X-/EPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-82-009 Sept. 1982
Project Summary
Effects of Selected Organic
Drinking Water
Contaminants on Male
Reproduction
Joseph F. Borzelleca and Richard A. Carchman
Because of the recent increase in
exposure of individuals to potentially
harmful chemicals, it has become
increasingly important to test the
potential of environmental chemicals
to cause adverse reproductive effects.
The Division of Toxicology within the
Department of Pharmacology, Med-
ical College of Virginia has responded
to this need by examining the abilities
of Kepone, hexachlorobenzene, 2,4-
dinitrotoluene, 1,2,3,4-tetrabromo-
butane, chloral hydrate, 1,1,2-trichlo-
roethylene, 1,2-dichloroethyleneK
1,2-dichloroethane, dibromochloro-
methane, trichloromethane, and 1,1,1-
trichloroethane to elicit harmful re-
productive effects. The following
tests were used to assess the extent of
these effects: analysis of effects upon
rat ejaculate volume and sperm mor-
phology, distribution studies in rats,
determination of the cellular sites of
action in a P388D! lymphoid neo-
plasm cell line, performance of a multi-
generation murine experiment which
included dominant lethal and terato-
logic studies, and analysis of the inhi-
bition of mouse testicular DNA syn-
thesis.
This Project Summary was devel-
oped by EPA's Health Effects Research
Laboratory, Research Triangle Park,
NC. to announce key findings of the
research project that is fully docu-
mented in a separate report of the
same title (see Project Report ordering
information at back).
Results
Inhibition of Mouse Testicular
DNA Synthesis
A group of structurally related com-
pounds were tested for their effects
upon mouse testicular DNA synthesis.
The compounds were administered
mtratesticularly, in two equal doses, one
dose per testis (0.02 ml/testis, 0.04
ml/mouse). One and one-half hours
later, tritiated thymidine was adminis-
tered (10 yuCi/testis, 20 //Ci/mouse).
The mice were sacrificed one-half hour
after the injections of tritiated thymidine,
and their testes surgically removed. The
testicular DNA was then isolated using
a modified Shibko method in which suc-
cessive PCA precipitations were used to
isolate DNA free of protein, lipids, and
contaminating RNA.
In each experiment, three fractions
obtained from the DNA isolation pro-
cedure were analyzed. The first fraction
was the homogenate which contained
both testes of an individual mouse,
homogenized in PBS buffer (0.15 M
KP04, in 0.85% NaCI, pH 7.2). The total
counts in the homogenate fraction
measured the tritiated thymidine re-
maining in the testes at the time of
sacrifice. The second fraction was the
supernatant fluid removed after centri-
f ugation of the homogenate. The counts
in this fraction were a measure of non-
PCA-precipitable tritiated thymidine, or
tritiated thymidine pool size. The third
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fraction analyzed contained relatively
pure DNA. The counts in this fraction
quantitated the amount of DNA synthe-
sized during the one half-hour after the
injections of the tritiated thymidine.
All results were analyzed using an
ANOVA, Dunnett's t-test, Duncan's
multiple range test, and linear regres-
sions of the dose-response curves of the
various fractions studied.
Chloral hydrate (CHL)
Doses ranging from 10 to 900 mg/kg
were tested. The 600 and 900 mg/kg
doses caused a significant increase in
the number of counts in the homogenate
fraction. These data imply a mechanism
of transport of tritiated thymidine out of
the testes. This mechanism is inhibited
at these doses of CHL, causing a greater
amount of total testicular counts,
relative to control values, to remain in
the testes at the time of sacrifice of the
animals.
Doses greater than or equal to 75
mg/kg caused a significant inhibition of
testicular DNA synthesis. The amount
of testicular DNA synthesis was 30%
and 3% of the vehicle control values at
75 and 300 mg/kg, respectively.
1,1,2-Trichloroethylene (TCE 2)
The range of doses tested was 24 to
1000 mg/kg. Doses greater than or
equal to 500 mg/kg caused an increase
in tritiated thymidine pool size, and this
was accompanied by a decrease in
testicular DNA synthesis. The amount
of DNA synthesis at 500, 700, and 1000
mg/kg was 40%, 18% and 2% of the
vehicle control values, respectively
1,1,2-Trichloroethane (TCE 1,2)
Doses ranging from 3 8 to 1200
mg/kg were tested. A dose-related
increase in tritiated thymidine pool size
and decrease in testicular DNA synthesis
was observed These deviations from
control values became statistically
significant at doses greater than or
equal to 300 mg/kg. At 1000 mg/kg,
DNA synthesis was inhibited to 2% of
the vehicle control value.
1,1,1-Trichloroethane (TCE 1,1)
Two doses of TCE 1,1 were tested,
100 and 1000 mg/kg. Both doses
caused an increase intritiatedthymidine
pool size accompanied by a decrease in
testicular DNA synthesis. The amount
of DNA synthesis at these doses was
60% and 27% of the vehicle control
values, respectively.
1,2-Dichloroethane (DCE 1,2)
DCE 1,2 was administered over a
dose range of 4 9-250 mg/kg. The only
significant effect observed was elicited
at 250 mg/kg where a decrease in DNA
synthesis to 47% of the vehicle control
value was effected.
Compound Comparisons (CHL,
DCE 1,2; TCE 1,1; TCE 1,2;
TCE 2)
In order to contrast the relative
capabilities of the above compounds to
inhibit testicular DNA synthesis, their
dose-response curves were compared
The mean percent DNA synthesis
versus log (milhmolar dose/kg) was
plotted. Four of the five compounds,
CHL; DCE 1,2; TCE 1,2, and TCE 2 were
found to have approximately equal
slopes, although the curve for CHL was
to the left of the others These observa-
tions suggest that these four compounds
may be causing their effects through the
same mechanism which is most sensi-
tive to CHL CHL differs from the other
compounds in that it contains an
aldehyde group. This could explain the
marked inhibition of testicular DNA
synthesis. As only two does of TCE 1,1
were tested, conclusions cannot be
drawn concerning the shape of the TCE
1,1 dose-response curve
2,4 Dinitrotoluene (DNT)
No dominant lethal effects were
caused by the oral administration of
either 60 or 180 mg DNT/kg/day for
five days in rats. The 60 mg/kg/day
dose produced no adverse changes in
male reproductive performance. The
moderately adverse effects of 180
mg/kg/day on mating and fertility
indices were reversible Severe repro-
ductive and moderate dominant lethal
effects were seen at the 240 mg/kg/day
level. These effects persisted for at least
eight weeks, indicating that this dose
was severely debilitating.
1,2,3,4-Tetrabromobutane
(TBB)
The oral administration of TBB at 10
and 40 mg/kg/day for five days in rats
failed to produce the classical picture of
dominant lethal effects elicited by the
positive control, triethylenemelamine
(TEM). TEM caused a mutagenic re-
sponse m the first few weeks of mating.
TBB induced significant effects in
weeks 6 and 7, but these were reversed
by week 14 of mating and were not
considered significant evidence of
dominant lethality. TBB, at both doses,
impaired male reproductive performance
as evidenced by a decrease in the
mating index (mating index = percentage
of mated females which became preg-
nant), but not as severely as TEM.
Kepone
Evaluation of Kepone in a dominant
lethal study (oral administration for five
days in rats at 3.6 and 11.4 mg/kg/day)
demonstrated no significant changes in
either male fertility or dominant lethal
mutations. The doses administered
produced tremors as previously reported
in the literature
Following a single oral administration
(40 mg/kg, 2.5^Ci/ml), Kepone and/or
its metabolites were found to distribute
in the ejaculate via the seminal vesicle
and appeared to bind to spermatozoa.
Both i.p. (18 and 36 mg/kg, single
acute dose) and p.o. (9 mg/kg/day for
14 days) del ivery of Kepone produced an
increase in the percent of morpholog-
ically abnormal sperm. The effect of
orally administered Kepone on sperm
morphology persisted for up to 7 weeks
post dosing.
Kepone interfered with cellular
energy production in P388Di cells in a
manner similar to the classical uncou-
pler of oxidative phosphorylation, dini-
trophenol (DNP). Kepone was approxi-
mately 80 times more potent than DNP
in inhibiting cellular proliferation and
stimulating oxygen consumption, sug-
gesting that the mitochondria may be
the ultimate site of subcellular toxicity.
Kepone also significantly altered the
mitochondrial calcium distribution and
the phagocytic process in the P388Di
cell.
Hexachlorobenzene (HCB)
HCB at 70 and 221 mg/kg/day p.o.
for five days in rats did not induce
classical dominant lethal events during
weeks 1-5 of mating. Statistically
significant effects during weeks 10-14
of mating were inconsistent in their
extent and direction, and were attri-
buted to weekly fluctuations in the test
groups. Male reproductive performance
(mating index) was impaired by HCB in a
dose-related manner.
Fourteen daily oral administrations of
35 mg/HCB/kg significantly altered
male body weight, ejaculate volume,
sperm count and the percent of morpho-
logically abnormal sperm for up to 7
weeks post-dosing.
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Dibromochloromethane
(DBCM) and Trichloromethane
(TCM)
In a two-generation study, impairment
of reproduction was observed among
mice ingesting 4 mg/ml DBCM or 5
mg/ml TCM (nominal doses of 685 and
855 mg/kg/day, respectively). Signifi-
cant decreases were observed in
fertility, litter size, and adult female
survival during gestation for both
compounds at these levels. Inconsistent
decreases were observed in postnatal
survival and body weight gains. No
significant adverse effects were ob-
served for either halomethane in
dominant lethal and teratology screening
studies.
Recommendations
1. A holistic approach to the evaluation
of potential toxicity should be con-
sidered. Test animals should all
receive the same doses and multiple
systems should be evaluated simul-
taneously.
2. The doses should be multiples of
maximum anticipated human expo-
sure (10X, 100X, 1000X, etc.).
3. Molecular mechanisms of adverse
effects should be determined.
4. Potential genotoxic effects should be
determined using the following test
systems: DNA damage, DNA repair,
sister chromatid exchange, adduct
formation, and DNA single strand
break analysis.
5. In vitro tests should be developed to
evaluate toxicity.
Joseph F. Borzelleca and Richard A. Carchman are with the Medical College of
Virginia. Virginia Commonwealth University. Richmond. VA 23298.
Kirby I. Campbell is the EPA Project Officer (see below).
The complete report, entitled "Effects of Selected Organic Drinking Water
Contaminants on Male Reproduction." (Order No. PB 82-259 847; Cost:
$13.50, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield. VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory—RTF, NC
U.S. Environmental Protection Agency
Cincinnati. OH 45268
o US. GOVERNMENT PRINTINO OFFICE-1MJ-559-017/0830
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United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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