg/kg/day, are presented in Table 8-34. The adjustment for partial lifetime in
the NCI study, 90 weeks versus 104 weeks in the NTP study, is made later.
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TABLE 8-34. DOSE CONVERSION FOR THE NCI BIOASSAY (1976) IN B6C3F1 MICE
male
Oral
experimental dose
(time-weighted)
Animal
metabolized dose
Human
equivalent dose3
dose (lifetime
average exposure)
Sex
mg/kg/day
mg/day
mg/day
mg/day
mg/m2/dayb
mg/kg/day
Tumor
incidence
0
1,169
2,339
0
38.58
77.19
0
30.90
58.77
(39.96)c
0
3,157.93
6,006.21
0
1,706.98
3,246.60
0
45.11
85.80
1/20
26/50
31/48
CO
0
female
0
869
1,739
0
22.59
45.21
0
18.49
35.89
(34.45)c
0
2,215.19
4,299.79
0
1,197.40
2,324.21
0
31.65
61.43
0/20
4/50
11/47
aHuman equivalent dose (mg/day) = animal metabolized dose (mg/day) x 5/7 x 78/90 x (WH/WA)2'3
where WA = 0.033 kg for male mice
= 0.026 kg for female mice
WH = 70 kg; weight for standard man.
The factors 5/7 and 78/90 reflect the treatment schedule of 5 days per week for 78 weeks of a 90-week lifetime.
bSurface area for standard man = 1.85 m2 (Diem and Lentner, 1970).
cAssuming urinary metabolites account for about 50% of total metabolism, the additional information provided by
the Buben and 0'Flaherty (1985) study would predict 40 mg/day (male) and 34.45 mg/day (females), a very small
difference, as the amount metabolized at the high doses (this is comparable to the amounts predicted by fitting
a curve to the Prout et al. [1984] data only, considering the differences in experimental conditions and mouse
strains).
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8.3.2.3 Summary—The parent chemical, TCI, is metabolized to electrophilic
intermediate products that can react readily with cellular macromolecules,
resulting in a spectrum of toxic responses that includes carcinogenesis. For
example, the epoxide metabolite is considered sufficiently capable of produ-
cing the toxic end point of carcinogenicity.
Thus, the experimental data base for TCI provides some support for deter-
mining an extrapolation base for laboratory animals to man of W2/3, and allows
estimates of daily animal exposure (fractional dose metabolized) contributing
to the tumorigenic response for the conditions of the NCI and NTP bioassays.
Estimates of equivalent human exposure can be calculated using the data
from the experimental animal studies. The appropriate interspecies scaling
is thus assumed to be on a surface area basis, i.e., W2/3. it is of some
interest also to view the estimates of equivalent human exposure that produced
hepatocellular carcinomas in mice in perspective with human body metabolic
loads from air exposure. Humans appear to metabolize inhaled TCI extensively,
as reported by various investigators (see Chapter 4). The studies in human
subjects indicate that at least 60% and possibly as much as 90% of the retained
TCI from vapor exposures of less than 500 ppm (1,690 mg/m^) is metabolized in
the body. Thus, human subjects undergoing light physical activity, exposed
for 8 hours to 150 ppm TCI in air, develop a body burden of about 2,500 mg
(Monster et al., 1976, 1979; Nomiyama and Nomiyama, 1977; Fernandez et al.,
1977). Nomiyama and Nomiyama (1977) observed that, at least for up to 300
ppm inhalation exposure, the metabolism of TCI is not saturated in humans.
(See Chapter 4 for a detailed discussion of these studies).
8.3.3 Choice of Risk Model
8.3.3.1 General Considerations—The data used by the CAG for quantitative
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estimation are of two types: 1) lifetime animal studies, and 2) human studies
in which 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 solidly acceptable scientific basis for any mathematical
extrapolation model that relates chemical carcinogen exposure to cancer risks
at the extremely low concentrations that must be dealt with in evaluating envi-
ironmental 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 car-
cinogenesis for guidance as to which risk model to use. At the present time
the dominant view of the carcinogenic process involves the concept that most
agents that cause cancer also cause irreversible damage to DNA. 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 quantal type of
biological response, -which is characteristic of mutagenesis, is associated with
a low-dose linearity and nonthreshold dose-response relationship. Indeed,
there is substantial evidence from mutagenicity studies with both ionizing
radiation and a wide variety of chemicals that this type of dose-response
model is the appropriate one to use. At higher doses there can be an upward
curvature, probably reflecting the effects of multistage processes on the
mutagenic response. The low-dose linearity and nonthreshold dose-response
relationship is also consistent with the relatively few epidemiologic studies
of cancer responses to specific agents that contain enough information to
make the evaluation possible (e.g., radiation-induced leukemia, breast and
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thyroid cancer, skin cancer induced by arsenic in drinking water, and liver
cancer induced by aflatoxins in the diet). There is also some evidence from
animal experiments that is consistent with the linear nonthreshold model (e.g.,
liver tumors induced in mice by 2-acetylaminofluorene in the large scale EDgi
study at the National Center for lexicological 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 nonthreshold model, which is
linear at low doses, has been adopted as the primary basis for risk extrapola-
tion to low levels of the dose-response relationship. The risk estimates made
with this model should be regarded as conservative, representing a plausible
upper limit for the risk, i.e., the true risk is not likely to be higher than
the estimate, but it could be lower.
The mathematical formulation chosen to describe the dose-risk 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-
risk response data, and it incorporates a procedure for estimating the largest
possible linear slope (in the 95% confidence limit sense) at low extrapolated
doses that is consistent with the data at all dose levels of the experiment.
The methods used by the 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
alternative models have not been used in the following analysis because, in the
opinion of the CAG, the evidence of carcinogenicity for TCI does not warrant
that complete an analysis. Furthermore, with the limited data available from
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these animal bioassays, especially at the high-dose levels required for test-
ing, 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 esti-
mates obtained by use of the low-dose linear nonthreshold 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,
where more than one acceptable study is available, a general approach is to use
the most sensitive responder, i.e., the data set that gives the highest estimate
of lifetime cancer risk, on the assumption that humans are as sensitive as the
most sensitive animal species tested. For TCI, four sets of bioassay data in
B6C3F1 mice, which produce comparable risk estimates, are used.
Extrapolation from animals to humans can be done on the basis of relative
body weights, surface areas, metabolic rates, or other measures. The general
approach is to use the extrapolation base (mg/kg, surface area, etc.) which
can be appropriately justified by the available experimental data from animals
and humans. However, it is usually not clear which extrapolation base is the
most appropriate for the carcinogenic response per se. When there is insuffi-
cient or no experimental data to determine an extrapolation base either direct-
ly or indirectly, the most generally employed and conservative method is used,
i.e., extrapolation from animal dose to a human equivalent dose on the basis of
relative surface areas (W2/3). For the TCI gavage studies in mice, the use of
an extrapolation based on surface area (W2/3) rather than body weight (W1*0)
would increase the unit risk estimates by a factor of 12 to 13. However, for
TCI, experimental data on metabolism and kinetics are used in dose extrapola-
tion from mouse to man.
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8.3.3.2 Mathematical Description of the Low-Dose Extrapolation Model—Let P(d)
represent the lifetime risk (probability) of cancer at dose d. The multistage
model has the form
P(d) = 1 - exp [-(q0 + q^ + q2d2 + ... + qkdk)]
where
qi j> 0, i = 0, 1, 2, ..., k
Equivalently,
. . _ . p i,
Pt(d) = 1 - exp [-(q^d + q2dc + ... + qkd^)]
where
- P(0)
1 -
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 GLOBAL83, developed by
Howe (1983). At low doses, upper 95% confidence limits on the extra risk and
lower 95% confidence limits on the dose producing a given risk are determined
from a 95% upper confidence limit, q^, on parameter q^. Whenever q^ > 0, at
low doses the extra risk Pt(d) has approximately the form Pt(d) = qi x d.
Therefore, q-i x d is a 95% upper confidence limit on the extra risk, and
is a 95% lower confidence limit on the dose, producing an extra risk of R. Let
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*
ll »
LQ be the maximum value of the log-likelihood function. The upper limit,
is then calculated by increasing qj to a value q1 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 - LI) = 2.70554
where 2.70554 is the cumulative 90% point of the chi-square distribution with
one degree of freedom, which corresponds to a 95% upper-limit (one-sided).
This approach of computing the upper confidence limit for the extra risk Pt(d)
is an improvement on the Crump et al. (1977) model. The upper confidence limit
for the extra risk calculated at low doses is always linear. This is conceptu-
ally consistent with the linear nonthreshold concept discussed earlier. The
slope, q^, is taken as an upper-bound of the potency of the chemical in
inducing cancer at low doses. (In the section calculating the risk estimates,
Pt(d) will be abbreviated as P.)
In fitting the dose-risk response model, the number of terms in the poly-
nomial 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 refitted 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
X2 . y <*, - N,P,
<• NiPi (1-Pi)
1 = 1
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is calculated where N.,- is the number of animals in the i^ dose group, X^ is
the number of animals in the i™ dose group with a tumor response, P^ is the
probability of a response in the ith dose group estimated by fitting the multi-
stage model to the data, and h is the number of remaining groups. The fit is
determined to be unacceptable whenever X^ is larger than the cumulative 99%
point of the chi-square distribution with f degrees of freedom, where f equals
the number of dose groups minus the number of non-zero multistage coefficients.
8.3.3.3 Adjustment for Less Than Lifespan Duration of Experiment—If the dura-
ation 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 multiply-
ing a factor (L/Le) . 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 demon-
strated 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)] 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/l_e)3.
This adjustment is conceptually consistent with the proportional hazard
model proposed by Cox (1972) and the time-to-tumor model considered by Daffer et
al. (1980) where the probability of cancer by age t and at dose d is given by
P(d,t) = 1 - exp [-f(t) x g(d)]
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8.3.4 Calculation of Unit Risk
8.3.4.1 Definition of Unit Risk—This 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 pollu-
tant is defined as the increased lifetime cancer risk occurring in a hypothe-
tical population in which all individuals are exposed continuously from birth
throughout their lifetimes to a concentration of 1 ug/m^ of the agent in the
air they breathe, or to 1 pg/L in the water they drink. This calculation is
done to estimate in quantitative terms the impact of the agent as a carcinogen.
Unit risk estimates are used for two purposes: 1) to 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.
8.3.4.2 Slope Calculation—Using the incidence data in Tables 8-33 and 8-34
and the corresponding human equivalent metabolized doses, the slope estimate,
q^, for TCI is calculated using the linearized multistage model, as shown in
Table 8-35. The q^ value is used to compare relative potencies of carcino-
gens and to calculate the unit risks for drinking water and air.
Since NCI mice were followed for only 90 weeks, rather than for 104 weeks
as in the NTP study, the risk estimates calculated on the basis of NCI mice are
multiplied by a factor (104/90)3 = 1.54. The rationale for such an adjustment
is given in Section 8.3.3..S. The slope estimates in the last column of Table
8-35 are expressed in terms of the ambient concentration. These values are
derived by using the calculated metabolized dose-response curve and the dose-
metabolism relationship previously established. Since both the metabolized
dose-response and dose-metabolism curves are linear at low doses, the resultant
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TABLE 8-35. SLOPE ESTIMATES (qt)a
BASED ON EXTRAPOLATION FROM DATA IN MALE AND FEMALE MICE
Data base
K
(animal)D
(mg metabolized
dose/kg/day)-l
(human)
(mg metabolized
dose/kg/day)-1
(human)
(mg administered
dose/kg/day)-l
NTP:
Male mice
Female mice
1.8 x ID'3
7.5 x 10-4
2.2 x lO-2
9.5 x 10-3
1.9 x 10-2
8.0 x lO-3
NCI:
Male mice
Female mice
Geometric mean
1.6 x ID'3
5.0 x 10~4
1.0 x ID'3
2.1 x ID'2
6.9 x 10-3
1.3 x ID'2
1.8 x 10-2
5.8 x 10-3
1.1 x 10-2
aq1 is defined as the 95% upper limit of the linear component (slope) in the
multistage model. Since the dose-response curve is virtually linear below
1 nig/kg/day, the slope is numerically equal to the upper limit of the incre-
mental lifetime risk estimates at 1 mg/kg/day. The slope values are used to
compare relative carcinogenic potencies and to calculate the unit risks for
drinking water and air.
bq1 (animal) is calculated using the daily lifetime average effective expo-
sure (LAE^).
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dose-response curve is also linear at low ambient doses. Because the slope
estimates from all four data sets are comparable, the geometric mean of these
four numbers is used to represent the slope, ql9 for TCI. The qj values
calculated from the NCI and NTP studies are presented in the last two columns
of Table 8-35, expressed in terms of metabolized dose and also in terms of
ambient dose after adjusting for metabolism. The geometric mean is qj =
1.3 x 10~2/ (mg/kg/day) in terms of the metabolized dose, or q^ = 1.1 x 10~2/
(mg/kg/day) in terms of the ambient dose. These q^ values are used to calcu-
late the unit risks for water and air.
8.3.4.3 Risk Associated with 1 ug/L of TCI in Drinking Water—The daily dose
(mg/kg/day) from consumption of 2 L of water containing 1 pg/L of TCI is cal-
culated as follows:
d = (1 yg/L) x (2 L/day) x (10-3 mg/yg)/70 kg
= 2.9 x ID'5 mg/kg/day
Here it is assumed that, on the average, humans weigh 70 kg and consume 2 L
of water each day. Therefore, the incremental lifetime risk associated with
of TCI in drinking water is:
p = 1.1 x 10-2 x 2.9 x lO-5
= 3.2 x 10-7
where 1.1 x 10-2 is the human carcinogenic potency in terms of adjusted ambient
dose, taken from Table 8-35.
8.3.4.4 Risk Associated with 1 ug/m3 of TCI in Air—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
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et al. (1978) study showing increased hepatocellular carcinomas in males. As
discussed in the qualitative section, both studies exhibited serious defects.
The Bell et al. (1978) study, which showed increased hepatocellular carcinomas
in male mice is used to calculate the unit risk for air. Because of the defi-
ciencies of this study, the risk estimate is used only to compare with those
calculated on the basis of gavage studies. In order to use the potency q^
from the gavage studies, it is necessary to estimate the amount metabolized
when a subject is exposed to 1 yg/m3 of TCI in air.
8.3.4.4.1 Unit Risk Estimate Calculated on the Basis of Human Metabolized
Dose. Although there are many studies on metabolism of TCI in humans, most of
the studies did not present the relationship between TCI uptake and the amount
metabolized. The study by Monster et al. (1976) is one which can be used for
estimating the amount metabolized when a subject is exposed to 1 yg/m3 of TCI
in air. The study presents the total uptake and the percentage excreted as TCI
in the exhaled air for four subjects who were exposed to 70 ppm and 140 ppm for
4 hours, including two half-hour 100-watt exercises. Since results from 70 ppm
and 140 ppm are comparable, the results for the 70 ppm group are used for risk
calculation. These results are as follows:
Percentage
of dose Total dose
Subject Uptake (mg) exhaled as TCI metabolized (mg)
A 500
B 450
C 470
D 660
9
17
10
8
455.0
373.5
423.0
607.2
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The median amount metabolized for the four subjects is 439 mg. Under the
assumption that the dose metabolized is linearly related to the intensity and
•duration of exposure, the dose metabolized corresponding to 1 yg/m3 of TCI in
air is
d = 439 mg x (24 hours/4 hours) / (70 ppm x 5,475 Pg/m3/ppm)
= 6.9 x 10~3 mg
where 1 ppm = 5,475 yg/m3. That is, under a continuous (24 hours) exposure to
1 yg/m3 of TCI in air, the body metabolic load is estimated to be
d = 6.9 x 10-3/70 = 9.9 x 10~5 mg/kg/day
Therefore, the unit risk for TCI in air is
P = 1.3 x ID'2 x 9.9 x lO-5
= 1.3 x ID'6
where 1.3 x 10~2 is the human carcinogenic potency in terms of metabolized
dose, taken from Table 8-35. This unit risk estimate is comparable to the
other three estimates to be calculated by different approaches and/or on dif-
ferent data bases. The CAG recommends that P = 1.3 x 10'6 be used as the unit
risk estimate for TCI in air because it is calculated on the basis of human
metabolized dose.
8.3.4.4.2 Unit Risk Estimate Calculated on the Basis of Animal Metabolized
Dose. Stott et al. (1982) exposed rats and mice to 10 ppm and 100 ppm of 14C-
TCI for 6 hours and estimated the amount metabolized by collecting the radio-
activity excreted in urine, feces, expired air, etc., and by determining TCI
in the expired air for 50 hours post-exposure. The results of 10 ppm expo-
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sure, which is more relevant to the risk calculation, are reproduced below from
Tables 8-30 and 8-31.
Mice Rats
TCI expired (mg) 0.003 0.03
Metabolized (mg) 0.36 (99%) 1.15 (98%)
Total uptake (mg) 0.36 1.18
These data can be used in two different ways to estimate human risk due
to 1 yg/m3 of TCI in ai r.
Approach 1: Since the body metabolic loads for mice after oral and inha-
lation exposure are known, it is possible to estimate the unit risk by inhala-
tion using the slope q^ (animal) = 1.0 x 10~3/(mg/kg/day), calculated on the
basis of a gavage study (Table 8-35). For mice, 10 ppm in air for 6 hours re-
sulted in a metabolized dose of 0.36 mg. Under the assumption that the amount
metabolized at low doses is linearly related to the intensity and duration of
exposure, the daily dose metabolized corresponding to 1 yg/m3 continuous
exposure of TCI in air is:
0.36 mg x (24 hours/6 hours) / (10 ppm x 5,475 Ug/m3/ppm)
= 2.63 x ID'5 mg
where 1 ppm = 5,475 yg/m3. That is, if a mouse is exposed continuously to
1 yg/m3 of TCI in air, its body metabolic load is 2.63 x 10~5 mg/day or 7.5
x 10~4 mg/kg/day for a mouse weighing 0.035 kg. Therefore, the lifetime incre-
mental risk for mice due to exposure to 1 yg/m3 of TCI in air is
P (mice) = 1.0 x 10'3 x 7.5 x 10'4
= 7.5 x 10-7
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To convert the risk estimate from mice to humans, the human concentration
C(yg/m3) that is equivalent to mouse exposure to 1 yg/m3 of TCI must be
determined. Under the assumption that the amount metabolized per body surface
area is equivalent between mice and humans, the human concentration C(yg/m3)
must satisfy the relationship
C x 20 m3/day = 1 x 0.043 m3/day
(70 kg)2/3 (0.035 kg)2/3
where 20 m-Vday and 0.043 m3/day are assumed to be, respectively, the volu-
metric breathing rates for a human weighing 70 kg and a mouse weighing 0.035
kg. As a consequence, C = 0.34 pg/m3. That is, the exposure concentration,
0.34 pg/m3, for humans is considered equivalent to that for mice at 1 y/m3.
Therefore, the unit risk estimate for humans is
P = 7.5 x 10-7/0.34
= 2.2 x ID'6
This value is comparable to that calculated on the basis of actual human data.
Approach 2: In Approach 1, only mouse data are used. As an alternative
approach, the relationship of the metabolized doses between mice and rats could
be used to infer the human metabolized dose. As demonstrated previously, the
relative amount of dose metabolized between mice and rats is approximately
equal to two-thirds of the ratio of body weights. Assuming that the same rela-
tionship holds for humans and mice, the human metabolized dose corresponding to
1 pg/m3 of TCI in air would be
d = 2.63 x lO-5 mg/day x (70 kg/0.035 kg)2/3
= 4.17 x 10"3 mg/day or equivalently
d = 4.17 x 10-3/70 = 5.96 x 10"5 mg/kg/day
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where 2.63 x 10"5 mg/day is the metabolized dose for mice corresponding to
1 yg/m3 of TCI in air, as calculated previously in Approach 1. Therefore,
the unit risk is estimated to be
P = 1.30 x lO-2 x 5.96 x 10~5
= 7.7 x 10-7
•k Q
where q^ = 1.30 x 10 /(mg/kg/day) is the slope for humans taken from Table
8-35. This risk estimate is again comparable to those calculated previously.
8.3.4.4.3 Unit Risk Estimate Calculated on the Basis of an Inhalation Study in
Mice. The Bell et al. (1978) study, which showed increased hepatocellular car-
cinomas in male mice, can be used to calculate the unit risk for TCI in air.
Because of the deficiencies in this study, the risk calculated from this study
should be considered crude and is to be used only for comparision with those
calculated on the basis of gavage studies. For both gavage and inhalation
studies, the animal species used in the experiment and the observed tumor site
and type are identical. Animals in the Bell et al. (1978) study were exposed
to TCI 6 hours/day, 5 days/week, for 24 months. The dose and response data are
as follows:
Experimental
dose (ppm)
0
100
300
600
LAD (ppm)a 0 17.86 53.57 107.14
Hepatocellular
carcinoma
incidence 18/99 28/95 31/100 43/97
aThe lifetime average dose (LAD) is calculated by
LAD = dose (ppm) x (5 days/7 days) x 6 hours/24 hours.
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Using the linearized multistage model, the carcinogenic potency (the 95% upper
limit of the linear coefficient in the multistage model) for animals is calcu-
lated to be
qj (animal) = 4.8 x 10~3/ppm
or
<\\ (animal) = 4.8 x l(T3/5475
= 8.8 x 10-7/(yg/m3)
using the fact that 1 ppm = 5475 yg/m3.
To convert the potency from animals to humans, it is assumed that dose
relative to body surface area is equivalent among species. As calculated
previously (see Section 8.3.4.4.2), the exposure concentration C (yg/m3)
for humans that is equivalent to 1 pg/m3 for mice is C = 0.34 yg/m3.
Therefore, the carcinogenic potency for humans is
q^ = 8.8 x 1(T7/0.34
= 2.6 x 10-6/(yg/m3)
The risk due to 1 yg/m3 of TCI in air is thus calculated to be
P = 2.6 x 10-6/(yg/m3) x 1 yg/m3
= 2.6 x ID'6
This risk estimate is also comparable to those calculated by means of different
approaches and on the basis of different data bases.
8.3.4.5 Discussion—To the extent possible, the available metabolism and
kinetic information for TCI has been used in the risk calculations. The use of
this information may or may not reduce the uncertainties that are associated
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with the various steps involved in risk calculations. There are three major
aspects of uncertainty in the risk assessment of TCI:
1. Extrapolation from high dose to low dose (i.e., low-dose
extrapolation)
2. Extrapolation from animals to humans (i.e., species con-
version), and
3. Extrapolation from gavage to inhalation (i.e., route-to-
route extrapolation.
In calculating the dose-response relationship for TCI, the amount meta-
bolized is considered to be an effective dose. The use of this surrogate
effective dose may not eliminate the uncertainty associated with the low-dose
extrapolation because the dose actually reaching the target sites may not be
linearly proportional to the total amount metabolized, and the shape of the
dose-response relationship is still unknown. However, it seems reasonable to
expect that the uncertainty with regard to the low-dose extrapolation would be
somewhat reduced by the use of the metabolized dose because the metabolized
dose better reflects the dose-response relationship, particularly within the
high-dose region.
To extrapolate from animals to humans, the amount metabolized relative to
body surface area is assumed to be equivalent (i.e., equally potent) among
species. This assumption is by no means supported by the empirical data. For
TCI, there is some evidence showing that, for a given dose in mg/kg (by the
oral route) or ppm (by the inhalation route), the amounts metabolized relative
to body surface area are approximately equal among species. However, there are
no observations which support the proposition that the metabolized dose relative
to body surface area is equally effective in inducing tumors among different
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species. An alternative approach would be to assume that mg metabolized dose/
kg/day is equivalent among species. If this assumption is made, the slope
factor q1, as calculated for TCI and expressed in terms of (mg/kg/day)"1, would
be reduced by approximately 10 times.
The slope calculated from the gavage study, using the metabolized dose,
provides a better basis for extrapolating a risk estimate from the gavage to
the inhalation route. This is so because the relationship between the ambient
air concentration and the amount metabolized for TCI is available for both
humans and animals. Therefore, the uncertainty due to route-to-route extrapo-
lation should be reduced by the use of metabolized dose.
8.3.4.6 Interpretation of Unit Risk Estimates—For several reasons, the
upper-limit unit risk estimate based on animal bioassays is only an approxi-
mate indication of the real risk in populations exposed to known carcinogen
concentrations. First, there may be important differences in target site
susceptibility, immunological responses, hormone function., dietary factors,
and disease. In addition, human populations are variable with respect to
genetic constitution and diet, living environment, activity patterns, and other
cultural factors.
The unit risk estimate can give a rough indication of the relative carci-
nogenic potency of a given agent compared with other carcinogens. The compara-
tive 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, although ordinarily, the risk should be independent of route of expo-
sure except in special circumstances, for example, nasal or lung carcinomas
with inhalation exposure, or forestomach tumors with gavage administration.
The quantitative aspect of carcinogen risk assessment is included here
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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 for TCI presented in this chapter should not be regarded
as immutable representations of the true cancer risks; 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. Table 8-35 gives the
upper-limit slope estimates, q^, from the linearized multistage model.
The slope estimate can be used to compare the relative carcinogenic potency of
TCI to that of other potential human carcinogens. The slope is also used to
calculate upper-bound incremental risks at low levels of exposure.
8.4 RISK ESTIMATION FROM EPIDEMIOLOGIC DATA
8.4.1 Selection of Epidemiologic Data Sets
Whenever possible, human data are used in preference to animal bioassay
data. If epidemiologic studies and sufficiently valid exposure information are
available for a compound, they may be used in several ways. If these data show
a carcinogenic effect, they are analyzed to give an estimate of the linear de-
pendence of cancer rates on lifetime average dose. If no carcinogenic effects
are seen in the epidemiologic studies when positive animal evidence is availa-
ble, the assumption is made that a risk does exist, but that it is too small to
be observable in the epidemiologic data. An upper limit to the cancer incidence
is then calculated, assuming hypothetically that the true incidence is below
8-126
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the level of detection in a human cohort of the size studied.
Very little information exists that can be used for extrapolating 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. Thus, the Axelson et
al. (1978) study provides data that may be used to calculate 95% upper-limit
estimates on relative risk, although this study does not provide evidence for
the carcinogenicity of TCI. Calculations may be based on the subcohort data
from the Axelson et al. study, with particular focus on the low-exposure group,
which had a latency period of at least 10 years.
8.4.2 Description of Risk Model
In human studies, the response is measured in terms of the relative risk
of the exposed cohort as compared to the control group. The mathematical model
employed assumes that for low exposures the lifetime probability of death from
a specific 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, 6^, 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
8-127
-------
= P = A + BH (xl + X2)
P0 A + BH X!
or
RP0 = A + BH (xi + x2)
where x^ = lifetime average daily exposure to the agent for the general popu-
lation, X£ = 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 + BH x^ and rearranging gives
BH = P0 (R - D/x2
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 U.S. Vital Statistics tables using the life table
methodology.
8.4.3 Calculation of Upper Limits of Risk
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
epidemiologic studies for providing upper limits when animal studies are
positive. Such use of negative epidemiologic studies can modify estimates of
potency from the positive animal studies.
Table 8-36 shows 95% upper-limit estimates on relative risk, as derived
from data in Table 8-24 obtained by Axelson et al. (1978). These upper-limit
estimates are calculated according to the following approach:
8-128
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TABLE 8-36. NUMBER OF WORKERS WITH CANCER DEATHS
Observed
Cohort X
Total cohort 11
Subcohort with
10-year latency
(high and low
exposure) 9
High exposure 3
Low exposure 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% upper limit
1.4
1.8
4.8
1.7
The 95% upper limit = u/expected number of cancer deaths, where u is the
solution to the sum of Poisson probabilities, as follows:
X
I e-" (u)1 -_ 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.
These upper-limit relative risks can be used to provide estimates of the
the upper-limit probability of death from cancer due to a lifetime exposure to
1 yg/rn^ of TCI. This upper-limit probability estimate is 1.7 x 10"^ per yg/m-*
of TCI in air, and it is approximately 10 times greater than the risk estimate
based on the NCI (1976) and NTP (1982) carcinogenicity gavage bioassays. In
showing these risk calculations below, the CA6 emphasizes that:
8-129
-------
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 period. Table 8-36 projects an upper-limit relative risk of 1.7.
Since "low exposure" referred to exposures not exceeding concentrations of 100
mg TCA per ml in urine on the average, which, according to Axelson et al.
(1978) corresponds to an ambient air exposure of no more than 30 ppm TCI in
air, 30 ppm is used as the 8-hour time-weighted average dose. This corresponds
to 30 ppm x 5,475 (yg/m3)/ppm - 1.64 x 105 yg/m3. A lower dose estimate
would provide an even higher upper-limit probability of death.
In the category of years exposed, in the absence of any other information,
10 years was chosen as a point assumed to be somewhere in the middle of the
range.
On the basis of the above information, the total lifetime dose of TCI to
the low-dose exposure workers is estimated as:
(0 ppm + 1.64 x 10s) x 240 days x 10 years = 7.7 x 103 yg/m3
2 365 days 70 years
The 1.64 x 105 yg/m3 TCI of the working day is averaged with the 0 yg/m3 of
the non-working day, assuming that half of the air breathed during a day will
8-130
-------
be during working hours. The value 7.7 x 10~3 yg/m3 is assumed to be a con-
tinuous exposure concentration. Thus, the upper-limit of the probability of
death from 1 yg/m3 TCI in air (continuous exposure) is estimated as:
p = Q - j = 0.19 (1.7- 1)1 , 1-7 x 1Q-5 /m3
d 7.7 x 103
where R = the relative risk, dj = 1 yg/m3, d2 = dose of workers, and PQ = the
background lifetime probability of death from all cancers.
Based on a comparison of this upper-limit estimate with that from the
animal studies, the Axelson et al . study is seen to provide no evidence that
the carcinogenicity potency of TCI is less for humans than for animals.
8.5 RELATIVE CARCINOGENIC POTENCY
8.5.1 Derivation
One of the uses of the concept of unit risk is to compare the relative
potencies of carcinogens. To estimate relative potency on a per mole basis,
the unit risk slope factor is multiplied by the molecular weight, and the
resulting number is expressed in terms of (mmol/kg/day)"1. 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 CAG as suspect carcinogens.
The actual data summarized by the histogram are presented in Table 8-37.
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.
8-131
-------
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i
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oo
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I I I I I I I I I I I I I I I I I | | I
x
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-------
TABLE 8-37. RELATIVE CARCINOGENIC POTENCIES AMONG 54 CHEMICALS EVALUATED BY THE CARCINOGEN ASSESSMENT GROUP
AS SUSPECT HUMAN CARCINOGENS
oo
CO
Level
of evidence3
Compounds
Acrylonitrile
Aflatoxin Bj
Aldrin
Ally! chloride
Arsenic
B[a]P
Benzene
Benzidene
Beryl 1 i urn
1,3-Butadiene
Cadmi urn
Carbon tetrachloride
Chlordane
CAS Number Humans '
107-13-1
1162-65-8
309-00-2
107-05-1
7440-38-2
50-32-8
71-43-2
92-87-5
7440-41-7
106-99-0
7440-43-9
56-23-5
57-74-9
L
L
I
S
I
S
S
L
I
L
I
I
Animal s
S
S
L
I
S
S
S
S
s
s
s
L
Grouping
based on
IARC
criteria
2A
2A
2B
1
2B
1
1
2A
28
2A
2B
3
SI opeb
(mg/kg/day)'1
0.24(W)
2900
11.4
1.19xlO-2
15(H)
11.5
2.9xlO-2(W)
234(W)
2.6
1.0xlO~1(I)
6.1(W)
1.30X10-1
1.61
7
Molecular
weight
53.1
312.3
369.4
76.5
149.8
252.3
78
184.2
9
54.1
112.4
153.8
409.8
Potency
index0
1x10+1
9xlO+5
4x1 0+3
9x10"!
2xlO+3
3xlO+3
2x10°
4xlO+4
2xlO+1
5x10°
7xlO+2
2xlO+1
7x10+2
Order of
magnitude
(Iog10
index)
+1
+6
+4
0
+3
+3
0
+5
+1
+1
+3
+1
+3
-------
TABLE 8-37. (continued)
CD
I—*
CA>
Level
of evidence3
Compounds
Chlorinated ethanes
1,2-Dichloroethane
hexachloroethane
CAS Number
107-06-2
67-72-1
1,1,2,2-Tetrachloroethane 79-34-5
1,1,2-Trichloroethane
Chloroform
Chromium VI
DDT
Dichlorobenzidine
1,1-Dichloroethylene
(Vinyl idene chloride)
Dichloromethane
(Methylene chloride)
Dieldrin
2,4-Dinitrotoluene
Diphenylhydrazine
Epichlorohydrin
Bis(2-chloroethyl )ether
79-00-5
67-66-3
7440-47-3
50-29-3
91-94-1
75-35-4
75-09-2
60-57-1
121-14-2
122-66-7
106-89-8
111-44-4
Humans
I
I
I
I
I
S
I
I
I
I
I
I
I
I
I
An imal s
S
L
L
L
S
S
S
S
L
L
S
S
S
s
s
Grouping
based on
IARC
criteria
2B
3
3
3
2B
1
28
2B
3
3
2B
2B
28
2B
28
SI opeb
(mg/kg/day)'1
9.1x10-2
1.42x10-2
0.20
5.73x10-2
7x10-2
41(W)
0.34
1.69
1.16(1)
6.3x10-4(1)
30.4
0.31
0.77
9.9x10-3
1.14
Molecul ar
weight
98.9
236.7
167.9
133.4
119.4
100
354.5
253.1
97
84.9
380.9
182
180
92.5
143
Potency
index0
9x10°
3x10°
3x10+1
8x10°
8x10°
4x10+3
1x10+2
4x10+2
1x10+2
5x10-2
1x10+4
6xlO+1
1x10+2
9x10-!
2x10+2
Order of
magnitude
(]og10
index)
+1
0
+1
+ 1
+1
+4
+2
v +3
+2
-1
+4
+2
+2
0
+2
-------
TABLE 8-37. (continued)
CO
co
en
Level
of evidence3
Compounds
Bis(chloromethyl ) ether
Ethyl ene dibromide (EDB)
Ethyl ene oxide
Heptachlor
Hexachlorobenzene
Hexachl orobutadi ene
Hexachlorocyclohexane
technical grade
alpha isomer
beta isomer
gamma isomer
Hexachl orodi benzodi oxi n
Nickel
Nitros amines
Dimethyl nitrosamine
Di ethyl nitrosamine
Dibutylnitrosamine
N-nitrosopyrrol idine
N-nitroso-N-ethylurea
CAS Number
542-88-1
106-93-4
75-21-8
76-44-8
118-74-1
87-68-3
319-84-6
319-85-7
58-89-9
34465-46^8
7440-02-0
62-75-9
55-18-5
924-16-3
930-55-2
759-73-9
Humans
S
I
L
I
I
I
I
I
I
I
L
I
I
I
I
I
Animal s
S
S
S
S
S
L
S
L
L
S
S
S
S
S
S
S
Grouping
based on
IARC
criteria
1
2B
2A
2B
2B
3
2B
3
2B
2B
2A
2B
2B
2B
2B
28
SI opeb
(mg/kg/day)-l
9300(1)
41
3.5x10-1(1)
3.37
1.67
7.75x10-2
4.75
11.12
1.84
1.33
6.2xlO+3
1.15(W)
25.9(not by ql
43.5(not by qj
5.43
2.13
32.9
Molecul ar
weight
115
187.9
44.1
373.3
284.4
261
290.9
290.9
290.9
290.9
391
58.7
') 74.1
f) 102.1
' 158.2
100.2
117.1
Potency
index0
1x10+6
8x10+3
2x10+1
1x10+3
5x10+2
2x10+1
1x10+3
3xlO+3
5x10+2
4x10+2
2x10+6
7x10+1
2xlO+3
4xlO+3
i O
9xlO+2
2x10+2
4x10+3
Order of
magnitude
(Iog10
index)
+6
+4
+1
+3
+3
+1
+3
+3
+3
+3
+6
+2
+3
+4
+3
+2
+4
(continued on the following page)
-------
TABLE 8-37. (continued)
CO
(—'
co
Compounds
N-nitroso-N-methylurea
N-nitroso-diphenyl amine
PCBs
Phenols
2 ,4 ,6-Tr i chl orophenol
Tetrachl orodi benzo-
p-dioxin (TCDD)
Tetrachl oroethyl ene
Toxaphene
Tri chl oroethyl ene
Vinyl chloride
CAS Number
684-93-5
86-30-6
1336-36-3
88-06-2
1746-01-6
127-18-4
8001-35-2
79-01-6
75-01-4
of
Level
evidence3
Humans Animals
I
I
I
I
I
I
I
I
S
S.
S
S
S
S
L
S
L/S
S
Grouping
based on
IARC
criteria
2B
2B
28
2B
2B
3
2B
3/2B
1
SI opeb
(mg/kg/day)'1
302.6
4.92x10-3
4.34
1.99x10-2
1.56x10+5
5.1xlO-2
1.13
1.1x10-2
1.75x10-2(1)
Molecular
weight
103.1
198
324
197.4
322
165.8
414
131.4
62.5
Potency
index0
3x1 0+4
1x10°
lxlO+3
4x10°
5x1 0+7
8x10°
5xlO+2
1x10°
1x10°
Order of
magnitude
(Io9in
index)
+4
0
+3
+1
+8
+1
+3
0
0
aS = Sufficient evidence; L = Limited evidence; I = Inadequate evidence.
^Animal slopes are 95% upper-bound slopes based on the linearized multistage model. They are calculated based on
animal oral studies, except for those indicated by I (animal inhalation), w (human occupational exposure), and H
(human drinking water exposure). Human slopes are point estimates based on the linear nonthreshold model. Not all
of the carcinogenic potencies presented in this table represent the same degree of certainty. All are subject to
change as new evidence becomes available. The slope value is an upper bound in the sense that the true value (which
is unknown) is not likely to exceed the upper bound and may be much lower, with a lower bound approaching zero.
Thus, the use of the slope estimate in risk evaluations requires an appreciation for the implication of the upper
bound concept as well as the "weight of evidence" for the likelihood that the substance is a human carcinogen.
cThe potency index is a rounded-off slope in (mmol/kg/day)-1 and is calculated by multiplying the slopes in
(mg/kg/day)"1 by the molecular weight of the compound.
-------
8.5.2 Potency Index
The potency index for TCI based on mouse hepatocellular carcinomas in
the NTP and NCI gavage studies is 1.4 x 10°. This is derived as follows: the
mean slope estimate from both studies, 1.1 x 10~2 (mg/kg/day)~l, is multiplied
by the molecular weight of 131.4 to give a potency index of 1.4 x 10°. Round-
ing off to the nearest order of magnitude gives a value of 1()0, which is the
scale presented on the horizontal axis of Figure 8-15. The index of 1.4 x 10°
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 uncertain-
ties involved in comparing estimates of potency for a number of chemicals based
on different routes of exposure in different species, using studies whose
quality varies widely. All of the indices presented here are based on estimates
of low-dose risk, using linear extrapolation from the observable range. These
indices may not be appropriate for the comparison of potencies if linearity
does not exist at the low-dose range, or if comparison is to be made at the
high-dose range. If the latter is the case, then an index other than the one
calculated above may be more appropriate.
8.6 SUMMARY
8.6.1 Qualitative Asssessment
8.6.1.1 Animal Studies —In a 90-week carcinogenicity 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, statisti-
cally significant increases in the incidence of hepatocellular carcinomas in
male and female mice compared to corn oil controls were found (NCI, 1976).
Statistically significant increases in hepatocellular carcinoma incidence in
8-137
-------
treated male and female mice compared to vehicle-control mice were also ob-
served 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 (NTP, 1982). In effect, this repeat study indicated that epoxide
stabilizers were not necessary factors in the TCI-induced increases in hepa-
tocellular carcinoma incidence in male and female B6C3F1 mice under the con-
ditions of these bioassays. It is noted that spontaneous hepatocellular tumor
formation occurs in the B6C3F1 mouse strain, particularly in the male.
A 110-week carcinogenicity study with male and female Osborne-Mendel rats
given technical grade, epoxide-stabilized TCI in corn oil by gavage at esti-
mated 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
terminal sacrifice (3/16) was calculated to be a statistically signifi-
cant 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 carcinogenic 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
8-138
-------
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/week 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 sponta-
neous incidence of malignant lymphomas in controls was high (30%), and it was
postulated in the study report that the increased incidence of malignant lym-
phomas in female mice could have been a result of an immunosuppressive response
capable 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
8-139
-------
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 puri-
fied 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
subcutaneous 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 carci-
nogenicity 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 carcinogenicity.
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 speci-
fied. Exposure of newborn Wistar rats for 10 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
8-140
-------
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 meta-
bolite 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 activ-
ity at estimated maximum tolerated doses.
8.6.1.2 Cell Transformation Studies—Cell 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 end point for predic-
ting carcinogenic activity; however, it is not certain whether these agents
elicited 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
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evaluation 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 (DiPaolo
and Doniger, 1982).
8.6.1.3 Human Studies—Three cohort studies of workers exposed to TCI did not
find that these workers experienced an excess risk of cancer (Axelson et al.,
1978; Tola et al., 1980; 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.
A case-control study of malignant lymphoma cases provided some suggestion
of an association of TCI exposure with malignant lymphoma (Hardell et al.,
1981). However, failure by the authors to adjust for age and the possibility
of inaccurate and biased exposure classification limit the usefulness of the
results. A study of 56 liver cancer cases found no evidence of TCI exposure
(Novotna et al., 1979); however, no controls were used in the study.
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. The author provided no indication of the
probability of finding a liver cancer case who had been an employee of the
plant and had been exposed to TCI. Also, the author provided no information on
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the distribution of workers at the plant exposed to TCI by length and time of
exposure. Furthermore, persons with liver cancer who may have worked at the
•plant but 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.
In summary, the epidemiologic data are inadequate for evaluating the car-
cinogenicity of TCI.
8.6.2 Quantitative Assessment
Four sets of gavage bioassay data on hepatocellular carcinomas in male
and female mice (NTP, 1982 and NCI, 1976) are used to calculate the upper-bound
slope estimate for TCI from the linearized multistage model. Since the upper-
bound slope estimates calculated on the basis of these data sets are comparable,
ranging from 5.8 x 10~3 to 1.9 x 10~2 mg/kg/day, the geometric mean, 1.1 x 10-2
mg/kg/day is used to calculate the incremental lifetime cancer risk (i.e., unit
risk) due to a unit exposure of trichloroethylene in drinking water and in air.
The upper-bound estimate of the cancer risk due to 1 yg/L of TCI in drinking
water is 3.2 x 10~7. The upper-bound estimate of the cancer risk due to 1 yg/
m^ of TCI in air is 1.3 x 10'^. The relevant information on metabolism and
kinetics for TCI by oral and inhalation exposures has been used to calculate
the unit risks for drinking water and air.
None of the epidemiologic studies reviewed in this report provide posi-
tive evidence from which to estimate a unit risk for exposure to TCI. As an
alternative, an upper-bound estimate has been calculated from one negative
study. The calculation on the basis of human data results in a greater risk
estimate than the estimate from animal data and thus does not appear to con-
tradict the risk estimate calculated from the animal data.
The potency index of TCI is 1 x 10°, ranking it in the lowest quartile of
54 chemicals that the CA6 has evaluated as suspect carcinogens.
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8.7 CONCLUSIONS
Evidence reviewed in this document for the carcinogenicity of TCI in
experimental animals includes: increases in the incidences of hepatocellular
carcinoma in male and female B6C3F1 mice (three studies), malignant lymphoma in
female Han: NMRI mice, and renal adenocarcinoma in male Fischer 344 rats. 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 role of
epoxide stabilizers in the induction of hepatocellular carcinomas. Similar
carcinogenic responses were observed in both studies. However, as discussed
earlier in this chapter, a third study in B6C3F1 mice, the inhalation study,
was weakened by deficiencies in its conduct. In the inhalation study in
Han:NMRI mice, there was a 30% incidence of spontaneous lymphoma in control
mice and the possibility of an indirect effect in treated mice; the gavage
treatment study in Fischer 344 rats was considered by the study's sponsor, NTP,
to be inadequate for a judgment of TCI carcinogenicity because of experimental
deficiences (See Section 8.1).
While six epidemiologic studies or related surveys have focused on TCI
exposure, the total body of evidence is considered to be inadequate to evaluate
the carcinogenic potential. Three cohort studies and two surveys showed no
excess risk of cancer resulting from exposure to TCI; however, in each case,
study deficiencies limited their usefulness and/or diminished their sensitivity
to detect a response. A case control study with a suggestive association
between exposure and malignant lymphoma has flaws which limit the usefulness
of the results.
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
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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, i.e., it requires
metabolic activation. 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 metaolite(s) can
react with cellular protein macromolecules in vivo and in vitro and with
exogenous DNA and RNA in vitro. There are no known differences among species
with regard to metabolic pathways or metabolic profiles for TCI. A study of
purified radio-labeled TCI binding to DNA in liver in vivo in male B6C3F1 mice
given a gavage dose of 1,200 mg/kg of TCI - 14C(7,500 yCi/animal) suggested
a low order of binding. Cell transformation with high doses of TCI oxide, a
putative direct-acting carcinogenic metabolite of TCI, has been observed j_n_
vitro; however, animal studies of initiating and complete carcinogenic poten-
tial of TCI oxide were negative.
TCI is positive for the induction of malignant tumors of the liver in both
male and female B6C3F1 mice in multiple studies. This constitutes a signal that
TCI might be carcinogenic in humans. The positive lymphoma response in females
of the Han:NMRI mouse strain does qualify as a separate strain, but that
result is merely 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. The kidney tumor results in the NTP rat study
are not strong indications 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 effect may be taking place.
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The TCI carcinogenicity results could be classified under the criteria of
the International Agency for Research on Cancer (IARC) as either "sufficient"
or "limited" depending on which of the differing current scientific views
about chlorinated organic compound induction of liver tumors in mice is chosen.
Since there are no adequate epidenriologic data in humans, the overall ranking
of TCI under the criteria of the IARC depends primarily upon the position taken
regarding the mouse liver tumor. Thus, the overall ranking could be either
Group 2B or Group 3. The more conservative public health view would regard
TCI as a probable human carcinogen (Group 2B), but there is also scientific
sentiment for regarding TCI as an agent that cannot be classified as to its
carcinogenicity for humans (Group 3).
The U.S. Environmental Protection Agency's Proposed Guidelines for Carci-
nogen Assessment (U.S. EPA, 1984) regard the mouse-liver-tumor-only response
as sufficient evidence for carcinogenicity in animals. A variety of factors
such as lack of mutagenicity, low malignancy, and other such measures, if
present, would downgrade this evidence; however, downgrading would not occur
with the TCI results. Thus, based on EPA's proposed cancer guidelines, the
overall evidence for TCI results in a classification of B2, i.e., a probable
human carcinogen.
Additional carcinogenicity studies on purified TCI (no detectable epox-
ides) including those sponsored by the NTP with lifetime gavage treatment in
four strains of rats (Osborne-Mendel, Marshall 540, August 28807, and ACI)
and those performed in the laboratories of Dr. Caesar Maltoni with lifetime
inhalation exposure in B6C3F1 mice, Swiss mice, and Sprague-Dawley rats are
not finalized or published. The Fukuda et al. (1983) and Henschler et al.
(1984) studies were not available at the time this document was submitted for
public comment and Science Advisory Board review; thus, these studies have not
8-146
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been included in the final document but will be incorporated into any updates
of this Health Assessment Document.
Gavage studies on hepatocellular carcinomas in mice provide a basis to
culate incremental lifetime cancer risks due to exposure to TCI. The develop-
ment of the risk estimates is for the purpose of evaluating the "what-if"
question: If TCI is carcinogenic in humans, what is the possible magnitude of
the public health impact? The upper-bound estimate of the incremental cancer
risk due to 1 yg/L of TCI in drinking water is 3.2 x 10~7. The upper-bound
estimate of the incremental cancer risk due to 1 yg/m^ of TCI in air is
1.3 x 10~6. The upper-bound nature of these estimates is such that the true
risk is not likely to exceed this value and may be lower.
The potency index of TCI is 1 x 10^, ranking it in the lowest quartile of
54 chemicals that the CAG has evaluated as suspect carcinogens.
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