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293 00891 93 379

This is to certify that the

dissertation entitled

TOXICOLOGY OF ORAL EXPOSURE TO TRICl-ILOROETHYLENE
0N MATERNAL REPRODUCTIVE FUNCTION IN NICE

presented by

NEAL COLEMAN COSBY

has been accepted towards fulfillment
of the requirements for

PH.D. degree in zoomev

 

 

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Majorrepofss

 

Date APRIL 23, 1991

 

 

 

 

 

 

 

 

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MSU Is An Affirmative ActiorVEqueI Opportunlty Institution

emunMI

 

 

TOXICOLOGY OF ORAL EXPOSURE TO TRICHLOROETHYLENE ,
ON MATERNAL REPRODUCTIVE FUNCTION IN MICE

By

Neal Coleman Cosby

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Zoology

1991

ABSTRACT

TOXICOLOGY OF ORAL EXPOSURE TO TRICHLOROETHYLENE
ON MATERNAL REPRODUCTIVE FUNCTION IN MICE

By

Neal Coleman Cosby

Trichloroethylene (TCE), an industrial solvent, is a soil and ground water contami-
nant found across the United States. The metabolism and carcinogenic potential of TCE have
been studied extensively in the past 15 years yet there is little information on the chemical’s
possible effects on reproduction. Recent studies have analyzed the metabolism of TCE in
mammals and show a species-specific production for the pathways of metabolites. No
reference to the reproductive effects in mice of TCE by oral administration exist in the
literature. In this study, female B6D2F1 mice were gavaged from Days 1 to 5, 6 to 10, or 11
to 15 (Day 1 = vaginal plug) with TCE in corn oil at O, l/ 10 and 1/ 100 of the oral LDSO. Weights
of mice were recorded throughout pregnancy and weaning. At this time, the maternal mice
were killed, and the livers and kidneys were weighed and preserved in 10% buffered
formalin. Litters were counted, sexed, and reduced to 6 on the day of birth. Litters were
weighed and measured for crown-rump length until weaning on Day 21 and reduced to 4 pups
(2 femalesz2 males) on this day, and these animals were allowed to develop to 6 weeks of age.
At this time, a minimum of 2 litters from each dose were killed and gonads removed, weighed,
and preserved in Bouin ’ s fixative. Litters were also assessed for developmental abnormalities
by observing for eye openings and lower incisor eruption, encephalies or spina bifidas, and
extra or missing digits. No maternal or reproductive effect of TCE was seen at either dose
level. TCE, administered consecutively on Days 1 to 5, 6 to 10, or 1 1 to 15 of pregnancy, does
not appear to be a potential reproductive toxin up to 1/10 the oral LDSO.

In a second series of studies, TCE and its metabolites dichloroacetic acid (DCA),

trichloroacetic acid (T CAA), and trichloroethanol (TCOH), were added to culture media to
assess the toxic effects on in vitro fertilization (IVF) in mice. Cumulus masses were collected
from superovulated B6D2F1 female mice and placed in medium containing one or a
combination of the above chemicals. A single epididymus was collected from mature
B6D2Fl male mice, the sperm were capacitated, and cultured with one cumulus mass in a 5%
C02 incubator. After a 24 hour period, oocytes were assessed for fertilization. TCE, up to 1000
ppm, showed no effect on IVF. DCA, TCAA, and TCOH each showed a dose-related effect
up to 1000 ppm. TCAA and TCOH did not exhibit a synergistic effect.

, To Paula

For all the reasons we know, and some only she knows.

ii

ACKNOWLEDGMENTS
I extend sincere thanks to my graduate committee: Drs. Richard Aulerich, Lynwood

G. Clemens, Richard Hill, and W Richard Dukelow. I appreciate the friendship of Bonnie,
Linda, and Gary, fellow “ERUs” through it all.

iii

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................ vi
LIST OF FIGURES .......................................................................................................... viii
INTRODUCTION ............................................................................................................ 1
LITERATURE REVIEW ................................................................................................. 4
Mouse Reproduction and Development. ................................................................. 4
Trichloroethylene .................................................................................................... 9
History and Use ....................................................................................................... 9
Metabolism .............................................................................................................. 12
Modeling Experiments ............................................................................................ 21
Carcinogenicity ....................................................................................................... 25
Mutagenicity ............................................................................................................ 29
Reproductive Toxicology ........................................................................................ 34
Vehicle ..................................................................................................................... 35
Fasting ...................................................................................................................... 37
Whitten Effect .......................................................................................................... 38
MATERIALS AND METHODS ...................................................................................... 39
In Vivo Fertilization Trials ...................................................................................... 39
Animals ....................................................... _. ..................................................... 39
Trichloroethylene .............................................................................................. 40
Dose ................................................................................................................... 40
Administration ................................................................................................... 40
Synchronous Pregnancy Induction .................................................................... 41

Data Collection ................................................................ 41
Solutions ............................................................................................................ 42
Statistical Analyses ............................................................................................ 42

In Vitro Fertilization Trials ..................................................................................... 42

' Animals ............................................................................................................. 42

iv

Trichloroethylene and Metabolites ................................................................... 42

Dose .................................................................................................................. 44

Administration .................................................................................................. 45

Superovulation .................................................................................................. 45

Culture Media. .................................................................................................. 45

Gamete Recovery .............................................................................................. 47

In Vitro Fertilization ......................................................................................... 47

Hoescht Nuclear Stain 33258 ............................................................................ 47

Statistical Anaylses ........................................................................................... 48

RESULTS ......................................................................................................................... 49
In Vivo Fertilization Trials ...................................................................................... 49 \

Synchronous Pregnancy Induction .................................................................... 49

Parturition ......................................................................................................... 49

Organ Weights .................................................................................................. 49

Litter Growth Parameters .................................................................................. 56

Litter Gonad Weights ........................................................................................ 56

In Vitro Fertilization Trials ..................................................................................... 56

Fertilization Rates for TCE and Metabolites .................................................... 56

DISCUSSION ................................................................................................................... 65

SUMMARY AND CONCLUSIONS ............................................................................... 72

BIBLIOGRAPHY ............................................................................................................ 73

APPENDICES .................................................................................................................. 87

Appendix A .............................................................................................................. 87

Appendix B .............................................................................................................. 88

LIST OF TABLES

Table Page
1. The physical properties of trichloroethylene (TCE) ................................................. 10
2. Components of two culture media used in the in Vitro fertilization trials of

IO.

trichloroethylene (TCE), trichloroacetic acid (TCAA), trichloroethanol
(TCOH), and dichloroacetic acid (DCA) .................................................................. 46

Fertility Index (FI) per replicate for female B6D2F1 mice used in the
in vivo fertilization study of trichloroethylene (T CE) ......... , .................................... 50

Gestation Index (GI) per replicate for female B6D2F] mice used in the
in vivo fertilization study of trichloroethylene (T CE) .............................................. 51

Maternal body weight of B6D2F] mice exposed to trichloroethylene (T CE)
by gavage on Days 1 to 5 of pregnancy .................................................................... 52

Maternal body weight of B6D2F 1 mice exposed to trichloroethylene (T CE)
by gavage on Days 6 to 10 of pregnancy .................................................................. 53

Maternal body weight of B6D2F 1 mice exposed to trichloroethylene (T CE)
by gavage on Days 11 to 15 of pregnancy ................................................................ 54

Liver and kidney weights of female B6D2F1 mice exposed to trichloro-
ethylene (TCE) by gavage during Days 1 to 5, Days 6 to 10, and Days 11
to 15 of pregnancy .................................................................................................... 55

Summary of data on litter size of female B6D2Fl mice exposed to trichloro-
ethylene (TCE) by gavage during Days 1 to 5, Days 6 to 10, and Days 11
to 15 of pregnancy .................................................................................................... 57

Mean pup weight and crown-rump (CR) length averaged per dam of B6D2F1
mice exposed to trichloroethylene (TCE) by gavage on Days 1 to 5
of pregnancy .............................................................................................................. 58

Table

11.

12.

13.

14.

15.

16.

17.

18.

Pup weight and crown-rump (CR) length averaged per dam of B6D2Fl
mice exposed to trichloroethylene ('1' CE) by gavage on Days 6 to 10

of pregnancy .............................................................................................................. 59

Pup weight and crown-rump (CR) length averaged per dam of B6D2F]
mice exposed to trichloroethylene (T CE) by gavage on Days 11 to 15

of pregnancy .............................................................................................................. 60

Relative gonad weights of a subset of littermates averaged per B6D2F]
dam at 6 weeks exposed to trichloroethylene (TCE) by gavage on Days 1

to 5, Days 6 to 10, and Days 11 to 15 of gestation ................................................... 61

In vitro fertilization of B6D2Fl mouse gametes in BMOC-3 culture

medium with trichloroethyene (T CE) ....................................................................... 62

In vitro fertilization of B6D2F] mouse gametes in BMOC-3 culture

medium with trichloroacetic acid (TCAA) ............................................................... 62

In vitro fertilization of B6D2Fl mouse gametes in BMOC-3 culture

medium with dichloroacetic acid (DCA) .................................................................. 63

In vitro fertilization of B6D2F1 mouse gametes in BMOC-3 culture
medium with trichloroacetic acid (TCAA), trichloroethanol (TCOH),

or both ....................................................................................................................... 64

Brand and trade names of trichloroethylene (T CE) .................................................. 86

vii

LIST OF FIGURES

Figure Page

1. Summary diagram of the proposed intermediary metabolism of
trichloroethylene (T CE) ............................................................................................ 13

2. Diagram illustrating the experimental protocol and data collection time
points for the in vivo fertilization trials .................................................................... 43

viii

INTRODUCTION

Trichloroethylene, commonly abbreviated as TRI or TCE in the literature, is a clear,
odorless liquid and has the chemical formula CZHCL. TCE has been used in industry as a
solvent and extractant, and it has been used in medicine as an inhalation anesthetic and for
the treatment of neuralgia. TCE is a halogenated hydrocarbon of low molecular weight and
poor water solubility. As such, TCE is referred to as a volatile organic compound (VOC). In
recent years, VOCs have been considered possible health hazards due to the levels of
production and waste in the United States and other industrialized countries.

Annual production of TCE in the United States in 1980 approximated 234,000 metric
tons (EPA, 1980). The volatilization in TCE production and use is the major source of
environmental levels of this compound. TCE has been detected in air, food, municipal
waters, ground water, rivers, seas, aquatic organisms, human breath, and human tissues. TCE
is not expected to persist in the environment because of its rapid photooxidation in air, its low
water solubility, and its volatility. Nevertheless, recent reviews illustrate the widespread use
and presence of TCE.

TCE, as most foreign compounds or xenobiotics, is metabolized by the mammalian
system if inhaled or ingested. Enzyme—catalyzed reactions occur in specific organs of the
animal to detoxify the xenobiotic. These reactions render the compound more water soluble,
and in some cases conjugate the compound for excretion. The major intermediate metabolite
of TCE is chloral or its hydrate. Chloral is converted to trichloroacetic acid (TCAA) and
trichloroethanol (TCOH). The levels and rates of formation of these compounds suggest
differential metabolism of TCE in mammals. Also, TCOH can be converted to TCAA. TCOH

can be conjugated to glucuronide and exhibits a relatively short excretion half-life. On the
other hand, TCAA persists much longer due probably to its ability to bind to protein and to
be converted from TCOH.

Research in the past decade has revealed additional metabolites that are species-
specific, such as dichloroacetic acid and a cysteine derivative in the mouse, generated
through metabolic pathways other than that for chloral.

A review of studies on TCE presents mixed results on the compound’s ability to act
as a carcinogen or mutagen. Most carcinogen studies have been inconclusive prompting the
International Agency for Research on Cancer (IARC) to classify TCE as a group 3 carcinogen
for inadequate evidence. The mutagenicity of TCE varies depending upon the test system
used (in vivo or in vitro) as the major factor. Briefly, when tested in animals for mutagenic
properties by either inhalation or oral administration, TCE only elicited tissue damage,
mostly in the liver, at high doses. When tested in vitro using liver fraction (microsome)
assays, TCE almost uniformly showed dose-related effects on test cells. In vivo and in vitro
toxicity testing differ fundamentally, and therefore, make direct comparisons of the two
testing systems impossible.

Reproduction is considered sensitive to environmental factors be they physical,
chemical, or emotional. Reproduction, the perpetuation of the species, is considered also to
be the single most important goal of a species. For any chemical compound, carcinogen,
mutagen or neither, it is important to know if and how it affects reproduction. It is well known
that some chemicals adversely affect reproduction: some chemicals are used for this single
purpose in humans and lower animals to control reproduction. Relatively little information
exists'on the effects of VOCs on reproduction in mammals. This study is proposed to test the
effects of orally ingested (gavage) TCE on pregnant mice for 5 consecutive days beginning
on either Day 1, Day 6, or Day 11.

Murine gestation is strain dependent, and in B6D2F] mice it predictably requires 19

days. In this study, Day 1 of pregnancy is indicated by the presence of a vaginal plug the
morning after placement with a male. The first 5 days of gestation are characterized by
sequential cleavage of the fertilized oocyte that generates a hatched blastocyst ready to invade
and attach to the maternal uterine endometrium. Implantation is completed on Day 5 and
from this point until parturition, the fetus remains physically connected to the mother. Days
6 to 10 are characterized by organogenesis. Traditionally, the fetus is thought to be the most
susceptible to toxic insult during this time although it has been shown that pre-implantation
insults are not always lethal and can result in serious detriment later in development
(Iannaconne et al., 1987). Finally, Days 11 to 15 are characterized by growth of the fetus.
These time periods were chosen in an effort to isolate the developmental time of detriment
if one is seen. Only pregnant female (F1) mice were treated, and they were mated to normal,
fertile males.

Additional experiments were undertaken using TCE and the metabolites DCA,
TCAA, and TCOH, to assess their effects on IVF of mouse gametes. Both oocytes and
spermatozoa are specialized cells inherently vital to the reproductive process. In the in vivo
studies, only fertilized oocytes within the pregnant females possibly endured a toxic insult
whereas in the in vitro studies, both oocytes and spermatozoa were challenged with the given
compound prior to fertilization. Both the parent compound, TCE, and its metabolites, DCA,
TCAA, and TCOH, were included in the in vitro studies as it was reasoned that gametes in
vivo would be exposed to the products of TCE metabolism by the first pass effect of the liver.

Finally, it is the hope of any experimenter to learn any new information, however
small and seemingly insignificant, in the course of any experiment. This work holds no

smaller ambition.

LITERATURE REVIEW

Mouse Reproduction and Development

Female mice exhibit a four to five day estrous cycle, and are receptive to mating
during estrus in the dark period around the time of ovulation. Secretions of the prostate,
seminal vesicles and coagulating glands of the male form a vaginal or copulatory plug in the
female. Presence of a vaginal plug indicates Day 1 of pregnancy.

Ovulated mouse oocytes are arrested at metaphase of the second meiotic division.
Activation, or release from this block, is triggered by a fertilizing sperrnatozoon. Ejaculation
occurs directly into the uterus but few sperm reach the ampullary region of the oviduct, the
site of fertilization. The fertilized (activated) oocyte (or zygote or embryo) initiates develop-
ment and remains a free entity within the lumen of the oviduct until contact is established with
the uterine epithelium. This is the preimplantation period. Four or five cleavage divisions
occur in the oviduct and the embryo enters the uterus 68 to 72 hours after ovulation at the
morula stage. After the first division, the embryo cleaves about every eleven hours until
implantation. The interval between ovulation and the first division is variable; the interval
between subsequent divisions is relatively constant.

Implantation or nidation is the establishment of physical contact between the embryo
and the uterus. Embryonic and maternal tissues join to form a placenta. Implantation is
directly under ovarian hormone control and is dependent upon the synchronization of
changes in both the embryo and the uterus. The events of implantation include spacing of the
blastocysts, loss of the zona pellucida, adhesion of the trophoblast to the uterine epithelium,

invasion of the endometrial stroma and decidualization. Recent work shows that in the

5

mouse, apoptosis, as opposed to necrosis, of the uterine epithelium occurs (Parr et al., 1987).
Apoptis is a form of cell death characterized by surface blebbing, shrinkage, and fragmen-
tation of the cells, condensation of the chromatin, and indentation and fragmentation of the
nuclei.

If ovulation occurs during proestrus on Day 1 of pregnancy, the embryo enters the
uterus on Day 3, continues dividing, forms a blastocyst and sheds the zona pellucida on Day
4, and trophoblast cells invade the uterine epithelium and decidualization is completed on
Day 5. Events associated with the embryos moving through the reproductive tract and
implantation in the uterus are under hormonal control. Recent work in other species indicates
that the process of maternal recognition of pregnancy depends upon discrete sequential
communication between the embryo and mother. Little information is known about maternal
recognition of pregnancy in the mouse. Implantation can be delayed if the mother is lactating
heavily or by other events affecting the hormonal balance.

Pregnancy, the period from fertilization to parturition, is approximately 19 days
depending upon the strain of mouse. The corpus luteum is required to maintain pregnancy in
the mouse. Establishment and continued maintenance of pregnancy is controlled by luteotro-
pic protein hormones of the pituitary interacting with the ovarian steriods. The placenta takes
over luteotropic activity around the eighth day of pregnancy. Prolactin, follicle stimulating
hormone (FSH) and luteinizing hormone (LH) are necessary for normal luteal function
during early pregnancy (Whittingham and Wood, 1983). .

A dramatic increase in embryo growth rate follows implantation, particularly in the
primitive ectoderm from which the fetus will deveIOp. Between Days 5 and 10, the basic body
plan is established within the primitive ectoderm. Mesoderm forms reiterated pairs of somite
blocks generating a segmented body pattern along the anterior-posterior axis. The neural
plate is induced and folds upward to become the neural tube. The placodes of the nose, ear

and eyes form. Neural crest cells migrate and the heart, circulatory system and limb buds

6

form. The genes controlling differentiation and morphogenesis of the adult organs play
important roles during this period. Between the tenth and fifteenth day of gestation, female
and male germ cells enter the genital ridge and the gonads become distinguishable at
approximately Day 12.5. Mitotic oogonia are seen between Day 10 and 13 and then entry
into meiosis occurs and proceeds in the female. In the male, protospermatogonia enter
mitotic arrest in G1 of the cell cycle and do not enter meiosis until later development (Hogan
et al., 1986).

Traditional belief in developmental toxicology is that any chemical insult prior to
implantation must result in the death of the embryo. However, recent research suggests that
this may be incorrect (See Iannaccone, 1984 and Iannaccone et al., 1987).

One potential source of disruption of human pregnancy is the exposure of women to
a toxic insult during very early periods of pregnancy. A paucity of data exists on the effects
of toxic exposure and early pregnancy in animals because of the traditional view that
preimplantation stages of development are refractory to the consequences of toxic insult. The
blastocyst stage of development has been shown to be sensitive to cyclophosophamide,
heavy metals and trypan blue (Spielman and Eibs, 1978). Actinomycin D prevents cell
cleavage but does not cause cell death until the blastocyst stage in mice (Epstein and Smith,
1978). Blastocysts are also more sensitive to methylmercury than morulae; methylmercury
interferes with development of the inner cell mass (Matsumoto and Spindle, 1982). Within
individual blastocysts, the inner cell mass and trophectoderm may have different sensitivities
to chemical insult. This has been demonstrated by Adams et a1. (1961) who administered
various chemicals during the preimplantation period and found that the inner cell mass was
affected more notably.

The deleterious effects of treatment during the preimplantation period range from
increased intrauterine mortality to fetal growth retardation and actual malformations. For

example, exposure to inhaled chloroform from Days 1 to 7 significantly impaired the ability

7

of female mice to maintain pregnancy but this was not significantly teratogenic (Murray et
al., 1979). Mean fetal body weight and crown-rump length were decreased significantly
among the surviving offspring.

Other studies illustrate the effects of a chemical challenge to preimplantation
development. Single i.p. injections of nickel chloride to pregnant mice on Day 1, 2, 3, 4, 5
or 6 of gestation resulted in decreased litter size, diminished fetal body weights, a larger
frequency of both early and late resorptions, and an increased frequency of stillborn and
abnormal fetuses, when compared with results in control groups (Storen g and Jonsen, 1981).

Takeuchi (1984) found that single i.p. injections of 10 mg/kg methylnitrosourea to
mice on Day 1, 2, 3 or 4 of gestation did not affect implantation but the mean fetal death rates
were significantly increased in treated groups compared with controls. The mean malformation
rates were also significantly increased in groups treated on Day 2, 3 or 4 as compared with
controls.

Cyproterone acetate treatment of pregnant mice (Eibs et al., 1982) on Day 2 resulted
in a significant increase in the rate of intrauterine mortality, exencephaly and heart
abnormalities, a dose-dependent increase in cleft palate and urinary tract abnormalities, and
a concentration-dependent decrease in fetal weight. Similar treatment with
medroxyprogesterone was followed by sporadic increase in dead and resorbed fetuses, a
decrease in fetal weight, and an increase in the rates of cleft palate and malformed or
abnormally developed fetuses (Eibs et al., 1982).

Storeng and Jonsen (1983) found a single i.p. injection of ricin to mice on Day 1, 2
or 3 of pregnancy had no effect on implantation frequency and the number of live fetuses.
However, the number of abnormal fetuses in the ricin-treated groups exceeded that of the
control groups, with exencephaly as one of the abnormalities. Exencephaly is believed to be
induced by some toxic agents during the period of organogenesis, but these workers

hypothesized it was due to an early effect of ricin on the preimplantation embryos with

subsequent expression in later stages of fetal development.

Various mechanisms could explain the deleterious effects of treatment during the
preimplantation period. Some chemicals may damage certain maternal tissues that are
necessary for the maintenance of normal embryonic development, or may cause reduced
maternal food intake. In either case retarded growth or malformed fetuses may result from
damaged maternal functions (Iannaccone et al., 1987).

To test whether cyclophosphamide interferes predominantly with the development of
the preimplantation embryo or with the decidualization of the uterus of the mother, rat
embryos from treated mothers were transferred to untreated recipients and embryos from
untreated mothers were transferred to pretreated foster mothers (Spielman and Eibs, 1978).
The results demonstrated that for treated embryos in untreated foster mothers, implantation
rate was unaffected but the percentage of live fetuses was significantly decreased and
resorption rate was significantly increased when compared with control groups.

In a study on the pharrnacokinetics of the herbicide 2,4,5-trichlorophenoxyacetic acid
(2,4,5-T) in pregnant mice, Bazare et a1. (1990) demonstrated a gestational status-dependence
of the chemical. There was an increase in the biological half-life and the area under the blood
concentration-time curve (AUC), and a decrease in clearance and total percent recovery.
Pregnant animals eliminated 2,4,5-T more slowly as gestation progressed, resulting in
potentially increased fetal exposure during the later stages of pregnancy.

A study on the effects of nitrous oxide on preimplantation mouse embryo cleavage
and development revealed that although brief exposure to the gas may be deleterious to
subsequent cleavage, the effect was highly dependent on the developmental stage at which
the exposure occurred. From 6 to 7 hours before the time of the first in vitro blastomere
division (2-cell to 4-cell), cleavage rate decreased with the presence of nitrous oxide. Four-
cell stage embryos and morulae similarly subjected to nitrous oxide were unaffected.

Developmental arrest at the 2-cell stage was the most common effect. Exposed embryos with

9

continued cleavage formed smaller blastocysts with less expanded blastocoel cavities
(Warren et al., 1990).

As TCE is a common and important volatile organic chemical found in dump sites
and ground water, the work reported in this dissertation is a study on the effect of this

chemical on the mammalian reproductive system.

Trichloroethylene
Although the term trichloroethylene (T CE) is the most common listing in referenced

journals, government publications, and review papers, according to Chemical Abstracts
Service (CAS) 9th Collective Index (1976) (Waters etal., 1977), the preferred name for this
compound is trichloroethene. This is also the name preferred by the International Union of
Pure and Applied Chemistry (IUPAC). A list of common names for TCE includes the
following: Acetylene trichloride, ethinyl trichloride, trichloroethylene, 1,1—dichloro-2-
chloroethylene, 1-ch10m2,2-dichloroethylene, and 1,1 ,2-trichloroethylene (—ethene). Brand
and trade names for TCE are listed in Appendix A.

Agency identifications of TCE include: Chemical Abstracts Service (CAS) registry
number 79-01-6, Wiswesser Line Notation (WIN) GYGUIG, and National Cancer Institute
(NCI) C 04546. The physical properties of TCE are given in Table 1.

History and Use

TCE was discovered by Fischer in 1864 during the preparation of tetrachloroethane
by reduction of hexachloroethane with hydrogen. The new volatile compound was patented
in 1906, and used as a degreaser for machinery and as a solvent for organic products. The first
medicinal use of TCE was in 1911 as a narcotic, dictated by analgesia produced in cases of
poisoning, e.g., to treat trigeminal neuralgia (Oljenick, 1928). Use of TCE as treatment for

trigeminal neuralgia has been discarded because this inhalant is not specific for the disease.

10

Table l: The physical properties of trichloroethylene (TCE).

 

Carbon = 18.28%
Hydrogen = 0.77%
Chloride = 80.95%

Boiling Range:
Chemical Family:
Density:
Description:

Distribution coefficients:

water/air
blood/air
plasma/air
fat/water
Evaporation rate:
Freezing range:
Melting point:
Molecular weight:
Reactivity:

Refractive index:
Solubility:
Soluble in:
Specific gravity:
Stability:

Vapor density:
Vapor pressure:
Viscosity:

87.14 to 87.55°C

halogen compound, aliphatic
1.4642 (d20/4)

non-flammable, colorless liquid

3 at 20°C; 1.6 at 37°C

18 to 22 at 20°C; 8 to 10 at 37°C
16 to 20 at 20°C

34.4

0.69 (CCl4 = 1.0)

-83 to -86.4°C

-73°C

131.4

reacts with sulfhydral groups; subject to autoxidation; not
hydrolyzed by water under normal conditions

n20/D = 1.4773

0.001 (w/w) in water at 25°C

acetone, benzene, chloroform, ethanol, ether, vegetable oils
1.465 at 204°C

slowly decomposed by light with formation of

hydrogen chloride

4.53 at 25°C

60 mmHg at 20°C; 94 mmHg at 30°C

0.55 centipoise

 

(Aviado et al., 1976; Waters et al., 1977)

11

The first use of TCE as a general anesthetic in a human was reported by Striker et a1. (1935).
TCE is an effective analgesic in the following clinical situations: for pregnant women early
in labor (Svandzhian, 1963), during proctoscopic examinations (Hovell et al., 1967), for
narcohypnosis (J oncev and Matinov, 1969) and for angina pectoris (Neel and Dupont, 1961).
The risks involved in the use of TCE as an anesthetic have not been completely evaluated
(Aviado et al., 1976).

During the 19705, the modern inhalational anesthetics halothane, methoxyflurane,
and fluroxene, replaced TCE for reasons such as the extent of muscular relaxation and the
incidence of postoperative complication (Chenowith, 1972).

The first commercial method of production of TCE was the dehydrochlorination of
acetylene-derived 1,1,2,2-tetrachloroethane by reaction with calcium hydroxide or by gas-
phase pyrolysis. Over 90% of the TCE produced in the United States today is prepared by the
chlorination and dehydrochlorination of ethylene dichloride. Output of TCE has been
decreasing since 1970, when a reported 277 million kg were produced by seven companies
(United States Tariff Commission, 197 2). This decrease in production was due primarily to
legislation restricting the use and emissions of TCE and to the closing of some acetylene-
based and ethylene-based plants. 7

Of the TCE consumed in the United States, about 90% is used for vapor degreasing
of metal parts in metalware factories, motors, machines, railway works and electrical works.
Additionally, 6% is used as a chain terminator for polyvinyl chloride production, and the
balance is used in a variety of household and commercial applications. The formation of
dichloroacetyl chloride and phosgene from TCE in the atmosphere of welding shops has
resulted in human deaths (Dahlberg and Myrin, 1971; Rinzema, 1971; Glass et al., 1971).

In the dry cleaning industry, TCE is one of the most widely used solvents. There is
strong evidence that TCE poisoning has occurred in this industry (Cary and Hepler, 1938;
Boume, 1959; Priete, 1965; Brancaccio et al., 1966; Veljanovski and Petrov, 1972).

12

TCE is also used in the boat, shoe, textile and chemical industries, as well as in
painting and enamelin g, photography, polishing of optical lenses, and extraction of caffeine
from coffee.

A partial list of selected products containing TCE has been made by Truhaut et al.
(1967) in France and by Gleason et a1. (1969) in the United States. Examples of these products
include cleaners for automobiles, buffing solutions, spot removers, rug cleaners, disinfec-
tants and deodorants, shampoos, and cleanser for false eyelashes and wigs. Two aerosol
products containing TCE and a fluorocarbon are a chimney sweep cleaner and a mildew

preventative (Aviado et al., 197 6).

Metabolism

Defense mechanisms for the ingestion of toxic substances can be divided into four
groups: elimination in unchanged form (e. g. , in expired air); chemical structural modification
(i.e., increased water solubility to facilitate excretion); structure modification for detoxifi-
cation regardless of whether water solubility is increased or not; and host defense mecha-
nisms (e. g., tolerance) (Norton, 1975).

Classically, enzymatic metabolic reactions have been considered to take place by four
chemical pathways: oxidative, reductive, hydrolytic and synthetic (conjugation), which
occur most often in the soluble, mitochondrial, or microsomal fractions of the liver. The first
three are classed as phase I reactions whereas conjugation is classed as a phase II reaction
(Waters et al., 1977). In the metabolism of TCE, almost all of these mechanisms are brought
into play. A summary diagram of the proposed intermediary metabolism of TCE is shown in
Figure 1. (See Waters et al., 1977; Hathway, 1980; Miller and Guengerich, 1983 for further
details on metabolism).

TCE can be absorbed into the animal system by inhalation, oral and dermal routes.

In humans, most exposure results from a combination of all three. TCE vapors are readily

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14
absorbed through the respiratory epithelium, absorption being greatest during the first
moments of exposure, then decreasing rapidly until equilibrium is reached in the blood. TCE
also readily passes across the gastrointestinal wall. Absorption of TCE through intact skin
is possible although cutaneous exposure is unlikely to result in absorption of dangerous
quantities of TCE (Sato and Nakajima, 1978). The route of absorption is key when
considering the animals’ capabilities to biotransform the compound. Thus, when adminis-
tered by the oral route, chemicals will be absorbed and transported first through the portal
circulation to the liver (Lukas et al., 1974). The lung, skin, and intestinal mucosa, although
generally less active than the liver, also have the capacity for biotransformation of foreign
organic chemicals (Alvares et al., 1973; Fouts, 1972; Lake et al., 1973; Wattenberg, 1972).

TCE is mostly water insoluble and, like most xenobiotics, is converted enzymatically
to more water soluble metabolites. Referring to Figure l, the first metabolic step involves the
oxidation of TCE through intermediates to chloral hydrate. It was determined that this step
is mediated by a reduced nicotinamide adenine dinuleotide phosphate/oxy gen (NADPH/Oz)
-dependent reaction taking place in liver microsomes (Leibman, 1965; Byington and
Leibman, 1965). Chemically, chloral hydrate formation requires migration of a chlorine atom
from the 2-carbon to the l-carbon position.

Using 36Cl-labeled TCE fed to rats, Daniel (1963) studied the metabolism of TCE by
determining the specific gravity of TCAA and TCOH. The specific gravity of these two
compounds was essentially the same as that of the administered TCE, which indicated an
intramolecular arrangement of TCE and, no exchange of chloride ions with the body chloride
ion pool.

Chloral hydrate, which is rapidly metabolized, has a short biological half-life in
humans, dogs and mice. Two possible pathways are reduction of chloral hydrate to
trichloroethanol (TCOH) by liver alcohol dehydrogenase (N ADH coenzyme), or oxidation
via “chloral hydrate dehydrogenase” (NAD coenzyme) to trichloroacetic acid (T CAA)

15

(Leibman, 1965). TCOH is most often conjugated with glucuronic acid before being excreted
in the urine as TCOH glucuronide or urochloric acid (Baselt, 1988; Muller et al., 1974).
Glucuronide formation takes place mainly in the liver. Thus the principal urinary metabolites
of TCE are TCAA, TCOH and TCOH glucuronide.

After inhalation exposure, about 16% of the absorbed TCE is exhaled unchanged. The
concentration of TCE in exhaled air declines very rapidly with time after discontinuation of
exposure, showing a multi-exponential curve of elimination. The remainder is gradually
metabolized and excreted in the urine and feces (Soucek and Vlachova, 1960; Nomiyama and
Nomiyama, 1971). High variations in reported values for mean lung retention exist (WHO,
1981). Some 51 to 64% is metabolized according to Bartonicek (1962) of which 45% is
TCOH glucuronide and 32% is TCAA in the urine with 8% of both in the feces. An additional
4% monochloroacetic acid is present in urine (Soucek and Vlachova, 1960). Others report
that 70 to 90% of the TCE is metabolized (Soucek and Vlachova, 1960; Fernandez, 1977;
Monster et a1. , 197 6; Muller et al., 1975). TCOH exhibits a relatively short half-life in humans
(approximately 12 to 13 hours) and excretion by the kidneys is rapid compared with a half-
life of 50 to 100 hours for TCAA because of the high protein-binding affinity of the acid
(Muller et al., 1974).

Following oral administration of 3“Cl-labeled TCE to rats, respiratory excretion ac-
counted for 72 to 85% of the dose and urinary excretion for 1 1 to 21% (Daniel, 1963). Urinary
metabolites were measured as TCOH (75%) and TCAA (25%). Radioactivity was detectable
18 days after dosing.

More sensitive investigations have yielded additional metabolites, not unexpectedly,
as well as novel molecular interactions, that account for some of the discrepancies between
the TCE administered and that excreted unchanged or in the form of the known metabolites.
Species differences have also been described (DeMatteis and Lock, 1987).

Formation of TCE-oxide is the first and rate-determining step in the metabolism of

16

TCE (Nomiyama and Nomiyama, 1979; Ikeda et al., 1980). The conversion of TCE-oxide
to chloral in vivo through intramolecular rearrangement of the C1 atom at 02 is remarkable,
as spontaneous transformation in aqueous solution of the moderately unstable TCE-oxide
through intramolecular rearrangement of a Cl atom at 01 gives exclusively dichloroacetic
acid, via dichloroacetyl chloride (Bonse et al., 1975). In studies with rabbit liver preparations,
epoxide formation occurred during microsomal oxidation of TCE (U ehleke et al., 1977b).
Apart from oxidation-reduction processes, chloral is relatively inert, and because of its lack
of protons, cannot yield imidazole-cyclization products with DNA nucleosides by two-point
attack and Schiff rearrangements, as vinyl chloride—derived chloroacetaldehyde does (Green
and Hathway, 1978). Clearly, TCE metabolism via chloral represents detoxification
(Hathway, 1980).

The usual rearrangement of TCE-oxide into chloral takes place in the smooth
endoplasmic reticulum (Hathway, 1980) and in contact with a catalytic center stimulating a
Lewis acid; in which case, the fast cytosolic biotran sformation of chloral would facilitate its
egress from smooth endoplasmic reticulum. In mice, on the other hand, rearrangement of the
epoxide into chloral would appear to be hindered. In this species, greater cytochrome P-450
activity in the liver and kidneys (Litterst et al., 1975) favors a faster rate of epoxidation, and
there is a tendency for the catalytic sites for chloroalkene oxide rearrangement into chloral
to become saturated as well as for the lower activity of the cytosol enzymes responsible for
chloral disposition in mice (Byington and Leibman, 1965; Waters et al., 1977) to become
rate-limiting. In an excess substrate to enzyme situation in mouse liver cells in vivo, the rest
of the TCE-oxide produced would be transformed into dichloroacetyl chloride at the cytosol
surface. The identification of dichloroacetic acid, as well as TCAA in the urine of TCE-
treated mice (p.o.), strongly supports the spill-over model that is now proposed for TCE
metabolism in mice, whereas according to Bonse et al., (1975), dichloroacetic acid is not

formed in other mammals.

l7

Radio-labeled TCE injected (i.p.) into male mice irreversibly bound to hepatic
protein with the highest levels after 6 hours in cytosolic and mitochondrial proteins and
maximal in microsomal proteins (U ehleke and Poplawski-Tabarelli, 1977a). Radio-labeled
TCE injected into mice (i.p.) bound RNA and DNA maximally in the spleen, and in vitro,
radioactivity was seen in DNA co-incubated with liver microsomes but not in non-
microsomal cultures. The interpretation was that the CC bond was split during in vivo
biotransformation (Bergman, 1983b).

Novel interpretations on the possible in vitro metabolism of TCE in isolated
hepatocytes, microsomes, and reconstituted enzyme systems containing cytochrome P-450
were reported by Miller and Guengerich (1983). The findings revealed that the major
pathways of metabolism depended upon the isozymes of cytochrome P-450 involved, which
could not be strictly correlated, and that conjugation of products with glutathione did not
appear to play a major role in TCE metabolism. TCE oxide was found not to be the metabolite
responsible for irreversible binding to DNA and protein. In agreement with other studies,
DNA and protein adducts formed were higher in isolated mouse (B6C3F1) hepatocytes than
in rat (Osborne-Mendel) hepatocytes.

‘ Chloral was the major microsomal metabolite found in this study, and TCE oxide,
glyoxylic acid and carbon monoxide(CO) were the minor microsomal metabolites. The
same authors established the year before that TCE was not metabolized to chloral via an
epoxide intermediate, and proposed instead that chlorine migration occurred within an
oxygenated TCE-P450 intermediate to form chloral. When human microsomal preparations
were used, TCE oxide was not detected. It was found that cytochrome P-450PB-B was the P-
450 isozyme that metabolized TCE to the greatest extent, based on the amount of chloral
formed. Other P-450 isozymes involved to a significant degree in the conversion of TCE to
chloral were P-4508NF-B and P-4SOBNF/ISF-G (Miller and Guengerich, 1983).

TCOH was the major product of metabolism, both conjugated and unconjugated, in

18

both the in vitro hepatocyte system and in vivo. Tissue liquid chromatography analysis
suggested that TCE-reduced glutathione (GSH) adducts were formed at very low levels, and
the lack of observation of urinary metabolites of TCE-GSH, such as mercapturic acids,
supported this view. The limited role of GSH in TCE metabolism contrasts directly with the
situation in two closely related haloalkenes, vinyl chloride and vinylidene chloride, which are
metabolized to mercapturic acids and excreted in the urine (McKenna et al., 1978; Watanabe
et al., 197 6).

Epoxides of chloroethylenes are frequently mutagenic (Greim et al., 1975) as are
chloral and chloral hydrate (Bignami et al., 1980). TCE-oxide and chloral are relatively
transient metabolites, and the rearrangement of epoxides is usually a non-enzymatic process,
independent of species. These then are not likely candidates for the increased risk from TCE
exposure in mice and rats (Prout et al., 1985).

The most striking differences in species were seen in the blood concentrations of the
end products TCOH and TCAA. Free TCOH was rapidly removed from blood by
glucuronidation whereas TCAA persisted at a relatively constant amount for up to 30 hours
following a single oral dose. Blood concentrations of TCAA were 10—fold those of TCOH
and up to 150-fold those of chloral. The blood concentrations of the major metabolites are
a direct consequence of the increased rate of metabolism of TCE in the mouse (Prout et al.,
1985). After i.p. injection in corn oil, CO2 excretion is greater in mice than rats (Parchman
and Magee, 1982). Single p.o. doses of COz-labeled TCE from 10 to 2000 mg/kg showed a
dose dependent expression in rats but not in mice (Prout et al., 1985).

A plot of the dose metabolized to TCAA and its metabolite CO2 against the dose of
TCE administered shows a linear relationship in the mouse (Elcombe et al., 1985). Recent
work has demonstrated the pharmacological significance of the high concentration of TCAA
found in the mouse.

A reinvestigation into the metabolism of TCE led to the discovery of an alternative

l9

mechanism which favors primary genotoxic events. The metabolism of TCE can lead to the
formation of a mercapturic acid which could only be explained by primary conjunction of
TCE with glutathione (Dekant et al., 1986a). The mercapturic acid, N-acetyl-S-l,2-
dichlorovinyl cysteine, is formed from the precursor, 1 ,2-dichlorovinyl cysteine, which itself
is the product of the well-studied metabolic processing steps of glutathione adducts. The
dichlorovinyl systems are established nephrotoxic compounds (Anders and Pohl, 1985) and
mutagens in in vitro systems (Green and Odum, 1985). The mutagenic potential may be
attributed to products formed by the B-lyase enzyme system specifically localized with high
activity in the brush border of kidney tubular-lining epithelial cells, which cleaves the sulfur
from cysteine to produce the chlorovinyl residue.

The biotransformation of TCE is dose-dependent in both mice and rats according to
Dekant et al., (1986b), and a higher rate of biotransformation is seen following induction of
hepatic mono-oxygenases by phenobarbital and polychlorinated biphenyls (PCBs) (e. g.,
Arochlor 1254). The major urine metabolites found in this study were TCAA, TCOH and
TCOH glucuronide (88.9 to 93.5%), but small amounts of N-(hydroxyacetyl)-aminoethanol
(4.1 to 7.2%), dichloroacetic acid (0.1 to 0.2%), oxalic acid (0.7 to 1.8%) and CO2 were seen
also. N-(hydroxyacetyl)-aminoethanol is a fragment of phosphatidylethanolamine, and
seems to indicate an attack of an electrophilic intermediate of TCE on the phospholipids of
the endoplasmic reticulum. The presence of dichloroacetic acid as a urinary metabolite in
mice after single, high TCE doses supported the spill-over hypothesis of Hathway (1980).

Dekant et al., (1986b) found that with increased dose of TCE, CO2 rose indicating a
dose—dependent formation of dichloroacetic acid and saturation of the deactivating mecha-
nisms for reactive intermediates. Mice were found also to display a higher overall biotrans-
formation rate and a higher TCOH-conjugating capacity. Saturation in mice occurs at higher
doses but in both species is reserved to the first step, the oxidative activation manifested in

the increase in the proportion of exhaled TCE. At the doses of 2, 20 and 200 mg/kg TCE in

20

mice (gavage), the urinary TCAA content was 19.6%, 14.4% and 8.3% respectively.

Specific conclusions about the metabolism of TCE in mice are that higher concentra-
tions of reactive intermediates were formed by oxidative metabolism (e. g., 2,2,3-
trichlorooxirane), and an increase with dose was seen in the formation of dichloroacetyl
chloride as a rearrangement product of the epoxide which also results in a higher steady-state
level of reactive intermediates. Both TCE-epoxide and dichloroacetyl chloride are rapidly
hydrolyzed in biological syStems; both compounds and the end-products of their hydrolysis
are highly cytotoxic (Henschler, 1979). This work supports the hypothesis of Stott et al.,
(1982), that high oral doses of TCE in mice may generate a non-specific, non-genetic
mechanism of a weak carcinogenic effect. Low amounts of TCE in both expired air and blood
in the mouse suggests extensive first-pass metabolism. This is supported also by the highest
concentrations of chloral and TCOH being seen soon (1 to 2 hours) after dosing (Prout et al.,
1985)‘.

Of interest is the interaction between TCE and ethyl alcohol. Intolerance of or
insensitivity to ethanol is a well-documented symptom of TCE exposure (Stewart et al. , 1974;
Jindrichova, 1970). Concurrent administration of ethyl alcohol and TCE to human volun-
teers resulted in a 2.5-fold increase in the concentration of TCE in blood and a 3 to 4-fold
increase in the TCE concentration in exhaled breath (Muller et al., 1975). These results
suggest a lower rate of TCE metabolism in the presence of ethyl alcohol with a subsequent
buildup of TCE concentration in the blood Muller et al. (1975) suggested that the mixed
function oxidases responsible for metabolizing ethanol were also responsible for metaboliz-
ing TCE, leading to competitive inhibition between the two for the enzyme and resulting in
a subsequent buildup of TCE concentration in the blood. No references were found as to a

synergistic or antagonistic effect of the metabolites of TCE either in in vivo or in vitro studies.

21
Modeling Experiments

An alternative method for studying the metabolism of TCE in the mammalian system
is by simulating the process with a mathematical model. Obviously such a model must rely
on physiological data from animal experiments but it has application to predicting the uptake,
distribution, metabolism, and excretion of varying doses of chemicals in various species of
animals.

Fernandez et a1. (1977) analyzed the simulated exposure of TCE in mice using a
mathematical model. The main reason for constructing the model was the difficulty in
understanding and defining the relationships between the degree of exposure and the
excretion of the solvent and its metabolites. Many assumptions were made for the model such
as: the distribution of TCE in the body occurred in three tissue groups - vascular tissues
(VRG), the muscles and skin (MG), and the adipose tissues (FG); TCE was transformed
quantitatively in the liver to chloral hydrate (CH), precursor of TCAA and TCOH; and the
evolution of these metabolites was studied using the corresponding rate constants for the
biotransformation and transfers which occurred.

The principal assumptions (e. g., solvent diffusion, arterial and venous blood concen-
tration, etc.) are listed in full in the original paper. Other assumptions of note included that
TCE biotransformation took place solely in the liver, and was an instantaneous process, such
that the biotransformation was limited by the blood flow through this organ. Also, the liver
was perfused by blood which had an arterial concentration of TCE. CH, formed in the liver,
was metabolized mainly by oxidation to TCAA and by reduction to TCOH. Further, it had
been established that TCAA was itself a metabolite of TCOH. It was noted at this time that
both TCOH and TCAA were eliminated by other biotransformations which were still
unknown, and that various studies had shown that the total amount of the two metabolites
eliminated in the urine represented only about half of the quantity of CH (Muller et al., 1974)
orTCE absorbed (Fernandez etal., 1977). Finally, since the kinetics offree TCOH and TCOH

22

glucuronide were similar (Breimer et al., 1974), no distinction was made between the two in
the mathematical model.

In the results, pulmonary excretion of TCE was predicted accurately by the model.
Close predictions were obtained for the urinary elimination rates of TCOH and TCAA. For
TCE exposures of 100 ppm from 1 to 8 hours, urinary elimination rates of TCOH and TCAA
were proportional to the concentration of exposure. A second finding was that the model
allowed one to predict the body burden of TCE and its metabolites in the tissue groups in
question. In the 8 hour exposure trial period at 100 ppm TCE, estimates of percent
metabolized, and the amount of TCAA and TCOH present at various times could be deduced.
By the end of the exposure period, 54% of the absorbed TCE was transformed to TCOH and
20% to TCAA, with only 26% unchanged (2% in VRG, 9% in MG, and 15% in FG). Just
after the end of the exposure period, 92% of the TCE had been metabolized, and the TCAA
amount increased to 27%.

Regarding the metabolites, TCOH body burden rose slightly for half an hour,
whereas that of TCAA rose progressively for about 30 hours. The urinary concentration of
TCAA increased also. In fact, the TCAA levels increased until approximately 22 hours post-
exposure. The delay in the TCAA elimination was attributed to a combination of the two
properties of the total system. The urinary elimination rate of TCAA is very slow owing to
its great affinity for proteins, and TCAA is formed not only from TCE though the
intermediary CH, but also by the biotransformation of TCOH.

The accumulation of TCAA in the body then would be predicted after repeated
exposures. It was expected that notable modifications to the total pattern of the processes
arising from the absorption of TCE would occur if the exposure was repeated day after day.
Therefore, Fernandez used the model to simulate a situation of this type, assuming the
behavior of the animal towards TCE was not significantly modified by repeated absorption
(Ikeda and Imamura, 1973).

23

The predicted alveolar concentration of TCE after repeated exposures at 100 ppm (5
days, 6 hours per day) showed that TCE concentration at the end of each exposure day
remained relatively constant. But when the fatty tissue (FG) was analyzed, the levels of TCE
rose progressively in a step-like manner each day because of the slow release of the solvent.

Metabolite behavior following the repeated exposure protocol was analyzed. The
excretion rate each day for TCOH rose only slightly and was characterized as a spike. On the
other hand, the TCAA excretion rate increased greatly each day and TCAAwas still
detectable at Day 16. It was noted that the form of the curves followed the variations in
concentration of the two metabolites determined experimentally in the blood (Muller et al.,
1974).

The conclusion was that the excretion rate of TCOH in urine was determined
principally by the most recent exposure, whereas the excretion rate for TCAA represented
the accumulation of all earlier exposures.

A final important consideration generated from the findings of Fernandez et a1.
(1977) was that the equivalence between the formation and elimination rate of TCAA was
reached earlier after repeated exposures. This considerable difference between isolated and
repeated exposure found using the model indicates that the results of urinary elimination of
TCAA obtained from isolated experimental exposures cannot be extrapolated directly for
considering industrial situations due precisely to the interdependence of these two metabolites.

A physiologically based-pharmacokinetic (PB-PK) model was developed by Reitz et
a1. (1988) for the halogenated hydrocarbon 1,1,1-trichloroethane commonly called
methylchloroform (MC) which shows implications for interspecies, high and low dose, and
administration route extrapolation. MC is similar in structure and chlorination number to
TCE. This model was developed and used to describe the disposition of MC in mice, rats, and
humans following exposure by the routes of inhalation, i.v. injection, bolus gavage, and

drinking water administration.

24

The metabolism of MC followed Michaelis-Menten kinetics in each species and the
Vmaxs were calculated from the allometric equation: Vmax = 0.419BW°'7, Km appeared to
be identical in each species (5.75 mg equivalents/liter). As with the Fernandez model, the
utility of the PB-PK model was derived to describe realistically the size of organ compart-
ments, partitioning of test material between blood and tissues, flow of blood and gases
through the organs, and rates of metabolic transformation of the test chemical. Similar
assumptions and physiologic data as for the Fernandez model were used in the PB-PK model.

In rat simulations, the PB-PK model was used to attempt a dose route extrapolation
from inhalation to i.v. injection exposure. The curve of the model closely predicted the
experimental results. Likewise, other dose-route extrapolations were predicted closely by
the model.

In mouse simulations, the PB-PK model was used for interspecies extrapolation by
predicting the pharmacological behavior of MC in male B6C3F1 mice after inhalation
exposure (from rat inhalation experiments). Only the appropriate physiological and
biochemical parameters were adjusted in the model. The PB-PK model predicted that MC
would be eliminated from the mouse much more rapidly than the rat, and this was consistent
with the observed data. -

The model worked well in human cases too. Concentration of MC in expired air was
well simulated by the model during exposure. Also, the PB-PK model predicted that4.30 mg
equivalents of MC would be metabolized during the 240 hours following a 6 hour exposure
to 35 ppm MC, and 4.32 mg equivalents of MC metabolized were actually recovered. When
the dose of MC was increased 10-fold, the PB-PK model prediction for MC metabolites was

similarly accurate.

25

Carcinogenicity

Chlorinated hydrocarbons came under scrutiny in the 19608 because of accidental
and intentional abuse. Although these chemicals are better solvents and are less inflammable
than the unsubstituted hydrocarbons, chlorinated hydrocarbons are generally more toxic. As
noted in a recent review of halogenated ethylenes by Cantelli-Forti and Bronzetti (1988), the
reactivity of the compound depends on the number and position of the substituted chlorine
atom The symmetric position renders the compounds stable, while the unsymmetric
substitution results in molecular instability with the formation of mutagenic epoxide (Greim
et al., 1975; Henschler and Bonse, 1977; Fishbein, 1976).

One of the first comprehensive studies of the chlorinated hydrocarbons was by
Klaasen and Plaa (1966) who quantitatively assessed liver and kidney damage in mice and
rats following i.p. injections of chloroform, 1,1,1-trichloroethylene, and carbon tetrachlo-
ride. Carbon tetrachloride and chloroform exerted the greatest effect on the liver. Chloroform
and 1,1,2-trichloroethane exerted the greatest effect on the kidney. TCE exerted weak
hepatotoxic and nephrotoxic effects. No significant effect was seen for TCE following
pretreatment with ethanol. These workers compared the doses required to produce organ
dysfunction (EDSO) to the doses required to produce death (LDSO), and ranked the chlorinated
hydrocarbons according to the ratio ED50:LD50. To illustrate, the potency ratio for carbon
tetrachloride was approximately 300 whereas for TCE it was approximately 2. As such, the
dose of TCE required to induce tissue damage is close to that which leads to death. This ability
to rank chemicals by potency is important in risk assessment.

Gehring (1968) performed a similar study in direct challenge to the above study of
Klaasen and Plaa (1966). The possible invalidity of the results obtained from animals dosed
i.p. prompted Gehring to repeat the experiments on the lethality and the anesthetic dose
required following inhalation of chlorinated hydrocarbon vapors. Although quantitative

26

differences were found, the same rank of liver dysfunction was found: carbon tetrachloride,
chloroform, 1,1,2-trichloroethane, tetrachloroethylene, TCE, dichloromethane, and 1,1,1-
trichloroethane.

The National Cancer Institute (NCI) conducted a gavage study with industrial grade
TCE, stabilized with 0.19% 1,2-epoxybutane and 0.09% epichlorohydrin, in BGC3F1 mice
and Osborne-Mendel rats (N C1, 1976). In mice, significant dose-related increases in primary
hepatocellular carcinomas were observed in both sexes.

Henschler et al. (1977) criticized the NCI study for using technical grade TCE
containing strong alkylating agents which confounded the results, and a new gavage study
was undertaken by the National Toxicology Program (NTP) in 1983 using highly purified,
epoxide-free TCE. In this study, increased numbers of hepatocellular carcinomas were found
in both male and female mice as compared with control groups. Also, the incidence of
hepatocellular adenomas was increased in mice of both sexes (NTP, 1983). Although highly
purified TCE was used in the NTP bioassay, the results obtained essentially confirmed the
findings of the earlier NCI study.

In rats, high purity, epoxide-free TCE administered orally for 52 weeks at lower doses
than either the N C1 or NTP studies showed no tumorigenic effect. Alterations of renal tubular
cells were observed in the high dose, male group (Maltoni and Maroni, 1977). Moslen et al.
(197 7) suggested that the hepatotoxic effects of TCE could be due to the inadequate
detoxification of its reactive intermediates. These studies were performed in rats, not mice,
and later studies reveal that a definite species-specific metabolism of TCE exists in rodents.

Pessayre et al. (1979) contrasted acute and repeated dosing effects of TCE on liver
function in rats. Following the acute administration of TCE, drug metabolism by the mixed
function oxidase system (MF OS) was competitively inhibited, and following repeated
administration of TCE, cytochrome P-450 was selectively destroyed and microsomal
enzymes were induced probably by a metabolite of TCE. In vitro, TCE activated glucuronyl

27

transferase.

Inhalation studies with TCE up to 500 ppm for 6 hours per day, 5 days per week for
18 months in rats, mice and Syrian hamsters of both sexes showed no significant increase in
tumor formation, except for malignant lymphomas in female mice. The strain of mice used,
NMRI, exhibit a high occurrence of lymphomas (Henschler et al., 1980), and the authors
concluded that no indication for a carcinogenic potential of TCE could be deduced.

A comprehensive toxicological study of TCE was reported in 1982 which assessed
the acute and subchronic effects of the chemical in mice using the oral dosing routes of gavage
and drinking water administration (Tucker et al., 1982). Acute studies with TCE estimated
the oral LD50 for female mice at 2443 mg/kg b.w., and estimated the oral LD50 for male mice
at 2402 mg/kg b.w. In 4 or 6 month administration trials of TCE up to a concentration of 5.0
mg/ml in drinking water, there was a decrease in body weight gain at the highest dose
attributable to a decrease in fluid consumption. The most significant effects attributable to
TCE were an increase in liver and kidney weight in both sexes accompanied by increases in
protein and ketones in the urine. No other adverse effects were noted. It was concluded that
at doses lower than the LD 50, TCE is not a potent hepatotoxin in the mouse. It was noted that
the elevated glutathione levels in the males may be important in the ability of these animals
to respond to the hepatotoxic effects of TCE.

A parallel study by the same group assessed the immune status of mice dosed with
TCE by gavage and in the drinking water (Sanders et al., 1982). TCE inhibited cell-mediated
immunity after a 14 day exposure by gavage. After 4 or 6 months exposure in the drinking
water, TCE inhibited humoral immunity at 2.5 and 5.0 mg/ml, and cell-mediated immunity
and bone marrow stem cell colonization were inhibited at all concentrations tested. Females
were affected the most, and males were unaffected, relatively, through both 4 and 6 month

exposure periods.

28

In further efforts to elucidate the role of epoxide stabilizers of TCE and its purported
carcinogenicity, Henschler et al. (1984) dosed male and female Swiss mice (ICR/HA) with
TCE daily by gavage for 18 months either with or without the addition of epichlorohydrin,
1,2-epoxybutane or both. A significant increase in the incidence of forestomach papillomas
and carcinomas was seen in all the epoxide-stabilized TCE test groups.

Inhalation studies with highly purified, epoxide-free TCE were performed in B6C3F1
mice by Maltoni et al. (1986). No increase in any tumor rate was observed in mice after eight
weeks of exposure. After 104 weeks of exposure, there was an excess of hepatocellular
carcinomas in both sexes of mice. Additionally, an increased frequency of lung tumors was
observed in female mice.

TCE was assayed as a tumor promoter in male B6C3F1 mice initiated by
ethylnitrosourea (Herren-Freund et al., 1987). TCE was administered in the drinking water,
and it did not result in an increased incidence of live tumors. The results were inconclusive,
nevertheless, because of the low dosage administered (6 mg/kg b.w.) which was necessitated
by the low solubility of TCE in water. In the same study, though, TCAA and DCA were found
to be complete hepatocarcinogens, increasing the incidence of hepatocellular carcinomas
without prior initiation by ethylnitrosourea.

A few skin application and subcutaneous injection studies of TCE in animals have
been performed but they do not present an adequate experimental model for detecting TCE
carcinogenisis because of the low pereutaneous absorption of the chemical and the lack of
liver metabolism in the subcutaneous injections (Crebelli and Carare, 1989).

In mice, oral and inhalational exposure to TCE results in an increased incidence of
hepatocellular carcinomas and lung tumors in both sexes of BGC3F1 and Swiss mice. The
hypothesis that contaminated, technical grade TCE resulted in earlier positive findings due
to the presence of epichlorhydrin and 1,2-epoxybutane can be ruled out on the basis of
repeated trials with highly purified TCE samples (NTP, 1983; Maltoni et al., 1986). The

29

reason for the negative outcome of the long term bioassay in ICR/HA mice was not
elucidated, but this strain may be insensitive to liver neoplasia (Crebelli and Carare, 1989).
On the basis of experimental results, the evidence for tumorigenicity in rodents can be
considered more firmly established at present, although the high dosages required to obtain

this evidence suggest that TCE is a weak carcinogen.

Mutagenicity

In cell free systems, l4C-TCE incubated with salmon sperm DNA and microsomal
preparations from B6C3F1 mice resulted in a dose-related covalent binding of TCE to DNA.
Binding was greater in the presence of microsomes from male mice, and was enhanced by
the in vivo pretreatment of mice with phenobarbital or by the addition in vitro of 1,2-epoxy-
3,3,3-trichloropropane (Banjeree and Van Duuren, 1978). In this study, binding was seen
also following coincubation with microsomes from mouse lung, stomach, and kidney.
Significant binding of l4C-TCE was seen in incubations of calf thymus DNA in the presence
of hepatic microsomes from phenobarbital-induced rats (DiRenzo et al., 1982).

' Liver microsomes from B6C3F1 mice, Osborne-Mendel rats, and humans were used
to study TCE metabolites and binding to macromolecules (Miller and Guengerich, 1983). In
all cases, a significant covalent binding of TCE metabolites to calf thymus DNA and proteins
was observed. It was also shown that whole hepatocytes of both untreated mice and
phenobarbital-induced rats and mice could activate TCE into metabolites able to covalently
bind exogenous DNA.

A different scenario is revealed from in vivo studies. In mice, after repeated i.p.
injections of 14c-rc13, radioactivity was detectable in the DNA and RNA of liver, lung,
spleen, kidney, pancreas, testes, and brain (Bergman, 1983a). However, the labeling was
shown to be due to metabolic incorporation of C-1 fragments, particularly in adenine and

guanine, rather than to DNA-adduct formation.

30

In another study of i.p. injection 1""C-TCE in male B6C3F1 mice at 10 to 250 mg/kg
b.w., high liver protein labeling was observed while very low DNA labeling was detected
(Bergman, 1983b). Also, very low DNA binding was found in B6C3F1 mouse liver after oral
administration of 1200 mg/kg b.w. TCE, a dose reported to be carcinogenic upon chronic
administration (Stott et al., 1982).

Whereas several studies demonstrate that TCE be converted in vitro by exogenous
metabolism into reactive metabolites able to covalently bind nucleic acids and proteins, in
vivo studies show equivocable evidence of DNA labeling attributable to covalent binding.
These results suggest either qualitative or quantitative differences in in vitro and in vivo TCE
metabolism, or the preferential binding in vivo to protein targets more easily accessible than
nuclear DNA (Crebelli and Carare, 1989).

In tests of TCE mutagenicity of mammalian cells in vitro, mixed results were
obtained. Two studies evaluated the possible induction of sister chromatid exchanges (SEC)
in Chinese hamster ovary cells (CHO) with one positive test (Galloway et al., 1987) and one
negative test (White et al., 1979). Different treatment protocols and dose levels were applied
in the studies; therefore, no direct comparison can be made to explain the apparent
discrepancy.

Studies performed to assess the induction of unscheduled DNA synthesis (UDS) by
TCE in different cell systems gave contrasting results. TCE samples tested on human
lymphocytes gave positive results only in assays with metabolic activation (Perocco and
Prodi, 1981). Tests of TCE samples with and without undefined stabilizers in hepatocyte
primary cultures showed negative results even at doses causing cell death (Shimada et al.,
1985).

Morphological transformations were studied in cellular systems with positive results
in all cases. TCE exposure transformed foci in Fisher rat embryo cells (Price et al., 1978).
TCE samples of undefined purity produced weakly positive results in Syrian hamster

31

embryo cells (Amacher and Zelljadt, 1983). TCE exposure resulted in a dose-related increase
in the BALB/c-3T3 cell transformation assay in the number of transformed type III foci (Tu
et al., 1985).

TCE has been assayed in several in vivo genotoxic studies in rodents. Duprat and
Gradiski (1980) reported highly significant and dose-related increases in micronuclei in
polychromatic and mature erythrocytes in CD-1 mice after oral administration of analytical
grade TCE. Sbrana et al. (1985) found a statistically significant increase in micronuclei in
bone marrow cells after a single oral administration of TCE to B6C3F1 mice. There was no
appreciable increase in structural chromosome aberrations. Prolonged exposure to 600 ppm
TCE for 7 hours per day, 5 days per week, for 10 weeks, did not increase the frequency of
structural chromosomal aberrations in bone marrow cells. These results suggested that pure
TCE is not appreciably clastogenic in mouse bone marrow and supports the hypothesis for
the involvement of a mechanism affecting chromosome segregation, rather than structural,
in micronucleus induction. To date, conclusive evidence of this possibility has not been
provided (Crebelli and Carare, 1989).

An inhalation study performed in male mice exposed up to 450 ppm TCE during 24
hours showed no mutagenic effect in the dominant lethal assay in which fertilization rate,
post-implantation loss, pre-implantation loss, and dominant lethal mutations were evaluated
(Slacik-Erbin et al., 1980). Significant effects were seen in the number of morphologically
abnbrmal sperm in mice after inhalation of an undefined TCE sample (Land et al., 1981).
Although this end point is believed to be related also to the induction of genetic damage, the
possible involvement of non genotoxic mechanisms does not allow regarding this result as
evidence for TCE mutagenicity at the germ cell level (Crebelli and Carare, 1989).

TCE does have an effect on its target organ the liver. In B6C3F1 mice following
repeated gavage of TCE, Stott et al. (1982) found an increase in DNA synthesis but very low
DNA binding, and severe histopathological changes of hepatocytes. In a second study using

32

the same mouse strain, acute exposure to TCE failed to induce UDS in an in vivofrn vitro
hepatocyte DNA-repair assay, but significantly increased hepatic cell proliferation (Mirsalis
et al., 1985). Similar results were obtained in a similar study using CD-1 mice by Doolittle
et al. (1987). Together these results support a possible relationship between induced cell
proliferation in the mouse liver and hepatocarcinogenicity at least in B6C3F1 mice.

Chloral hydrate (CI-l), the major intermediate metabolite of TCE, caused increased
incidence of nondisjunction in mouse spermatogenesis following i.p. injection of doses up
to 413.5 mg/kg b.w. (Russo et al., 1984). The prevalent action of TCE was localized to the
mitotic spindle. In vivo, CH, injected i.p. to mice on 5 consecutive days up to 2500 mg/kg
b.w., was negative in bone marrow micronucleus and sperm abnormality assays (Bruce and
Heddle, 1979).

TCAA was tested in 3 cytogenetic assays, bone marrow chromosome aberration,
micronucleus and sperm-head abnormality in the mouse (Swiss). The chemical was admin-
istered in vivo either p.o. or i.p. and in acute or fractionated (5 consecutive daily) doses.
Results showed a variety of anomalies in all tested groups compared to controls. Single acute
dosing was more effective cyto genetically, and results were route and time-dependent but not
dose-responsive (Bhunya and Behera, 1987).

When chloral was tested up to 250 mM for its ability to form DNA-protein crosslinks,
results were negative although chloroacetaldehyde (47 mM) and acetaldehyde (200 mM) did
form crosslinks indicating a very strong hydration of chloral. Interestingly, mice given 800
mg/kg 14C-chloral (i.p.) after pretreatment with 1500 mg/kg TCE (gavage) for 10 days had
no detectable covalent binding of 14C to DNA in the liver. These results do not support a
genotoxic theory of carcinogenesis for TCE mediated through chloral (Keller and Heck,-
1988).

The interaction of TCE, metabolites, and hepatic DNA was investigated by Nelson

and Bull (1988). B6C3F1 mice and Sprague-Dawley rats were dosed with TCE in vivo.

33

Doses of TCE of 22-30 mmole/kg were required to produce single strand breaks (SSB) in
DNA in rats, whereas a dose of 11.4 mmole/kg was sufficient to produce SSBs in mouse
DNA. Doses of TCE above 22.9 mmole/kg were lethal to mice. TCAA, CH, and dichloroacetic
acid (DCA) also induced 8835 in hepatic DNA in a dose-dependent manner in both species.
Importantly, 8833 were observed at doses that produced no observable hepatotoxic effects
as measured by serum aspartate aminotransferase and alanine aminotransferase levels. The
rank of TCE metabolites that induced SSBs in mouse hepatic DNA was TCAA, DCA, and
CH. The order for SSBs in rat hepatic DNA by TCE metabolites was DCA, TCAA, CH, and
TCOH. Also, the dose-response curves were different for each metabolite and were different
in each species. Pretreatment with phenobarbital increased 8835 suggesting again that
metabolic activation is necessary for DNA damage to occur. Pretreatment with ethanol had
no effect. Variation in the relative potency of TCAA in rats and mice parallels the species
specificity of the hepatocarcinogenic effects of TCE. The induction of SSBs appeared to be
independent of the hepatotoxicity of these chemicals since increased damage occurred in the
absence of significant elevation in serum aspartate aminotransferase and alanine amino-
transferase levels.

A summary of all the mutagenicity studies of TCE and its metabolites allow a few
conclusions to be drawn. Liver microsomal preparations of rodents can metabolically convert
TCE into reactive species capable of covalent binding in vitro to exogenous DNA and
proteins. The reactive TCE metabolites have not been identified. Pathways that can form
reactive intermediates from halogenated hydrocarbons (i.e., glutathione conjugation) do not
play an important role in TCE metabolism in the liver (Miller and Guengerich, 1983; Rouisse
and Chakrabarti, 1986). Purified, epoxide-free TCE is weakly mutagenic after exogenous or
endogenous metabolic activation in several in vitro mutagenicity assays, including mamma-
lian cell transformation. Evidence for TCE activity in other assays in mammalian cells is

equivocal. Epoxide-free TCE is genotoxic in vivo to mouse bone marrow cells, where it

34
induces high rates of micronuclei but no structural chromosome aberrations. Acute or
repeated TCE exposure produces an increase in hepatic cell proliferation in the mouse but no
definite sign of DNA binding, damage, or repair. CH is weakly mutagenic in vitro and
induces chromosome malsegregation in vivo. TCAA is an effective clastogenic agent to

mouse bone marrow after in vivo exposure.

Reproductive Toxicology

In mammals, inhalation exposure up to 300 ppm of TCE for 7 hours on Days 6 to 15
of gestation did not result in any maternal toxicity or in any teratogenic effect in Swiss-
Webster mice orin Sprague-Dawley rats (Leong et al., 1975; Schwetz etal., 1975). In another
study, Lon g-Evans rats were exposed to up to 1800 ppm TCE for 6 hours per day, 5 days per
week for two weeks before mating, and during pregnancy. Significant elevations in skeletal
and soft-tissue anomalies, indicating developmental delay rather than teratogenesis, were
observed in animals exposed during pregnancy only. Additionally, a post-natal reduction in
body weight was seen in the offspring of mothers exposed pregestationally (Westergren et
al., 1984).

Two studies of oral administration of TCE effects on reproductive function have been
performed in the rat: one in males and one in females. Zenick et al. (1984) administered TCE
up to 1000 mg/kg b.w. for 5 days per week for 6 weeks to male Long-Evans rats. No
spermatotoxic effects were observed. A transient alteration in copulatory behavior was seen
in the high dose group, which was attributed to the narcotic effects of TCE. Manson et al.
(1984) exposed female rats of the same strain to the same dose of TCE for 2 weeks before
mating and throughout pregnancy. Fertility was not affected in any group. In the high dose
group, though, significant depression of weight gain throughout the treatment period and
lower neonatal survival were observed.

In conclusion, exposure of rats, and to a degree mice, to TCE during pregnancy

35

resulted in a developmental delay of litters with no concurrent signs of severe embryotoxicity
or teratogenicity. No impairment of reproductive function and fertility was observed in
treated animals.

TCAA treatment by gavage of pregnant rats resulted in dose-dependent reduction in
fetal weight and length, and an increase in congenital anomalies (Smith, 1988). TCAA, a
major metabolite of TCE, has been found in the amniotic fluid of pregnant mice exposed to
TCE (Ghantous et al., 1986).

No reports on the reproductive effects of TCOH were found.

Vehicle

A concern in administering a chemical orally to animals for toxicology is the vehicle
used. Studies in the past decade have illustrated the relative effect the vehicle plays in the
toxic response of animals to chemicals.

The effect of administration vehicle on oral 1,1-dichloroethylene (DCE) toxicity was
addressed by Chieco et al. (1981). Using fasted (16 to 18 hours) and fed rats, the vehicles
mineral oil, corn oil, and aqueous Tween-80 were assessed for their ability to affect a toxic
response to DCE. Results showed that in fasted rats, mineral oil and corn oil use was
associated with massive liver damage whereas with aqueous Tween-80, there was only
moderate liver damage. In all fed groups, liver injury was slight. The administration vehicle
did not affect the total amount of DCE exhaled (approximately half the dose), nor did the
vehicle affect the initial rapid phase of DCE exhalation. In contrast, the later slow phase of
DCE exhalation was predictably affected by the administration vehicle; t1/2 values for the
fasted and fed groups were most prolonged with mineral oil, intermediate with corn oil, and
most brief with aqueous Tween-80.

The results agree intuitively with the fact that the gastrointestinal (GI) tract handles
each vehicle differently, and thus should alter the rate of DCE transfer from the GI tract.

36

Mineral oil consists of intermediate length paraffins and is not taken up extensively by the
gut. Corn oil consists predominantly of triglycerides and is digested in the small intestine,
and does not prolong uptake as mineral oil does. Aqueous Tween— 80 should produce the most
brief duration of uptake due in part to the poor retention of DCE in an aqueous medium
compared to the more retentive soil media. This influence was similar in the fasted and fed
animals despite the difference in distention and physiology of the GI tracts (Chieco et al.,
1981).

Enhancement of the hepatotoxicity of chloroform in mice by corn oil was demon-
strated by Bull et al. (1986). In mice administered chloroform in corn oil versus chloroform
in 2% Emulphor® the following were observed: significant increases in serum glutamate
oxalacetate transaminase (SGOT) levels, small increases in blood urea nitrogen (BUN), and
significant decreases in serum triglyceride (T G) levels. Although body weight and liver
weight decreases occurred in both vehicles, the effects in corn oil vehicle were significantly
greater. The general conclusion was that administration of chloroform in corn oil by gavage
resulted in more marked hepatotoxic effects than when administered as an aqueous
suspension.

In two companion studies on the effect of oral dosing of the vehicle on the acute
hepatotoxicity of CC14 in rats, it was found that liver damage was less pronounced when corn
oil was used as the vehicle (Kim et al., 1990a; Kim et al., 1990b).

In the first study, fasted rats administered CC14 in corn oil showed reduced extent of
injury relative to that when the chemical was given undiluted or as an aqueous emulsion.
Dose-dependent increases in serum enzyme levels and pathological changes, and dose-
dependent decreases in microsomal P-450 and glucose-6-phosphatase activity were ob-
served in each vehicle group indicating an effect due to CCl4. Yet at each dose of CC14 in
corn oil, acute hepatotoxicity was less pronounced (Kim et al., 1990a).

The effects of dosing vehicles on the pharmacokinetics of orally administered CCl4

37

were assessed in the companion study. Fasted rats were administeredCCl4 by gavage either
in corn oil, as an aqueous Emulphor® emulsion, in water, or as undiluted chemical. Peak
blood levels of CC14 were higher after administration in aqueous emulsion and water groups
than those in undiluted CC14 group, and much higher than those in the corn oil group. Corn
oil markedly delayed the absorption of CC14 from the GI tract producing secondary peaks
in the blood concentration profiles. It was concluded that CC14 was less acutely hepatotoxic
in corn oil due to the delay and prolongation of CC14 absorption, resulting in marked decrease
in the concentration of the chemical in the arterial blood (Kim et al., 1990b).

Taken together, the results of these studies suggest that corn oil has sufficient effect
on the pharrnacokinetics of orally administered CC14 to require an appraisal of its use in
studies of the acute oral toxicity of CC14 and other VOCs. It was suggested that aqueous
Emulphor® emulsion would serve as an appropriate vehicle in studies of VOC contaminants
in drinking water, as the emulsion did not substantially alter the pharrnacokinetics or

hepatotoxicity of CC14 from that ingested in water.

Fasting

In a study aimed at assessing the effects of 24 hour fasting on mice used in toxicology
tests, Strubelt et al. (1981) showed that such fasting altered the results to an unpredictable
degree. Results showed increased CC14 content in the liver of 30%, increased extent of CC14
bound in vivo to hepatic protein, decreased liver weight of 29%, decreased hepatic glycogen
of 93%, decreased hepatic glutathione (GSH) of 50%, and increased liver triglycerides of
162% in fasting mice. No unique explanation could be made for the increased susceptibility
of mouse liver to toxic injury induced by fasting but some factors were suggested. Depletion
of both hepatic glycogen and glutathione as well as hepatic lipid accumulation could play an

important role.

38

In acute ingestion exposure animal toxicity studies, mice should be fasted for about
4 hours whereas fasting times are greater for rats and other mammals. It has been noted that
LD50 values may differ by a factor of 2 when gavaged doses are administered to non-fasted
animals (Boyd, 1959; Boyd et al., 1965).

Whitten Effect

. Group-housed female mice, when introduced to a male, will undergo a synchronized
estrous cycle with a characteristic pattern of mating where the majority of animals mate on
the third day. This phenomenon is termed the Whitten effect.

Pregnant mice that mated on the third day and that were exposed to diethylstilbestrol
(DES) were significantly more susceptible to DES-induced abortion than animals that mated
on any other day (Jackson and Wordinger, 1990). A significant increase in the ratio of
embryos between uterine horns was observed in the animals that mated on Day 3 (2.13)
compared to the animals mating on other days (1.23 to 1.48). Although Jackson and
Wordinger concluded that the cause of increased sensitivity to DES in Day-3 mated animals
was unclear, it was postulated that the induction of ovulation associated with the Whitten
effect could have caused unequal embryo distribution leading to functional differences in

sensitivity of the pregnant mice to exogenous stimuli.

MATERIALS & METHODS

In Vivo Fertilization Trials

Animals: Male and female C57BL/6 x DEA/2 (B6D2F1) mice, aged 4 months and 6 to 8
weeks, respectively, were used. Mice were purchased from The Jackson Laboratory (Bar
Harbor, ME).

A number of mice were removed from the Endocrine Research Center during January
of 1991. A random population of these animals were tested for the presence of pathogens. All
randomly-chosen subjects tested positive for mouse hepatitis virus (MHV). MHV is
characterized by inapparent infection, wasting disease in nude or thymectomized mice,
diarrhea (often fatal) in suckling mice, ruffled fur, inactivity, incoordination, and paresis
(Conliffe-Beamer and Les, 1987). In addition, the age of the animal and the strain of the virus
influence the clinical signs of MHV. Although the remaining mice showed no symptoms of
the disease, it is assumed that our animals would test positive and the necessary steps were
taken to eradicate the carrier mice from the colony.

The in vivo fertilization trials were completed with no indication of MHV infection
in the animals. There was no decrease seen in bleedin g litter size or in experimental litter size,
and no effects were evident in any other parameters in the experimental animals. The in vitro
fertilization trials were carried out prior to and after the possibility of MHV infection. As the
virus is unable to cross the zona pellucida of the mouse oocyte (A.L. Smith, personal

communication), there is no reason to believe that the in vitro fertilization trials were affected.

39

4o

Trichloroethylene: Spectrophotometric grade trichloroethylene (TCE) was purchased from
the Aldrich Chemical Company (Milwaukee, WI). The chemical formula for TCE is
ClCH=CCL2, and catalog #25,642-O and lot #007 23AP was used.

Dose: The dose of administered TCE was based upon the 50% lethal dose (LDso) value of
2402 mg/kg body weight (b.w.) in mice taken from the Registry for Toxic Effects of Chemical
Substances (RTECS) (NIOSH, 1988). Two doses of TCE were tested which were 1/10 and
l/100 of the LD50, Control doses consisted of pure corn oil. Doses were calculated per
replication as follows:

LDso (mg/kg) x average b.w. (g) x 1x10‘5 (kg/mg) = LDso (g).

[LD50 (g) x ml]/ 1.464 g = LDso (ml).

High dose = 0.1 x LD50 (ml).

Low dose = 0.01 x LDso (ml).

The volume of liquid given per animal was 0.2 ml and doses were constituted in 100
ml volumes, 100% corn oil vehicle + TCE, to contain the proper concentrations of test
chemical.

Doses were prepared in clean, reverse-osmosis (RO) water rinsed and autoclaved
glass bottles wrapped in foil. Teflon coated magnetic stir bars were used for initial mixing

and prior to daily dosing. Doses were made up immediately preceeding the first dosing.

Administration: Dosing per mouse was performed using an 18 gauge, 3.8 cm curved gavage
needle (Perfektum, New Hyde Park, NY) attached to a graded, 3cc glass syringe. The gavage
needle was inserted into the esophagus taking care not to enter the trachea, and the test
solution was extruded quickly. Food was withheld from all animals for 4 hours prior to daily
gavagin g, and administration was performed between 1300 and 1500 hrs. Mice received the

41

gavage for 5 consecutive days beginning on Day 1, Day 6 or Day 1 1, with Day 1 representing
the first day following conception confirmed by the presence of a vaginal plug.

Synchronous Pregnancy Induction: Female-only housed mice were placed one to one with
male mice beginning on the first afternoon of arrival to maximize mating success on the third
evening by the Whitten effect (Whitten, 1971). Successful matings were assessed by the
presence of a vaginal plug between 0800 and 0900 hr. the following morning. A minimum
of 15 females were used per replication, 5 animals per dose level, and a treatment consisted
of 2 replications. Only animals exhibiting synchronous (same day) or near synchronous
(within one day) vaginal plugs were used. Individual animal weight on Day 1 (vaginal plug)
was used to allocate animals to dose levels and to block any weight bias in the trials beginning
on Day 1 or Day 6. Animal weight on Day 11 was used to block for weight bias in the Day
11' to 15 trial.

Data Collection: Dosed female (F1) mice were weighed on Days 1, 8, and 15 of pregnancy
and on Days 1, 8, 15 and 22 following parturition in Day 1 to 5 and Day 6 to 10 trials. In the
Day 11 to 15 trial, prepartum weights were taken only on Day 11 and Day 15. In all cases
parturition occurred on Day 19 with conception as Day 1. After the last weight was recorded,
the F1 females were starved for one hour, killed and the liver and both kidneys excised,
weighed and preserved in 10% buffered formalin. One kidney was partially cut in the sagittal
plane, the other in the frontal plane. Spleen length was recorded also. Data were collected
from the pups of each F1 female. Pup weight was recorded on Days 1, 8, 15, and 22, and
crown-rump (CR) length was recorded on Days 1, 8, and 15. In addition, the incidence of
gross abnormalities on Day 1, lower incisor eruption on Day 11 and eye opening on Day 15
were recorded. Measurements were taken for the whole litter on Day 1, and then litters were

reduced to 6 at a 1:1 sex ratio when possible. On Day 22, each litter was weaned, reduced

42

further to 4 pups, and allowed to develop until 6 weeks of age. On Day 43, two randomly
chosen litters per dose level were weighed, killed and the ovaries and testes removed for
weighing and preservation in Bouin’s fixative. See Figure 2 for a diagram illustrating the

experimental protocol and data collection time points.

Solutions: Buffered formalin was made by combining 100 ml of 37% formaldehyde, 900 ml
of RO water, 4.0 g sodium phosphate monobasic and 6.5 g sodium phosphate dibasic
(anhydrous). Bouin’s fixative was made by combining picric acid, saturated in aqueous

solution, and glacial acetic acid at a ratio of 5 to 1.

Statistical Analyses: Statistical methods were taken from Gill (1978). Where statistical
significance was found, confidence levels were indicated. One-way analysis of variance
(ANOVA) was used to test for differences of treatment means with significance at the 5%
level, except for the sex ratio data which were analyzed by a binomial probability test. Data

transformation was used when original data showed heterogeneous variance.

In Vitro Fertilization Trials

Animals: Male and female C57BL/6 x DEA/2 (B6D2Fl) mice, aged 4 months and 3 to 5
weeks, respectively, were used. Mice were bred on site from parental strains obtained from

The Jackson Laboratory (Bar Harbor, ME).

Trichloroethylene and Metabolites: TCE used for the in vitro fertilization trials was the same
as reported in the in vivo fertilization subsection of the materials and methods section.
Dichloroacetic acid (DCA), trichloroacetic acid (TCAA), and trichloroethanol (TCOH) were
purchased from the Aldrich Chemical Co. (Milwaukee, WI). The chemical formula for DCA

@9555 95 eoswmoa 858 can 555 H+O
85. v 9 e852 5:: mi
035 o 9 e852 5:: Tm
82030 mew—25 emu m
.3385 55:5 .5505 .532 H
coinage 5335.552“ <
3958.— 5w=2 558-5595 MD
9555— 93 50>: v74
8282 ans; 3 ”>5.

 

 

9+0 3. E
m _ <
3 3 5? 5? 5? 5
a mi 3 3 3 3 Hanna:
xxxxx
xxxxx
xxxxx amaze
MVooo\\eooNN oooooo n“ ...... w ...... fl. Goa?“
G~ . . . W“ ...... w ...... fl %O§=w£
5Q

 

.255 55398 95> 5 05 5.5 352— 0:5
550:8 fine 95 585.5 355598 05 wee—ESE 5835 .N nun—mi

44

is C12CHC02H. 99+% grade DCA with a boiling point of 194°C, melting point of 9-11°C,
nD20=1.4663, d=1.563, m.w.=128.94, catalog #D5,470-2, and lot #06726AX was used. The
chemical formula for TCAA is CC 13C02H. 99+% A.C.S. grade TCAA with a boiling point
of 196°C, melting point of 54-56°C, m.w.=l63.39, catalog #25,139—9 and lot#10608HX was
used. The chemical formula for TCOH is CC13CH20H. 99+% grade TCOH with a boiling
point of 151°C, melting point of 17.8°C, nD=1.4900, d=1.557, m.w.=l49.40, catalog
#T5,480-1 and lot #11104DX was used.

Dose: TCAA was constituted in culture medium to yield concentrations of 100 and 1000 ppm
on a vol./vol. basis. The control dose consisted of culture medium only. Doses were

calculated per replication as follows:

 

 

 

40 ul TCAA x 1 ml = 1 part TCAA = high dose
40 ml culture 1000 ul 1000 part culture
medium medium

2 ml of the above solution was then diluted with 18 ml of culture medium to make 1 part
TCAA in 10,000 parts culture medium (100 ppm) for the low dose.

A hypothetical exposure of 1000 ppm TCE was used in the dose calculation for the
factorial trial of metabolites TCAA and TCOH. As TCE is metabolized to approximately
25% TCAA and 75% TCOH, maximum exposure to the organism would be 250 ppm TCAA
and 750 ppm TCOH. The following calculations were used to convert a hypothetical TCE
exposure to pl of TCAA and TCOH. Conversion factors were derived from the molecular

weight and density values for the three chemicals as follows:

TCAA = 163.4 gm/mole, d=1.629 gm/ml ......................... 100.31 ml/mole
TCOH = 149.4 gin/mole, d=1.557 gm/ml ........................... 95.95 ml/mole
TCE = 131.4 gm/mole, d=1.465 gm/ml ........................... 89.69 mllmole

45

Conversion factor for TCAA, 100.31 ml/mole = 1.118
89.69 ml/mole

 

Conversion factor for TCOH, 95.95 ml/mole = 1.070.
89.69 ml/mole

 

The conversion factor equates the ml per mole value of exposure to metabolite, i.e., for 1 ml
TCE, 1. 1 18 ml of TCAA is potentially generated if all TCE is converted to TCAA. Therefore,
for a hypothetical exposure of 1000 ppm TCE, or 40 pl chemical per 40 ml medium:

TCAA; 40 pl x 1.118 x 0.25 = 11.20 pl and
TCOH; 40 pl x 1.070 x 0.75 = 32.10 pl.
Administration: TCAA and TCOH were mixed in culture medium in sterile glassware,

washed and rinsed in R0 water using teflon coated stir bars. Culture dishes were loaded with

1.0 ml of treated medium.

Superovulation: Female B6D2F] mice were superovulated by subcutaneous (s.c.) injection
with 8 IU pregnant mare’s serum gonadotropin (PMSG) and 8 IU human chorionic
gonadotropin (HCG) (Sigma Chemical Co., St. Louis, MO) 48 to 51 hours later (Cosby et al.,
1989).

Culture Media: The culture medium used for the in vitro fertilization (IVF) trials was
Brinster’s medium for oocyte culture (BMOC-3) (Gibco, Grand Island, NY). An alternate
medium was constituted in the laboratory and used in the outer well of Falcon organ tissue
cultm'e dishes (Becton-Dickinson, and Co., #3037, Cockeysville, MD) during incubation.
This medium was termed BMOC. The components of BMOC and BMOC-3 are listed in
Table 2.

In a laminar flow hood, 1.0 ml of BMOC-3 and 3.0 ml of BMOC were placed in the inner

46

Table 2. Components of two culture media used in the in vitro fertilization trials of
trichloroethylene (TCE), trichloroacetic acid (T CAA),trichloroethanol
(TCOH), and dichloroacetic acid (DCA).

 

 

Component BMOC' BMOC-3‘
NaCl 119.37 mM 94.90 mM
Na-lactate --- 20.11 mM
Na-pyruvate 1.02 mM 0.51 mM
KCl 4.78 mM 4.78 mM
CaClz-ZHZO 1.71 mM 1.28 mM
KH2PO4 1.20 mM 1.20 mM
MgSO4-7HZO 1.19 mM 1.19 mM
NaHCO3 25.07 mM 25.07 mM
Bovine serum albumin (BSA) -—- 5 gm/L
Glucose 5.55 mM 5.55 mM

 

'Brinster’s medium for oocyte culture GBrinster, 1971).

47

and outer wells of each culture dish, respectively, per epididymal sperm sample to be
collected (one per male). Chemicals of interest were mixed in BMOC-3 and placed in the
inner wells of the appropriate culture dishes. Culture dishes were placed in a sin gle-chamber

incubator of 5% COflair at 37°C approximately 15 hours prior to IVF.

Gamete Recovery: For each IVF procedure, 2 to 3 male mice were killed by cervical
dislocation for sperm donation. With each mouse, a single cauda epididymus was excised,
placed in BMOC-3 and poked repeatedly with a 25 gauge needle. Each epididymal
suspension was replaced to the incubator for 1 to 1.5 hours.

Beginning 1 hour after sperm collection, 6 female mice were killed by cervical
dislocation for cumulus mass donation. Both oviducts and uteri were excised, placed in
BMOC—3 and stripped of their cumulus masses. Each cumulus mass was washed and placed

in 3a preincubated, treatment culture dish.

In Vitro Fertilization (IV F): Fifty pl of sperm suspension were placed in each cumulus mass
culture so that sperm from each male inseminated cumulus masses across treatment groups.
Similarly, no two cumulus masses from the same female were used in the same treatment
group. Treatment and control group cultures were replaced to the incubator for a 24 hour

incubation period.

Hoescht Nuclear Stain #33258: Immediately prior to the 24 hour assessment of NF cultures,
a stock solution of the vital nuclear stain Bisbenzimide H 33258 fluorochrome, trihydrochloride,
pentahydrate (Calbiochem-Benning, La Jolta, CA) was constituted. Stain (0.002 gm) was
placed in 10 ml of BMOC. From this, 0.1 ml was added to each culture dish and then replaced

to the incubator for a 30 minute period to allow for uptake and interchelating of the stain in

48

the DNA. At the end of the incubation period, each culture dish was scored for percent oocytes
fertilized. Oocytes were considered fertilized by the presence of 2 cells with 1 polar body or
1 cell with 2 pronuclei, or oocytes were considered unfertilized by the presence of 1 cell with
1 pronucleus or if degenerate. Cultures were analysed on a Nikon Diaphot inverted
microscope equipped with fluorescent objective lenses (20X and 40X). Fluorescence was
visualized using a Nikon epifluorescence filter set (365/10 exciter, 400 dichroic mirror, 400

barrier filter) at a 365 nm wavelength with a 50 watt mercury arc lamp.

Statistical Anaylses: Statistical methods were taken from Gill (1978). Where statistical
significance was found, confidence levels were indicated.

Results of the NF trials were analysed by multiple contingency tables for a single
compound at 3 dose levels, or by factorial contingency tables for 2 compounds either alone

or together.

RESULTS

In Vivo Fertilization Trials
Replicate treatment data sets were pooled for tests of statistical significance.
Tables 3 and 4 show the reproductive performance of the female B6D2F1 mice as
calculated by a Fertility Index (FI) and a Gestation Index (GI), respectively. The data in these
tables are presented per replicate. Fertility Index is defined as the number of pregnant females
divided by the number of mated females times 100. Similarily, Gestation Index is defined as
the number of litter-bearin g females divided by the number of pregnant females.

A total of 214 female B6D2F] mice were mated one-to-one with a pool of fertile, male
B6D2F1 mice. Matings were assessed for up to four days after cohabitation of female and
male mice. On the first, second, third and fourth nights, 41, 41, 40 and 13 mice became
pregnant. Of the 135 pregnant mice, 111 were allocated to experimental groups. Seventy-
nine mice failed to become pregnant and were not used. Table 4 shows per replicate which

animals bore litters.

Maternal: Body weight of dams was recorded two or three times prior to parturition and
beginning on the f'u'st day of pregnancy at 7 day intervals until weaning of the litters. Mean
values (iSEMs) are listed in Tables 5, 6 and 7 for Days 1 to 5, Days 6 to 10, and Days 11 to
15, respectively. Weight gain of female litter-bearin g mice was not significantly different
across treatment doses and periods of administration when compared on the day of weaning.

Maternal liver and kidney weights were recorded and tissue samples were preserved for
possible histopathological examination at a later date from a random subset of animals per

treatment at approximately 12 weeks of age. See Table 8 for mean weight values (iSEMs).

49

50

Table 3. Fertility Index ‘ (Fl) per replicate for female B6D2F1 mice used in the in vivo
fertilization study of trichloroethylene (T CE).

 

 

Replicate " Day after placement with male F1
1 First 20.0 (8/40)
Second 28. 1 (9/32)
2 First 2.5 (1/40)
Second 2.6 (1/39)
Third 55.3 (21/38)
3 First 33.3 (8/24)
Second 43.8 (7/16)
4 First 5.0 (2/40)
Second 34.2 (13/38)
Third 36.0 (9/25)
5 First 8.6 (3/35)
Second 3.1 (1/32)
Third 32.3 (1031)
Fourth 61.9 (13/12)
6 First 54.3 (19/35)
Second 62.5 (IO/16)

 

‘ Fertility Index = number of pregnant females x 100%
number of mated femalg
" Replicates 1+2, 3+4, and 5+6 represent Days 1 to 5, Days 6 to 10, and Days 11 to 15 of

pregnancy respectively.

 

51

Table 4. Gestation Index ‘ (GI) per replicate for female B6D2F1 mice used in the in vivo
fertilization study of trichloroethylene (TCE).

 

 

Control 1/ 100 LDso 1/10 LDso
Replicate

1 40.0 (2/5) 80.0 (4/5)” 80.0 (4/5)
2 71.4 (5/7) 85.7 (6/7) 100.0 (7/7)
3 100.0 (5/5) 80.0 (4/5) 80.0 (4/5)
4 85.7 (6/7) 100.0 (7/7) 100.0 (7/7)
5 85.7 (6/7)c 100.0 (7/7) 85.7 (6/7)d
6 100.0 (6/6) 83.3 (5/6)° 100.0 (6/6)

 

‘ Gestation Index = number of litter-bearing females x 100%
number of pregnant females
" One female died on the second day of treatment.
° One female was killed during the gavage procedure on Day 15; 8 fetuses were found in
the uterine horns.
" One female died on the fifth day of treatment.
° One female died on the third day of treatment.

 

52

Table 5. Maternal body weight of B6D2F] mice exposed to trichloroethylene (TCE) by
gavage on Days 1 to 5 of pregnancy.‘

 

 

Control 1/ 100 LDso 1/10 LDs0
Prepartum
Day 1 18.6104 18.4104 18.6103
Day 8 21.0105 21.2105 21.3102
Day 15 32.3109 31.5109 31.4106
Postpartum
Day 1 25.6106 25.3103 24.8102
Day 8 29.0107 28.9105 28.5103
Day 15 31.2109 30.6107 31.1105
Day 22 29.4109 28.7107 28.0103

 

' Values represent the mean 1 SE in grams. The LDS0 used was 2402 mg/kg body weight.

Table. 6. Maternal body weight of B6D2F] mice exposed to trichloroethylene (TCE) by

gavage on Days 6 to 10 of pregnancy.‘

 

Prepartum
Day 1
Day 8
Day 15

Postpartum

‘ Day 1
Day 8
Day 15

Day 22

 

Control 1/100 1.1)so 1/10 1.1350
18.6102 18.7103 18.7104
19.9103 20.2103 20510.4
30.0105 30.6105 31.5103
23.5105 24.0103 24.1105
24.8125 27.7103 28.2104
28.7105 29.4106 29.4106
25.4104 26.2104 29.4106

 

' Values represent the mean 1 SE in grams. The LDso used was 2402 mg/kg body weight.

Table 7. Maternal body weight of B6D2F1 mice exposed to trichloroethylene (T CE) by

gavage on Days 11 to 15 of pregnancy.‘

 

Prepartum
Day 11
Day 15

Postpartum
Day 1
Day 8
Day 15

Day 22

 

Control 1/ 100 LDso 1/ 10 LDS0
23.7104 24.0106 23.5104
28.5106 28.9107 29.2104
24.0103 24.0106 24.1104
28.0104 27.6105 27.9103
31.6103 31.3106 31.4105
27.9105 27.7107 27.8106

 

' Values represent the mean 1 SE in grams. The LDS0 used was 2402 mg/kg body weight.

55

Table 8. Liver and kidney weights of female B6D2F1 mice exposed to trichloroethylene
('1' CE) by gavage during Days 1 to 5, Days 6 to 10 and Days 11 to 15 of

 

 

 

 

pregnancy.‘
LIVER
Control 1/ 100 LDso 1/10 LDSo
Replicate
1+2 Absolute 1.95 10.05 1.89 10.07 1.83 10.05
Relative 6.501014 6.57 10.10 6.49101 1
3+4 Absolute 1.65 10.06 1.74 10.06 1.801004
Relative 6.45 10. 14 6.63 10.14 6.5410. 14
5+6 Absolute 1.901012 1.83 10.08 1.821007
Relative 6.801012 6.501014 6.47 10.10
KIDNEY
Control 1/100 LD 50 1/10 LDS0
Replicate
1+2 Absolute 0.39 10.02 0.401001 03910.01
Relative 1.301002 1.38 1003" 1.391002"
3+4 Absolute 0.321001 0.34 10.01 03410.01
Relative 1.27 10.02 1.29 10.03 1.221002
5+6 Absolute 0.33 10.02 03210.01 0.321001
Relative 1.201005 1.141010 1.171002

 

 

 

' Mice were killed at approximately 12 weeks of age. Absolute values are mean 1 SE in
grams, and relative values are mean percent body weight 1 SE in grams. The LDso used
was 2402 mg/kg body weight. Replicates 1+2, 3+4, and 5+6 represent Days 1 to 5, Days
6 to 10, and Days 11 to 15 of gestation, respectively.

" Treated group relative kidney weights were significantly different (P<005) than the
control group.

56

Relative kidney weights collected from low and high dose group females treated during Days
1 to 5 were significantly different (P<0.05) than the control group. Absolute and relative
weights tended to decrease with increasing period of pregnancy when treated. Intra-replicate
variation was less than inter-replicate variation. No other trend in organ weights was seen.

Litter size and sex ratio were recorded on the day of birth and no effect was seen following
treatment at any of the three periods of pregnancy. A grand total of 878 pups were delivered,
of which 438 were female and 440 were male (Table 9). Binomial probability showed no
significant difference in the sex ratio during the three treatment periods of pregnancy. Mean

litter size ranged from 9.1 to 9.8 pups per litter.

Fl Generation: Litter weight and CR length values were recorded and averaged per dam.
Tables 10, 1 1 and 12 list the mean body weight values (1SEMs) for Days 1, 8, 15 and 22, and
the mean CR values (1SEM) for Days 1, 8 and 15 postpartum. No significant differences
were seen in any treatment. Inna-replicate variation of body weight and CR length was less
than inter-replicate variation.

Ovary and testis weights were recorded for a subset of litterrnate pups at six weeks
of age (within the set of maternal liver and kidney subjects) for each treatment. Table 13 lists
the mean ovarian and testicular weights (1SEMs). No significant difference was seen in the

relative gonad weights.

In Vitro Fertilization Trials

Table 14 shows the in vitro fertilization rate for B6D2F] mouse gametes placed in a
culture medium with TCE. Seventy-eight percent of the oocytes in the high dose group (1000
ppm) were fertilized. This amount, although less than both the control and low dose group,
was not significant.

Table 15 shows the in vitro fertilization rate for oocytes and sperm placed in culture

medium containing TCAA. Only 53.1% of the oocytes exposed to 1000 ppm TCAA were

57

Table 9. Summary data on litter size of female B6D2F] mice exposed to trichloro-
ethylene (TCE) by gavage during Days 1 to 5, Days 6 to 10 and Days 11 to 15

 

 

 

 

of pregnancy.‘
Control 1/ 100 LDSO 1/10 LDS0 Total

Replicate 1+2

No. of litters 7 10 11 28

Females 28 38 47 l 13

Males 37 53 54 144

Total 65 91 101 257

Mean1813 9.3105 9.1104 9.2103
Replicate 3+4

No. litters 1 1 l 1 11 33

Females 57 48 62 167

Males 43 53 46 142

Total 100 101 108 309

Mean1SE 9.1104 9.2104 9.8104
Replicate 5+6

No. litters l 1 l2 1 1 34

Females 47 58 53 158

Males 54 51 49 154

Total 101 109 102 312

MeaniSE 9.2103 9.1104 9.3102

 

' The LDso used was 2402 mg/kg body weight. Replicates 1+2, 3+4, and 5+6 represent
Days 1 to 5, Days 6 to 10, and Days 11 to 15, respectively.

58

Table 10. Mean pup weight and crown-rump (CR) length averaged per dam of B6D2F]
mice exposed to trichloroethylene (TCE) by gavage on Days 1 to 5 of

 

 

pregnancy.‘
Control mm ID,() 1/10 LD50

Weight (gm)

Day 1 1.261001 1.261002 1.281002

Day 8 5.231017 5.041013 5.051009

Day 15 8.791009 8.841015 8.671014

Day 22 11.051027 10.861027 10.871015
CR length (mm)

Day 1b 27.3101 27.5101 27.3101

Day 8b 40.8104 41.3103 41.5102

Day 15 50510.3 50810.3 50.1102

 

‘ Values represent the mean 1 SE. The LDSo used was 2402 mg/kg body weight.
b Data for CR length are from replicate 2 only.

59

Table 11. Pup weight and crown-rump (CR) length averaged per dam of B6D2Fl mice
exposed to trichloroethylene (TCE) by gavage on Days 6 to 10 of pregnancy. '

 

 

Control 1/ 100 LDSo 1/ 10 LDso
Weight (gm)
Day 1 1.271002 1.281002 1.271003
Day 8 5.431006 5.521008 5.591011
Day 15 9.131015 9.321017 9.361026
Day 22 11.981019 12.121019 12.041030
CR length (mm)
Day 1 26.41013 26.61016 26.61020
Day 8 40.410.20 41.611.01 40.810.30
Day 15 52.41040 52.81039 53.31055

 

‘ Values represent the mean 1 SE. The LDso used was 2402 mg/kg body weight.

Table 12. Pup weight and crown-rump (CR) length averaged per dam of B6D2F1 mice
exposed to trichloroethylene (T CE) by gavage on Days 11 to 15 of pregnancy.‘

 

 

Control 1/100 LD so 1/ 10 LD 5o

Weight (gm)

Day 1 1.251002 1.271002 1.251003

Day 8 4.801016 4.961005 5.021007

Day 15 9.171019 9.211025 9.311014

Day 22 11.651027 11.901027 11.861019
CR length (mm)

Day 1 26.0102 26.1101 25.9102

Day 8 38.7106 39.8102 39.8102

Day 15 50.8106 51.2105 51.7103

 

‘ Values represent the mean 1 SE. The LD so used was 2402 mg/kg body weight.

61

Table 13. Relative gonad weights of a subset of littermates averaged per B6D2F] dam at
6 weeks exposed to trichloroethylene (T CE) by gavage on Days 1 to 5, Days

6 to 10, and Days 11 to 15 of gestation.‘I

 

 

Control 1/ 100 LDso l/10 LDso

Replicate 1+2

Female 0.041 10.003 0.03610002 0.033 10.002

Male 0.648 1 0.014 0.77010049 0.68110034
Replicate 3+4

Female 0.033 10.005 004010002 0.038 10.003

Male 0.745 10.053 0.707 10.043 0.72110009
Replicate 5+6

Female 004010.002 0.03910003 0.031 10.004

Male 0.693 10.069 0.663 10.035 0.775 10.061

 

' Values represent the mean percent of body weight 1S5 in grams.The LDoo used was
2402 mg/kg body weight.

62

Table 14. In vitro fertilization of B6D2F1 mouse gametes in BMOC-3 culture medium

 

 

with trichloroethylene (TCE).
DOSE (ppm)
Control (0) Low (100) High (1000)

No. of fertilized

embryos 44 38 39
No. of incubated

embryos 52 44 50
% fertilized 84.6 86.4 78.0

 

Table 15. In vitro fertilization of B6D2F1 mouse gametes in BMOC-3 culttu'e medium
with trichloroacetic acid (TCAA).

 

 

DOSE (ppm)
Control (0) Low (100) High (1000)
No. of fertilized
embryos 122 1 14 93
No. of incubated
embryos 148 142 175
% fertilized 82.4 80.6 53.1‘I

 

' Significantly different (P<0001) from control and low dose groups.

63

fertilized; this value was significantly less (P<0.001) than the control and low dose groups.

The results of the in vitro fertilization trials using DCA-containing culture medium
are shown in Table 16. Only 78.1% of the high dose (1000 ppm) and 68.1% of the low dose
(100 ppm) group oocytes were fertilized compared to 87.0% for the control group. Exposure
of oocytes and sperm to DCA significantly lowered the fertilization rate for the low dose
group (P<0.025) but the fertilization rate for the high dose group was not significantly
(P2010) different.

When oocytes and sperm were placed in medium containing 250 ppm TCAA, 77.3%
were fertilized. In 750 ppm TCOH, 60.0% of the oocytes were fertilized. Oocytes and sperm
placed in culture medium with both TCAA and TCOH at the above concentrations resulted
in only 47.3% being fertilized. See Table 17 for the results of of the factorial combination of
TCAA and TCOH. There was no interaction.

Table 16. In vitro fertilization of B6D2F1 mouse gametes in BMOC—3 culture medium
with dichloroacetic acid (DCA).

 

 

DOSE (ppm)
Control (0) Low (100) High (1000)
No. of fertilized
embryos 4O 35 28
No. of incubated
embryos 46 52 39
% fertilized 87.0 67.3' 71.8b

 

' Significantly different (P<0.025) from control dose group.
" Significantly different (P<0.10) from control dose group.

Table 17. In vitro fertilization of B6D2F1 mouse gametes in BMOC-3 culture medium
with trichloroacetic acid (TCAA), trichloroethanol (T COH), or both. ‘

 

No. of fertilized embryos

 

 

Treatment No. of incubated embryos (%)
Control 93/ 106 (87.7)
TCAA 85/110 (77.3)b
TCOH 54/90 (60-0)c
TCAA + TCOH 43/91 (47.3)

 

' Based upon hypothetical exposure to 750 ppm TCOH and 250 ppm TCAA.
b Significantly different (P<0.025) from control dose group.
° Significantly different (P<0.001) from com'rol dose group.

DISCUSSION

The results of this study demonstrated that oral dosing of B6D2F1 mice with TCE on
Days 1 to 5, 6 to 10, or 11 to 15 of gestation had no adverse effect on reproduction. TCE was
tested at 1/10 and 1/ 100 of the published oral LDso value for mice (Klaasan and Plaa, 1966;
Aviado et al., 1976; NIOSH, 1988; Tucker et al., 1982). It has been argued that reactive
intermediate metabolites of TCE may act epigenetically and be cytotoxic to the liver and
therefore any effects of TCE should exhibit a threshold (Kimbrough et al., 1985). We chose
1/ 10 the oral LDso as our maximum dose in order to not generate a toxic maternal effect while
observing for possible reproductive effects.

. Recent publications (Parchman and Magee, 1982; Stott et al., 1982; Elcombe et al.,
1985; Green and Prout, 1985; Prout et al., 1985; Nelson and Bull, 1988) clearly demonstrate
that mice and rats metabolize TCE differentially. Not only are the dose and route important
parameters, but mice, in contrast to rats and humans, have been shown to convert TCE to an
additional metabolite, dichloroacetic acid, which could perpetrate the mutagenic and
tumorogenic effects of the parent compound (Hathway, 1980). This could result from the
rearrangement of the 2-haloalkene-derived haloepoxides into chloroacetyl chloride and
dichloroacetyl chloride (Kimbrough et al., 1985). In rats and humans, TCE becomes simply
chloral and further products of its metabolism render it relatively non-toxic in these species
(Kimbrough et al., 1985). Radio-labeled TCE experiments have shown that mice metabolize
more of the compound than do rats to a reactive intermediate capable of binding to
macromolecules in hepatic and renal tissues (Kimbrough et al., 1985).

The carcinogenicity of TCE in animals is unclear and it has been designated a group

65

66

3 carcinogen by the IARC (1987). Epidemiological studies of exposed humans do not
support the carcinogenicity of TCE. A host of in vitro studies have added to the controversy
on the possible carcinogenicity of TCE as discussed previously. In essence, TCE tested
negative, weakly positive, and positive in mutagenicity and transformation assays. The
consistent transformation results (all positive) in contrast with the inconsistent mutagenicity
results lend support to the growing concept that TCE may act through an epigenetic
mechanism. A recent study of gossypol in mice and chicks showed this chemical to be
embryotoxic with no evidence of mutagenicity in the Ames test (Li et al., 1989).

Tucker et al. (1982) found an oral LD50 for TCE in mice to be 2443 mg/kg in females
and 2402 mg/kg in males. They then dosed the drinking water with 0.1, 1.0, 2.5 and 5.0 mg/
ml of TCE for 4 for 6 months. Body weight gain was decreased at the highest dose due to
decreased water consumption. An increase in liver and kidney weight was seen in both sexes.
In another trial, Tucker et al. (1982) dosed male CD- 1 mice orally with 240 and 24 mg/kg TCE
in 10% Emulphor® solution. Neither body weight nor most organ weights were affected.
Only a dose-dependent increased liver weight in the high dose group was seen. The results
of our study are in agreement with Tucker et al. (1982). Although the same dose levels were
used in both studies, they used drinking water administration of TCE emulsified in
Emulphor® and dosed male mice. Sanders et al. (1982) studied the effects of TCE on the
immune system and found in females that humoral immunity was inhibited at 2.5 and 5 mg/
ml, whereas cell-mediated immunity and bone marrow stem cell colonization were inhibited
at all concentrations tested (0.1, 1.0, 2.5 and 5 mg/ml). Recent studies indicate an important
role of the immune system in early pregnancy and such a suppression or interference could
have profound effects on reproduction.

Stott et al. (1982) studied the pharmacokinetics and molecular interactions of TCE in
mice and rats. In male B6C3F1 mice, liver/b.w. ratios were significantly increased in the 500

to 2400 mg/kg dose levels. There was a concomittant significant decrease in pg DNA/g

67

hepatic tissue. As expected, histopathology was most pronounced in the high dose group and
was dose related. No renal effects were noted. TCE did not readily bind hepatic DNA in vivo.
The authors suggested from these results, and those of prior experiments, that TCE acted
through an epigenetic mechanism of tumorigenicity by enhancing the normal cellular
mutation rate during regenerative activity (increased DNA synthesis) in response to a
cytotoxic challenge by a chemical.

In the study reported here, no effect was seen in liver/b.w. ratio as in the Stott et al.
(1982) study below 500 mg/kg TCE. This and other studies support a hypothesis that TCE
must be available at relatively high doses, near the LD50, for it to be tumorigenic. Again, the
study herein involved dosing female pregnant B6D2F] mice, and the strain, gender, and
reproductive status of the animals could be significant factors in the effects of TCE.

Two complimentary studies compared the species differences in mice and rats in
response to TCE with respect to pharmacokinetics and biotransformation. Prout et al. (1985)
tested single oral doses of C-labeled TCE at dose levels from 10 to 2000 mg/kg in rats and
mice. The results showed a marked dose-dependence in rats but not in mice. At high closes,
the mouse was exposed to significantly higher concentrations of TCE metabolites than the
rat. Blood level kinetics of TCE and its metabolites confu'med a faster rate of metabolism
in the mouse than in the rat. It was concluded that the high TCAA blood amounts, peroxisome
proliferation, and the link between peroxisomes and liver cancer were the basis of species
differences in response to TCE.

When assessing for a species-specific biotransformation of TCE, Green and Prout
(1985) failed to detect a difference. Although a greater proportion of the dose was
metabolized in mice than in rats, the relative proportions of the major metabolites were
similar in both strains and were unaffected by the dose amount. These workers reported
finding DCA in the urine of both rats and mice at less than 1% of the dose. Again, little

evidence was found for the formation of chemically reactive species from TCE in either rats

68

or mice and none that could be the basis of a major species difference. Echoing the conclusion
of the companion study, it was proposed that the increased rate of metabolism in the mouse,
the high blood concentrations of TCAA, and stimulation of hepatic peroxisome proliferation
appeared to be the major species difference.

In previous reproductive studies, Schwetz et al. (1975) found no maternal, embryonal
or fetal toxicity following TCE inhalation in mice or rats at 300 ppm for 7 hr. daily during
days 6- 15 of gestation (vaginal plug=day 0). There was no evidence of teratogenicity either,
although there were 3 incidences of fetal defects. The mice used were Swiss Webster White
(SWW), weighing 25-30 g, of an unspecified age.

In two inhalation studies, TCE was found to exert no maternal, fetal or teratogenic
effects in female rats (Dorfmueller et al., 1979) but did affect sperm after exposure of male
mice to TCE anesthetic (Land et al., 1981). Dorfrnueller et al. (1979) dosed females before
and during pregnancy in a 2 by 2 factorial design. Their aim was to compare the effects of
dosing for the 14 days prior to and the first 20 days of pregnancy with dosing during either
time period alone. They found significant increases in skeletal and soft tissue anomalies
indicating developmental delay in maturation and not teratogenesis in the group exposed
during pregnancy alone. A reduction in postnatal body weights was seen in litters from
mothers with pregestational exposure. They concluded that no treatment-related maternal
toxicity, embryotoxicity or severe teratogenicity occurred. Land et al., (1981) looked at
epididymal spermatozoa morphology following TCE-induced anesthesia (inhalation) for 4
hrs per day for 5 days at concentrations of 0.2 and 0.02% (MAC=1.0 and 0.1). A significant
increase in abnormal spermatozoa was seen with the high dose of TCE. They did not test the
ability of treated male mice to fertilize normal females or the in vitro fertilizing capacity of
the treated sperm.

The single rodent (rat) gavage study of TCE on reproduction revealed no maternal
effects although whole body effects were noted at the high dose level of 1000 mg/kg/day

69

(Manson et al., 1984). In male rats, TCE was administered by gavage up to 1000 mg/kg for
5 days per week for 6 weeks (Zenick et al., 1984). Copulatory behavior and semen
evaluations were conducted the first week and the last week as well as 4 weeks post-exposure.
Also, some animals were sacrificed at the end of exposure and levels of exposure of rats to
TCE and its metabolites measured in various organs and blood. TCE-related effects were
seen primarily in the high dose group as reduced b.w. gain, elevated liver/b.w. ratios, and
impaired copulatory behavior. Intrauterine exposure of rats to TCE resulted in significant
alterations in exploratory and locomotor activity after birth (Taylor, 1985). Studies in mice
and rats suggest that brain myelination or other maturational processes may be inhibited by
intrauterine exposure to TCE (Westergren et al., 1984; Isaacson and Taylor, 1989). Again,
most studies show that TCE biotransformation and bioactivation differs between rats and
mice making comparisons difficult.

’ The results of the study reported here support, in essence, the conclusions of both
types of prior TCE reproductive studies in female mice. An increase in liver and kidney
weight was seen in TCE-dosed animals but was not significant except for the relative weights
in the Days 1 to 5 trial (See Table 8 of Results). No other effects were seen, and histopathology
of kidney, as well as liver, was negative. It is concluded, therefore, that this significant
difference is most likely a Type II statistical error. A decrease in pup weight at Day 22 showed
possible developmental delay but not teratogenicity. As evidenced by the percent females
that became pregnant, the percent females dropping litters, mean litter size, weight and GR
lengths, TCE did not affect implantation and was, therefore, neither a maternal nor a fetal
toxin. Lack of incidence of any developmental abnormalities such as retarded eye opening
and lower incisor eruption, encephalies, spina bifidas, and extra or missing digits, does not

support TCE as a potential teratogen.

70

In Vitro Fertilization Studies

Results of the in vitro fertilization studies of TCE and the metabolites DCA, TCAA,
and TCOH support what was previously known about these chemicals. No information is
available on the reproductive effects of TCOH at present. Both DCA and TCAA are known
mutagenic and toxic compounds. The fact that DCA and TCAA interfere with the fertilization
process in vitro is not surprising.

. An interest in using in vitro studies to characterize potential developmental toxins has
generated active discussion in the field of toxicology (Nau, 1990). These recent reviews help
to highlight the advantages and disadvantages of the in vitro methods. Using an in vitro
system to study a potential toxicant is less expensive, less time consuming, and entails fewer
animals than an in vivo system. On the other hand, an in vitro system differs remarkably from
a whole animal in vivo study. Pharmacokinetics is the major difference between the two
methods of toxicology. In vivo, an animal absorbs, metabolizes, distributes, and eliminates
a given compound. In vitro, the added drug persists unless it is unstable, volatile, or is reacted
upon by enzymes in the medium. In an in vivo study, a result depends on maternal absorption,
the route of administration, and the first pass effect of the liver. In an in vitro study, there is
no absorption if the compound is dissolved in the medium, unless poorly soluble. There is
no fu'st pass effect, there is no distribution, and there are no elimination pathways. Depending
on the study, often there is a lack of drug metabolizing enzymes. In light of the inherent
differences between in vivo and in vitro testing systems, a direct comparison of results from
the two is impossible. The two methods, can, on the other hand, be used to compliment each
other to shed knowledge on the particular aspects of the compound’s effect in controlled
experiments.

Further studies may be warranted to test TCE in the range between our high dose and
the oral LD50 value of 2402 mg/kg, but any reproductive effects seen following an oral
dosing of TCE greater than the high dose reported here would be secondary to a whole body

71

effect. An alternative approach would be to use a multi-generational study and test the
reproductive capacity of the F2 generation males and females. Future studies to consider
would include dosing female mice prior to conception, and dosing male mice followed by the

ability to in vitro fertilize oocytes and in vivo fertilize normal females.

SUMMARY AND CONCLUSIONS

TCE is a common halogenated hydrocarbon used mainly as an industrial solvent. Not
unsurprisingly, it is a common dumpsite and ground water contaminant. Research in animals
suggests that TCE is a weak carcinogen and mutagen; research on the reproductive effects
of TCE has been inconclusive.

When administered by gavage to B6D2F1 mice for a period of 5 consecutive days
through Day 15 of pregnancy up to 240 mg/kg b.w., TCE showed no effect on reproduction.
When added to culture medium in vitro up to 1000 ppm, the metabolites DCA, TCAA, and
TCOH, but not TCE, affected the in vitro fertilization of mouse gametes. The following
conclusions were drawn from the data:

1) Oral administration of TCE to pregnant B6D2F1 mice for 5 consecutive days beginning
either on Day 1, Day 6, or Day 1 1, did not affect implantation, organogenesis, maturation,
growth or development as evidenced by litter size and number, pup weight and CR length,
a lack of developmental abnormalities, and timing of lower incisor eruption and eye
opening.

2) The F2 generation grew into normal adults as evidenced by growth rate and gonad weight
at 6 weeks of age.

3) TCE did not permanently affect maternal B6D2F1 mice as no liver damage was evident
3 weeks after the delivery of pups.

4) TCE is not detrimental to the in vitro fertilization of B6D2F1 mouse gametes.

5) The major metabolites TCAA and TCOH, and a minor metabolite, DCA, inhibited the in

vitro fertilization of B6D2F1 mouse gametes.

72

BIBLIOGRAPHY

BIBLIOGRAPHY

Adams, C.E., M.F. Hay, and C. Lutwak-Mann. The actions of various agents upon the
rabbit embryo. .1. EM Earp. MthQL 9:468-491, 1961.

Alvares, A.P., S. Leigh, A. Kappas, W. Levin, and AH. Conney. Induction of aryl carbon
hydroxylase in human skin. 12mg M21312. 1.215125251115211 1:386-390, 1973.

Amacher, D. E., and I Zelljadt. The morphological transformation of Syrian hamster
embryo cells by chemicals reportedly nonmutagenic to Salmonella 01911111111111.1111.
Wu. 4: 291 -,295 1983.

Anders, M.W. and LR. Pohl. Halogenated alkanes. In: Bloagmmign of Eqmjgn Cam;
mugs. M.W. Anders, ed., Academic Press, New York, pp. 284-316, 1985.

Aviado, D.,M. S. Zakhari, J..A Simaan, andA..G Ulsarner. In: Memylghlgmflmnand

I‘richloroethxleneinthefinmnment LGoldbcrg. ed., CRC Press. Inc.
Cleveland, OH, pp. 47-102, 1976.

Banjeree, S., B.L. Van Duuren. Covalent binding of the carcinogen trichloroethylene to
hepatic microsomal proteins and exogenous DNA in vitro. Cancer Res. 38:776—
780, 1978.

Bartonicek, V. Metabolism and excretion of trichloroethylene after inhalation by human
subjects. Em. L Ind Med. 19:134-141, 1962.

Baselt. R.C. W Moniming Mmhnds for Industrial Chsmiszals. PSG Publishing
Company, Inc., Littleton, MA, pp. 290-293, 1988.

Bazare, Jr., J .J ., C.J. Nelson, and J.F. Young. Pharmacokinetics of 2,4,5-T in mice as a
function of dose and gestational status. Rem 1951901. 4: 137- 144, 1990.

Bergman, K. Applications and results of whole-body autoradiography in distribution
studies of organic solvents. Cm. 8mm 12:59-118, 1983a.

Bergman, K. Interactions of trichloroethylene with DNA in vitro and with RNA and
DNA of various mouse tissues in vivo. Arch ngimL 54:181-193, 1983b.

73

74

Bhunya, SR, and BC. Behera. Relative genotoxicity of trichloroacetic acid (T CA) as
revealed by different cytogenetic assays: bone marrow chromosome aberration,
micronucleus and sperm-head abnormality in the mouse. Mata}. Ros, 188:215-
221, 1987.

Bignami, M., G. Conti, L. Conti, R. Crebelli, B. Misuraca, A.M. Puglia, R. Randazzo,
G. Sciandrello, and A. Carere. Mutagenicity of halogenated hydrocarbons in
Salmonella typhimurium, Streptomyces coelicolor, and Aspergillus ridulans.

Chem..-Bi01. Interact. 309-23. 1980.

Bonse, G., T. Urban, D. Reichert, and D. Henschler. Chemical reactivity, metabolic
oxirane formation and biological reactivity of chlorinated ethylenes in the isolated
purified rat liver preparation. Bighorn. EhamaooL 24: 1829- 1834, 1975.

Bourne, R.G. Solvent hazards in dry-cleaning industry. Mod, L Anon 1:879, 1959.

Boyd, E.M. The acute oral toxicity of acetylsalicylic acid. Ioaiool. Aool, Ehannaool.
1:229-239, 1959.

Boyd, E.M., M. Dolman, L.M. Knight, and RE. Sheppard. The chronic oral toxicity of
caffeine. Cam .L EhysjoL EhamaooL 43:995- 1007, 1965.

Brancaccio, A., U. Fermariello, and N. Castellino. Riscio lavorativo nelle lavanderie a
secco con impiego di tricloroetilene. Eolia Mod (Naples) 49:729, 1966.

Breimer, D.D., H.C.J. Ketelaars, and J.M. Van Rossum. Gas chromatographic determina-
tion of chloral hydrate, trichloroethanol and trichloroacetic acid in blood and urine
employing head-space analysis. L (llll'omatt 88:55-63, 1974.

Brinster, R.L. In vitro culture of the embryo. In: Balm to Conoeotjom A.I. Sherman,
ed., Charles C. Thomas, Springfield, IL, pp. 245-277, 1971.

Bruce, W.R., and J .A. Heddle. The mutagenic activity of 61 agents as determined by the
micronucleus, Salmonella and sperm abnormality assays. Can. .L Gong, anL
21:319-334, 1979.

Bull, R.J., J.M. Brown, E.A. Meierhenry, T.A. Jorgenson, M. Robinson, and J.A. Stober.
Enhancement of the hepatotoxicity of chloroform in B6C3F1 mice by corn oil:
implications for chloroform carcinogensis. Enflrom Hoalm Romp, 69:49-58,
1986.

75

Byington, K.H., and KC. Leibman. Metabolism of trichloroethylene in liver microsomes.
H. Identification of the reaction product as chloral hydrate. MoL EharmaooL
1(3):247-254. 1965.

Cantelli-Forti, G., and G. Bronzetti. Mutagenesis and carcinogenesis of halogenated
ethylenes. 1&1. Aoagl, SoL 534:679-689, 1988.

Cary, W.H., Jr., and J .M. Hepler. Health hazards in the dry cleaning industry. Ana L
mono Health 28:1029. 1938.

Chenowith, M.B. Ed. Modom lnhflatlon Anosthotios. Springer-Verlag, Berlin, p. 116,
1972.

Chieco, P., M.T. Moslen, and ES. Reynolds. Effect of administration vehicle on oral
lldichloroethylene toxicity. oniooL Aool. EharmaooL 57:146-155, 1981.

Conliffe-Beamer, TL, and ER Les. The laboratory mouse. In: Ibo LIEAW mm

on the Care and Management of Lahoram Animals. siXth edition. T. Poole. cd..
Longman Scientific and Technical, Great Britain, pp. 304-306, 1987.

Cosby, N .C., K. Chou, and W.R. Dukelow. Embryo production in B6D2F1 mice using
two superovulatory regimens. Lao, Anim. Sol, 39:249-250, 1989.

Crebelli, R., and A. Carere. Genetic toxicology of 1,1,2-trichloroethylene. Mata; Ros.
221:11-37, 1989.

Dahlberg, J.A., and L.M. Myrin. The formation of dichloroacetyl chloride and phosgene
from trichloroethylene in the atmosphere of welding shops. Ann. Qoom Hyg.
14:269, 1971.

Daniel, J .W. The metabolism of C136-labeled trichloroethylene and tetrachloroethylene in
the rat. Bjoohm EhamaooL 12(8):795-802, 1963.

Dekant, W., M. Metzler, and D. Henschler. Identification of S-1,2-dichloro-vinyl-N-
acetyl-cysteine as a urinary metabolite of trichloroethylene: a possible explanation
for its nephrocarcinogenicity in male rats. Bioohom. Eharmaool. 35:2455-2458,
1986a.

Dekant, W., A. Schulz, M. Metzler, and D. Henschler. Absorption, elimination and
metabolism of trichloroethylene: a quantitative comparison between rats and
mice. Xonooiotioa 16:143-152. 1986b.

76

DcMatteis. F.. and EA. Lock. 5912901110 and 11491991113: Mechanisms of 1081910
MacMillan Publishing Company, New York, pp. 171-181, 1987.

DiRenzo, A.B., A.J. Gandolfi, and LG. Sipes. Microsomal bioactivation and covalent
binding of aliphatic halides to DNA. oniooL Lott. 11:243-252. 1982.

Doolittle, D.J., G. Muller, and HE. Scribner. The in vivo-in vitro hepatocyte assay for
assessing DNA repair and DNA replication: studies in the CD-l mouse. Eogl

Chem. 192119.91. 25:399-405. 1987.

Dorfrnueller, M.A., S.P. Henne, R.G. York, R.L. Bornschein, and J .M. Manson. Evalua-
tion of teratogenicity and behavioral toxicity with inhalation exposure of maternal
rats to trichloroethylene. Iozgjoology 14: 153-166, 1979.

Duprat, D., and D. Gradiski. Cytogenetic effect of trichloroethylene in the mouse as
evaluated by the micronucleus test. IRCS, Moi SoL 8:182, 1980.

Bibs, H.G., H. Spielman, and M. Hagele. Teratogenic effects of cyproterone acetate and
medroxyprogesterone treatment during the pre- and postimplantation period of
mouse embryos. I. Ioratology 25:27-36, 1982.

Elcombe, C.R., M.S. Rose, and LS. Pratt. Biochemical, histological, and ultrastructural
changes in rat and mouse liver following the administration of trichloroethylene:
possible relevance to species differences in hepatocarcinogenicity. IoalooL Aool,
W 79:365-376, 1985.

Environmental Protection Agency. Environmental sources of trichloroethylene exposure:
source contribution factors. EPA-560/11-80-009. Office of Toxic Substances,
Washington, DC, 1980.

Epstein, C.J., and SA. Smith. Handling of actinomycin D by preimplantation mouse
- embryos. Emflollfioa. 111:117-126, 1978.

Fernandez, J.G., P.O. Droz, B.E. Humbert, and LR. Caperos. Trichloroethylene exposure.
Simulation of uptake, excretion, and metabolism using a mathematical model. EL

1. Ind. Med. 34:43-55. 1977.

Fernandez, J.G., B.E. Humbert, and PO. Droz. Exposition au trichloroethylene, bilan de
l’absorption de le’excretion et du metabolism sur des sujets humains. A1311. MaL
Prof, 36:397-407, 1975.

Fishbein, L. Industrial mutagens and potential mutagens. I. Halogenated aliphatic deriva-
tives. Mum Rog 32:267-308, 1976.

77

Fouts, J.R. Some comments and studies on hepatic and extrahepatic microsomal
toxication-detoxification system. Emdron, Hoalth Rom 2:55-66, 1972.

Galloway, S.M., M.J. Armstrong, C. Reuban, S. Cloman, B. Brown, C. Cannon, A.D.
Bloom, F. Nakamura, M. Ahmed, S. Duk, J. Rempo, B.H. Margolin, M.A.
Resnick, B. Anderson, and E. Zeiger. Chromosome aberrations and sister
chromatide exchanges in Chinese hamster ovary cells: evaluations of 108 chemi-

cals. Em MoLMmagon. 10:1-175, 1987.

Gehrin g, PJ. Hepatotoxic potency of various chlorinated hydrocarbon vapors relative to

their narcotic and lethal potencies in mice. IQZtiQQL Apol, Pharmaool. 13: 287-
298. 1968.

Ghantous, H., B.R.G. Danielsson, L. Dencker, J.Gorczak, and O. Vesterberg. Trichloro-
acetic acid accumulates in murine anmiotic fluid after tri- and tetrachloroethylene

inhalation. Aota Eharmaool, onjooL 58:105-114, 1986.

Gill. 1L. DesignandAnalxsisofExnenmemsintheAnimalandMedicalSflenm.
Vol. I. The Iowa State University, Ames, Iowa, 1987.

Glass, W.I., E.A. Hanis, and R.M.L. Whitlock. Phosgene poisoning: case report. 111,2,
Mod, L 742386, 1971.

Gleason, M.N., R.E. Gosselin, H.C. Hodge, and R.P. Smith. Cljnjoal Iozgjoology of
Commoroial Moots. Acute Poisoning. 3rd Ed., Williams and Wilkins,
Baltimore, 1969.

Green, T., and DE. Hathway. Interaction of vinyl chloride with rat liver DNA in vivo.
M 1m. 22:11-15, 1978.

Green, T., and J. Odum. Structure/activity studies of the nephrotoxic and mutagenic
' action of cysteine conjugates of chloro- and fluoralkenes. Chomfilol. 1m.
54:15-21, 1985.

Green, T., and M.S. Prout. Species differences in response to trichloroethylene. II.
Biotransformation in rats and mice. oniooL AooL EhannaooL 79:401-411, 1985.

Greim, H., G. Bonse, Z. Radwan, D. Reichert, and D. Henschler. Mutagenicity in vitro
and potential carcinogenicity of chlorinated ethylenes on a function of metabolic
oxirane formation. Mom. EharmaooL 24:2013-2017, 1975.

78

Hathway, D.E. Consideration of the evidence for mechanisms of 1.1.2-trichloroethylene
metabolism, including new identification of its dichloroacetic acid metabolites in
mice. Canoor Lon. 8:263-269, 1980.

Henschler, D. Metabolism and mutagenicity of halogenated olefins. A comparison of
structure and activity. Enyim Hoalm Roma 21:61-64, 1979.

Henschler, D., and G. Bonse. Metabolic activation of chlorinated ethylenes. Dependence
of mutagenic effect on electrophilic reactivity of the metabolically formated

epoxides. Arm oniool. 39:7-12, 1977.

Henschler, D., E. Eder, T. Nevdecker, and M. Metzler. Carcinogenicity of trichloro-
ethylene: fact or artifact? A1521]. Ioniool, 37:233-236, 1977.

Henschler, D., H. Elsasser, W. Romen, and E. Eder. Carcinogenicity study of trichloro-
ethylene, with and without epoxide stabilizers in mice. L Canoor Boo, Clinl QngoL
107:149-156, 1984.

Henschler, D., W. Romen, H.M. Elsasser, D. Reighert, E. Eder, and Z. Radwan. Carcino-
genicity study of trichloroethylene by longterm inhalation in three animal species.
Aron, oniooL 43:237-248. 1980.

Herren-Freund, S.L., M.A. Pereira, M.D. Khoury, and G. Olson. The carcinogenicity of
trichloroethylene and its metabolites, trichloroacetic acid and dichloroacetic acid,

in mouse liver. IoniooL Aool. EharmaooL 90:183-189. 1987.

Hogan, B., F. Costantini, and E. Lacy, eds. Manipulating Iho Mooso Emlnyo. Cold
Spring Harbor Institute, Cold Spring Harbor, NY, pp. 19-24, 1986.

Hovell, B.C., A.H.B. Masson, and J. Wilson. Trichloroethylene for post-operative
analgesia. A clinical assessment of trichloroethylene for the relief of post-opera-
tive pain. Anamhosia 22:284, 1967.

Iannaccone, P.M. Long term effects of exposure to methylnitrosourea on blastocyst
following transfer to surrogate female mice. Canooi: Ros. 44:2785-2789, 1984.

Iannaccone, P.M., N.L. Bossert, and CS. Connelly. Disruption of embryonic and fetal
. development due to preimplantation chemical insults: A critical review. Ann L
Qboton Moi, 157:476-484, 1987 .

lkeda, M., M. Yoshio, M. Ogata, and S. Ohmori. Metabolism of trichloroethylene.
Bioohom. EhannaooL 29:2983-2992, 1980.

79

Ikeda, M., and T. Imamura. Biological half-er of trichloroethylene and tetrachloro-
ethylene in human subjects. lno Aroln mm 31:209-224, 1973.

International Agency for Research on Cancer (IARC). Overall evaluations of carcino-
genicity: an updating of IARC monographs volumes 1 to 42. IARC Monogi; Exal.
Casinos. Risk Chem. Hum. SUPPI- 7. 364-366. 1987.

Isaacson, LG, and DH. Taylor. Maternal exposure to 1,1,2-trichloroethylene affects
myelin in the hippocarnpal formation of the developing rat. Brain Boo, 4882403-
-407, 1989.

Jackson, FL, and R]. Wordinger. Sensitivity to diethylstilbestrol-(DES)-induced abor-
tions is influenced by the Whitten effect. Room Wool. 4: 177-181, 1990.

Jindrichova, J. Poisoning by trichloroethylene 1n the production of brake linings. limo.
Loki 22(3): 103-107, 1970.

Joncev, V., and L. Marinov. Narcohypnosis with trilene. Eolia Moi (Plovdiv) 11:281,
1969.

Keller, D.A., and HA. Heck. Mechanistic studies on chloral toxicity: relationship to
trichloroethylene carciongenesis. oniool, Lon. 42:183- 191, 1988.

Kim, H.J., S. Odend’hal, and J .V. Bruckner. Effect of dosing vehicles on the acute
hepatotoxicity of carbon tetrachloride in rats. oniooL AooL Ehannaool, 102:34-
49, 1990a.

Kim, H.J., J .V. Bruckner, C.E. Dallas, and J .M. Gallo. Effect of dosing vehicles on the
pharmacokinetics of orally administered carbon tetrachloride in rats. Ioniool,

A1221. Ehannasol. 102:50-60, 1990b.

Kimbrough, R.D., F.L. Mitchell, and V.N. Hook. Trichloroethylene: an update. L
oniool. Emdron. Health 15:369-383. 1985.

Klaasan, C.D., and G.L. Plaa. Relative effects of various chlorinated hydrocarbons on
liver and kidney function in mice. Ioniool. ApoL Ehannaool. 9: 139-151, 1966.

Lake, B.G., R. Hopkins, J. Chakraborty, J .W. Bridges, and V.W. Parke. The influence of
some hepatic enzyme inducers on extrahepatic drug metabolism. 12mg Momml,

Disposition 1:342-349, 1973.

80

Land, D.C., B.L. Owen, and H.W. Linde. Morphologic changes in mouse spermatozoa
after exposure to inhalation anesthetics during early spermatogenesis.

Anosthosiol. 54:53-56, 1981.

Leibman, K.C. Metabolism of trichloroethylene in liver microsomes. 1. Characteristics of
the reaction. MoL Ehannaool, 1(3):239-246, 1965.

Leong, B.K.J., B.A. Schwetz, and P.J. Gehring. Embryo- and fetotoxicity of inhalated
trichloroethylene, perchloroethylene, methylchloroform, and methylene chloride

in mice and rats. Ioniool. AopL Ehannaool. 33:136-145, 1975.

Li, Y.F., G.M. Booth, and RE. Seegmiller. Evidence for embryotoxicity of gossypol in
mice and chicks with no evidence of mutagenic activity in the Ames test. Room,

' M35962, 1989.

Lin, T.P., and I.W. Monie. The development of mouse blastocysts injected with, or
cultured in, trypan blue solution. LRoonogL Emil. 32:149-151, 1973.

Litterst, C.L., E.G. Mimnaugh, R.L. Reagan, and TE. Gram. Comparison of in vitro drug
metabolism by lung, liver, and kidney of several common laboratory species.

12mg MetaooL Disoosiiion 3:259-267. 1975.

Lukas, G., S. Bindle, and P. Greengard. The route of absorption of intraperitoneally
administered compounds. L EhannacoL m Ihorao, 178:562-566, 1971.

Maltoni, C., G. Lefemine, and G. Cotti. Experimental research on trichloroethylene

carcinogenesis. In: Archixcs and Research on Industrial Carcinogenesis Moi 5.
CV. Maltoni and M.A. Mehlman, eds. Princeton Scientific Publishing Co., Inc.,

New Jersey, pp. 1-393, 1986.

Maltoni, LC, and T. Maroni. Long term bioassay of carcinogenicity of trichloroethylene:
preliminary results. Coo. Vita. 4: 108-1 10, 1977.

Manson, J.M., M. Murphy, N. Richdale, and M.K. Smith. Effects of oral exposure to
trichloroethylene on female reproductive function. Ionioology 32:229-242, 1984.

Matsumoto, N., and A. Spindle. Sensitivity of early mouse embryos to methylmercury
' toxicity. WWW 64:108-117,1982.

McKenna, M.I., J .A. Zempel, E.o. Madrid, and W.H. Braun. Metabolism and pharmaco-
kinetic profile of vinylidene chloride in rats following oral administration.

Toxicol. AcoLEhannacoL 45:281-288, 1978.

 

81

Miller, R.E., and F.P. Guengerich. Metabolism of trichloroethylene in isolated hepato-
cytes, microsomes, and reconstituted enzyme systems containing cytochrome
P-450. Canoor Ros. 43:1145-1152, 1983.

Mirsalis, J.C., C. Kimerly-Tyson, E.N. Low, K.L. Steinmetz, J .P. Bakke, C.N. Hamilton,
D.K. Spak, and J .W. Spalding. Induction of hepatic cell proliferation and
unscheduled DNA systhesis in mouse hepatocytes following in vivo treatment.

Cminogonosis 621521-1524, 1985.

Monster, A.C., G. Boersma, and WC. Duba. Pharmacokinetics of trichloroethylene in
volunteers, influence of workload and exposure concentration. Int, A1911. Qoouo.

Enxirom Hoallh 38:87-102, 1976.

Moslen, M.T., E.S. Reynolds, P.J. Boor, K.Bailey, and S. Szabo. Trichloroethylene-
induced deactivation of cytochrome P-450 and loss of liver glutathione in vivo.

ResComChemEatLEharmaooL 16:109-114. 1977.

Muller, G., M. Spassovski, and D. Henschler. Metabolism of trichloroethylene in man. 11.
. Pharmacokinetics of metabolites. Aron, Ioniool. 32:283-295, 1974.

Muller, G., M. Spassovski, and D. Henschler. Metabolism of trichloroethylene in man. 11.
Interaction of trichloroethylene and ethanol. Aron. 192115291. 33: 173-189, 1975.

Murray, F.J., B.A. Schwetz, J.G. McBride, and RE. Staples. Toxicity of inhaled chloro-

form in pregnant mice and their offspring. oniooL Aool. EharmaooL 50:512-522,
1979.

National Cancer Institme. Carcinogenesis Bioassax of Trichloroethylene. CAS No.
79-01-6, NCI-CG-TR, 1976.

National Institute for Occupational Safety and Health. Trichloroethylene. Registry for
Toxic Effects of Chemical Substances (RTECS), July, #KX4550000, 1988.

National Toxicology Program. NTP technical report on the carcinogenesis studies of
trichloroethylene (without epichlorohydrin) in F344/N rats and B6C3F1 mice
(gavage studies). NIH publication no. 83-1799. Research Triangle Park, NC:
Department of Health and Human Services, 1983.

Nau, H. Pharmacokinetic aspects of in vitro teratogenicity studies: Comparison to in
vivo.In:In¥iqueihodsinDcxclonmemalonicologxtllseinDefining
Moohanismo and Risk Earamotors. G.L. Kimmel and DM. Kochhar, eds., CRC
Press, Boca Raton, FL, pp. 29-43, 1990.

82

Neel, J .L., and P. Dupont. Etude de test du papier au trichloroethylene chez les sujets

coronariens (sur 128 cas). Amh. Mal. Cooun yaios. (Suppl. 3, Rev. Atheroscler.)
3:46, 1961.

Nelson, M.A., and RJ. Bull. Induction of strand breaks in DNA by trichloroethylene and
metabolites in rat and mouse liver in vivo. oniooL Apol. EhannaooL 94:45-54,
1988.

Nomiyama, K., and N. Nomiyama. Metabolism of trichloroethylene in human. Int. ArolL
Amnamogl. 28:37-48, 1971.

Nomiyama, H., and K. Nomiyama. Pathway and rate of metabolism of trichloroethylene
in rats and rabbits. lngL Hoalth 17:29-37, 1979.

Norton, T.R. Metabolism of toxic substances. In: Ionioology. Iho Basio Soionoo of
Poisons. L.J. Casarett and J. Doull, eds., Macmillan, New York, pp. 45-142, 1975.

Oljenick, I. Trichloroethylene treatment of trigeminal neuralgia. .[AMA 91:1085, 1928.

Parchman, LG, and RN. Magee. Metabolism of [14C] trichloroethylene to 14C02 and
‘ interaction of a metabolite with liver DNA in rats and mice. L Ioniool, Enyiron.
floalth 9:797-813, 1982.

Parr, B.L., H.N. Tung, and MB. Parr. Apoptosis as the mode of uterine epithelial cell
death during embryo implantation in mice and rats. Biol. Room 36:211-225,
1987.

Perocco, P., and G. Prodi. DNA damage by haloalkanes in human lymphocytes cultured
in vitro.Canoo[Lon.13:213-218, 1981.

Pessayre, D., H. Allemand, J.C. Wandscher, V. Descatoire, J.Y. Artigou, and J .P.
Benhamou. Inhibition, activation, destruction, and induction of drug-metabolizin g

enzymes by trichloroethylene. Ioniool. Aool. Ehaunaool. 49:355-363. 1979.

Price, P.J., C.M. Hassett, and 1.1. Mansfield. Transforming activities of trichloroethylene
and proposed industrial alternatives. ln Minn 14:290-293, 1978.

Priete, E. Indagini sulle condizioni igienico-sanitarie di alcune lavanderie a secco della
citta di Modena. Rio. Ital. lg. 25:205. 1965.

Prout, M.S., W.M. Provan, and T. Green. Species differences in response to trichloro-
ethylene. I. Pharmacokinetics in rats and mice. Ioniool, Aool, Ehannaool, 79:389-
400, 1985.

83

Reitz, R.H., J .N. McDougal, M.H. Himmelstein, R.J. Nolan, and A.M. Schumann.
Physiologically based pharmacokinetic modeling with methylchloroform: Impli-
cation for interspecies, high dose/low dose, and dose route extrapolation. oniooL
Anal. Eharmaool. 95:185-199. 1988.

Rinzema, L.C. Behavior of chlorohydrocarbon solvents in the welding environment. Ina

ArchArbeitsrnedi28:151.l97l.

Rouisse, L., and SK. Charkrabarti. Dose-dependent metabolism of trichloroethylene and
its relevance to hepatotoxicity in rats. Enyirom Ros, 40:450-458, 1986.

Russo, A., F. Pacchierotti, and P. Metalli. Nondisjunction induced in mouse spermato-
genesis by chloral hydrate, a metabolite of trichloroethylene. Enxiron. Mntagon.
- 6:695-703, 1984.

Sanders, V.M., A.N. Tucker, K.L. White, Jr., B.M. Kaufrnann, P. Hallett, R.A.Carchman,
J.F. Borzelleca, and A.E. Munson. Humoral and cell-mediated immune status in
mice exposed to trichloroethylene in the drinking water. oniooL AooL

EhannaooL 62:358-368, 1982.

Sato, A., T. N akajima. Differences following skin or inhalation exposure in the absorp-
tion and excretion kinetics of trichloroethylene and toluene. Elli. L 1nd,. Mod.
35:43-49, 1978.

Sbrana, 1., D. Lascialfari, and N. Loprieno. TCE induces micronuclei but not chromo-
somal aberrations in mouse bone marrow cells. IV ICEM, Stockholm, Abstracts,
p.163,l985.

Schwetz, B.A., B.K.J. Leong, and P.J. Gehring. The effect of maternally inhaled tri-
chloroethylene, perchloroethylene, methyl chloroform, and methylene chloride on

embryonal and fetal development in mice and rats. oniool, Apol. Ehannaool.
32:84-96. 1975.

Slacik-Erbin, R., R. Roll, G. Frank, and H. Uehleke. Trichloroethylene vapours do not
produce dominant lethal mutations in male mice. Aron. Toxicol. 45:37-44, 1980.

Shimada, T., A.F. Swanson, P. Leber, and G. Williams. Activities of chlorinated ethane
and ethylene compounds in the Salmonella/rat microsome mutagenesis and rat
hepatocyte/DNA repair assays under vapor phase exposure conditions. Coll BioL
oniool. 1:159-179, 1985.

Smith, A.L. Personal communication, 1991.

84

Smith, M.K., J .L. Randall, and J .A. Stober. Developmental effects of trichloroacetic acid
in Long-Evans rats. Iormology 37 :495, 1988.

Soucek, B., and D. Vlachova. Excretion of trichloroethylene metabolites in human urine.

EL .1. Ind. Med. 17:60-64. 1960.

Spielman, H., and HG. Eibs. Recent progress in teratology. A survey of methods for the
study of drug actions during the preimplantation period. Am. 12mg Ros,
28:1733-1742, 1978.

Stewart, R.D., C.L. Hake, and J .E. Peterson. “Degreasers’ flush” : Dermal response to
trichloroethylene and ethanol. Aron Enyiron. Hoalm 29:1-5, 1974.

Storeng, R., and J. Jonsen. Nickel toxicity in early embryogenesis in mice. Ionioology
20:45-51, 1981.

Storeng, R., and J. Jonsen. Inhibitory effect of ricin on the development of preimplanta-

tion mouse embryos. L oniooL Enyinom Hoalth 12: 193-202, 1983.

Stott, 'W.T., J .F. Quast, and RC. Watanabe. The pharmacokinetics and macromolecular
interactions of trichloroethylene in mice and rats. logical. Ann]. RharmaooL 62:
137-151, 1982.

Striker, C., S. Goldblatt, I.S. Warm, and DE. Lackson. Clinical experiences with use of
trichloroethylene in production of over 300 analgesias and anesthesias. Anosth.

Analg 14:68. 1935.

Strubelt, O., E. Dost-Kempf, C.P. Siegers, M. Younes, M. Volpel, U. Preuss, and LG.
Dreckrnann. The influence of fasting on susceptibility of mice to hepatic injury.

onicoLAooLEhatrnacol. 60:6077, 1981.

Svandzhian, E.P. Trichloroethylene (narcogen) for anesthesia and analgesia. m Khin
AnostozioL 8:60, 1963.

Takeuchi, I. Teratogenic effects of methylnitrosourea on pregnant mice before implanta-
tion. W40879-881, 1984.

Taylor, D.H. Effect of trichloroethylene on the exploratory and locomotor activity of rats
exposed during development. Soi. Iom Enxinon. 47 :415-420, 1985.

Truhaut, M.R., C. Boudene, N. Phy-Lich, and H. Catella. Contribution a l’etude du ‘
dosage du trichloroethylene dans les solvants complexes. Anon. Mal, Erof, Mod.

Irax. Soon Sm 28:369, 1967.

85

Tu, A.S., T.A. Murray, K.M. Hatch, A. Sivak, and H.A. Milman. In vitro transformation
of BALB/c-3T3 cells by chlorinated ethanes and ethylenes. Canooi: Lora 28:85-
92, 1985.

Tucker, A.N., V.M. Sanders, D.W. Barnes, T.J. Bradshaw, K.L. White, Jr., L.E. Sain, J.F.
Borzelleca, and A.E. Munson. Toxicology of trichloroethylene in mice. Ioniool,

Anal. Ehannacol. 62:351-357. 1982.

Uehleke, H., and S. Poplawski-Tabarelli. Irreversible binding of C-labeled trichloro-
ethylene to mice liver constituents in vivo and in vitro. Aron. Ioniool. 37 :289-
294, 1977.

Uehleke, H., S. Tabarelli—Poplawski, G. Bonse, and D. Henschler. Spectral evidence for
2,2,3-trichloro-oxirane formation during microsomal trichloroethylene oxidation.

Amt]. oniool. 37:95-105, 1977.

United States Tariff Commission, 1972.

Veljanovski, A., and T. Petrov. Prilog zastiti radnika u hemijskim cistionicama.

Moinosanin Rzogl, 29:183, 1972.

Warren, J.R., B. Shaw, and MP. Steinkampf. Effect of nitrous oxide on preimplantation
mouse embryo cleavage and development. Biol. Room 43: 158-161, 1990.

Watanabe, P.G., G.R. McGowan, and P.J. Gehring. Fate of [14C] vinyl chloride after
. single oral administration in rats. IoniooL Aool, RhannaooL 36:339-352, 1976.

Waters, E.M., H.B. Gerstner, and LE. Huff. Trichloroethylene. I. An overview. L
oniool. Enxiron. Health 2:671-707, 1977.

Wattenberg, L. Dietary modification of intestinal and pulmonary aryl hydrocarbon
hydroxylase activity. oniooL Apol, RhannaooL 23:741-748, 1972.

Westergren, I., P. Kjellstrand, L.E. Linder, and BB. Johansson. Reduction of brain
specific gravity in mice prenatally exposed to trichloroethylene. IoniooL Lon, 23:
223-226, 1984.

White, A.E., S. Takehisa, E.I. Eger, S. Wolff, and WC. Stevens. Sister chromatid ex-
changes induced by inhaled anesthetics. Anoslhosiology 50:426-430, 1979.

Whitten, W.K. Modification of the oestrus cycle of the mouse by external stimuli
associated with the male. Changes in the oestrus cycle determined by vaginal
smears. LEngloorinoL 17:307-313, 1958.

86

Whittingham, D.G., and M.J. Wood. Reproductive physiology. In: Ibo Monso in Rio;
modioal Rosoaooh, Vol. III. Academic Press, Inc., New York, pp. 138-164, 1983.

Zenick, H., K. Blackburn, E. Hope, N. Richdale, and M.K. Smith. Effect of trichloro-
ethylene exposure on male reproductive function in rats. Ionioology 31:237-250,
1984.

APPENDICES

APPENDIX A

Table 18. Brand and trade names for trichloroethylene (TCE).

 

Algylen
Aramenth
Alk-Tri
Blacosolv
Chlorilen
Chlorylen
Circosolv
Crauhaspol
Dow-Tri
Dukeron
Ex-Tri
Fleck-Flip
Gamalgene
Hi-Tri
Landin
Lethorin
Narcogen
Narcosoid
New-Tri
Nialk
Perm-A-Chlor
Petzinol
Philex (Phillex)

Threthylen(e)
Trethylene
TRI

Triad
Trial
Trianan
Triasol
Trichlor(an)
Trichloren
Triclene
Tri-Clene
Triclene
Trielin(e)
Triklone
Trilin(en)
Triman
Trimar
Triol
Trisan
Vestrol
Vestrosol
Vitran
Westrosol

 

Name:
Born:

Birthplace:

Formal Education:

Degrees Received:

APPENDIX B

VITA
Neal C. Cosby
May 4, 1964
Richmond, Virginia

St. Christopher’s School
Richmond, Virginia 1976 - 1982

Michigan State University
East Lansing, Michigan 1982 - present

Bachelor of Science, Physiology
Michigan State University, 1986

Master of Science, Bioscience
Michigan State University, 1989

Publications by the Author

In xigo dcxelonment of preimplantation mouse emootos microencaosiilated in sodium
alginato. Master’s Thesis, Michigan State University, East Lansing, 1989.

Embryo production in B6D2F1 mice using two superovulating regimens. Neal C. Cosby,
Karen Chou, and W. Richard Dukelow. Lab, Anim. SoL, 39:249-250, 1989.

Microencapsulation of single, multiple and zona pellucida-free mouse preimplantation
embryos in sodium alginate and their development in xino. N .C. Cosby and W. Richard
Dukelow. L_. mm, 90:19—24, 1990.

Cell differentiation as determined by micromanipulation of mouse embryos. N.C. Cosby,
W.E. Roudebush, L. Ye, and W.R. Dukelow. Biol. Rm 38 (suppl. l):128, 1988.

(abstract)

88

89

APPENDD( B (cont’d)

VITA

Timed-mating and gestation. W.R. Dukelow, S.D. Kholkute, Ye Lian, N.C. Cosby, W.E.
Roudebush, and S. Bruggeman. Int. L 2111118191.. 8:558, 1988. (abstract)

Development of multiple encapsulated mouse embryos in sodium alginate. N.C. Cosby,
L. Ye, and W.R. Dukelow. Biol. Rom 40 (suppl. l):110, 1989. (abstract)

Encapsulation of mouse embryos in calcium-alginate matrix as an aid for environmental
toxicology testing. N.C. Cosby and W.R. Dukelow. Abstract presented at the Central
Great Lakes Regional Chapter, Society of Environmental Toxicology and Chemistry,
Sept. 22, 1989.

Capacitation and the acrosome reaction in squirrel monkey (Saimiri sciureus) spermatoza
evaluated by the chlortetra-cycline fluorescence assay. S.D. Kholkute, Y. Lian, N.C.
Cosby, and W.R. Dukelow. Am. Lanam, 18 (2):151, 1989. (abstract)

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