EPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-81-056 Sept. 1981
Project Summary
Evaluation of Mutagenic
Effects of Diesel Emissions:
I. Tests for Heritable and
Germ-Cell Effects in the Mouse
L. B. Russell, W. M. Generoso, W. L Russell, and E. F. Oakberg
In order to assess potential risk from
heritable effects in human populations,
mice were exposed by inhalation to
whole diesel exhaust, and a number of
genetic endpoints were studied. Ex-
posure times in different groups varied
from 5 to 10 weeks. In the maximally-
exposed group, the total intake of
diesel exhaust per mouse was about
85 times the 30-year (generation
length) intake by a person in an
average U.S. environment.
The battery of assays was chosen to
detect several types of genetic end-
points, namely, point mutations in
males (specific-locus test), chromo-
some damage in males (dominant-
lethal and heritable-translocation
tests), and chromosome damage in
females (dominant-lethal tests). An-
cillary studies were carried out to look
for direct reproductive damage in both
sexes; thus, various parameters were
used to assess reproductive perform-
ance in females, and hlstological
analyses of germ-cell survival were
done in males.
The results of all genetic assays in
both sexes were negative. In the
ancillary tests, small but unequivocal
effects on the reproductive perform-
ance of females of one strain could be
observed, consisting of a decrease in
the number of ovulations and an
increase in the interval between
mating opportunity and copulation.
There was no detectable effect of
diesel exposure on the number and
distribution of cell types in the testis.
The absence of genetic effects could
indicate either that no active metabo-
lites reached the gonads, or that the
germcells have an efficient repair
system against induction of mutational
events by such metabolites. Thus,
transmitted genetic effects appear not
to be a major hazard from exposure to
diesel exhaust. The findings reported
must, however, not be used to draw
any conclusions concerning possible
risks to the exposed individual him/
herself.
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).
Introduction
Although non-mammalian and in
vitro assays can provide evidence on
presence or absence of mutagenicity,
they have limitations in addressing the
complexities associated with repro-
ductive-cell targets in intact organisms
The assessment of risk from heritable
damage must therefore utilize in vivo
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mammalian germlme mutagemcity
tests.
The effectiveness of inhaled whole
diesel exhaust in inducing heritable
effects in mammals was studied by a
battery of tests in the mouse. The assays
chosen were designed to detect a
number of genetic endpomts, namely
chromosome breakage, chromosome
interchange, and point mutations.
Ancillary studies involved germcell
survival and reproductive performance.
Effects were looked for in both sexes
and in a variety of germcell stages.
Several of the test systems had been
developed at Oak Ridge, and all experi-
ments are carried outthere. Exposure to
the diesel exhaust took place at the EPA
laboratory at Cincinnati. In the maxi-
mum-exposure group, the total intake of
exhaust per mouse was about 85 times
the 30-year (generation length) intake
by a person in an average U.S environ-
ment.
Overall Conclusion
The results of all genetic tests in both
sexes were negative Small but un-
equivocal effects on the reproductive
performance of females of one strain
could be observed, consisting of a
decrease in the number of ovulations
and an increase in the interval between
mating opportunity and copulation. It is
no' known whether these effects were
the result of damage directly to the
ovary or to some other endocrine organ
(e.g., pituitary). There was no detectable
effect of diesel exposure on the number
and distribution of cell types in the
testis.
The absence of genetic effects could
indicate either that no active metabolites
reached the gonads, or that the germ-
cells have an efficient repair system
against induction of mutational events
by such metabolites In experiments
with chemical .agents, one does not
expect—and, in fact, does not find—
good correlation between transmitted
damage induced in mammalian germ-
cells in vivo and results from other test
systems. It is the former result that is
pertinent to transmission of genetic
lesions to future generations of human
beings; and the work summarized in this
report thus indicates that transmitted
genetic effects are not a major hazard
from exposure to diesel exhaust. The
findings reported here must, however,
not be used to draw any conclusions
concerning possible riskstothe exposed
individual himself.
General Procedure
All mice for the experiment were bred
at Oak Ridge, shipped by air-conditioned
van to the EPA laboratory at Cincinnati
for exposure to diesel exhaust air, and
returned to Oak Ridge for the
subsequent genetic experiments. The
exposure dates are listed separately for
the individual projects, but all fell within
the period of March 21 to June 4, 1979.
Because of the danger of introducing
pathological conditions into the valuable
Oak Ridge Mammalian Genetics facility,
mice returning from Cincinnati were
placed into a quarantine facility in
which initial matings were carried out
for some of the projects, and the entire
procedure took place for others. Animals
were monitored for key diseases before
their departure to Cincinnati and, again,
after their return to the Oak Ridge
quarantine building. The first group to
return to Oak Ridge was found to carry
three viruses not present before they
were shipped to Cincinnati. We there-
fore had to utilize a building separate
from the Mammalian Genetics facility
(as well as from the quarantine building)
in which to complete all experiments.
At Cincinnati, mice were housed 3, 4,
or 12 animals to a cage and cages were
placed into 100 cf exposure chambers,
having a horizontal cross section of 5 x 5
feet. One of these chambers received
CBR-filtered and -conditioned air, while
the other chamber was connected by
piping to an automobile diesel engine
exhaust dilution system. The six-
cylinder Nissan engine was operated
under load on the Federal Short Cycle,
and the exhaust diluted with filtered and
conditioned air at the ratio of 1.18. The
diesel paniculate concentration in the
chamber averaged 6/mg/m3 during the
exposure period of eight hours per day
and seven days of the week. All engine
operations, aerometry measurements,
and animal care were performed by the
EPA staff at Cincinnati. [A few animals
were lost during the exposure period
(presumably m the process of transfer to
clean cages), and a few others during
the overnight storage between final
removal from the chambers and return
shipment to Oak Ridge (when some of
the cardboard shipping boxes were
chewed through)].
We calculate that, during 10-week
exposure under these conditions, the
total intake of exhaust per mouse was
about 85 times the 30-year (generation-
length) intake by a person in an average
U.S. environment (urban-rural) This
calculation is based on paniculate
concentration (mouse, 6 mg/m3 vs.
man 0.3 /ug/m3), length of exposure
(mouse, 10 x 7 x 8/24 = 23.3 days vs
man 30 years - 10,957 days), and
pulmonary ventilation rate (mouse 2x
man).
Tests were carried out for several
types of genetic effects, namely, point
mutations in males (specific-locus test),
chromosome damage in males (domi-
nant-lethal and heritable translocation
test), and chromosome damage in
females (dominant-lethal test). In
addition, we looked for reproductive
effects in females, and for changes in
testis histology. The scope of the
experiment did not include chemical
determinations of what, if any, sub-
stances reached the gonads. If active
material from diesel exhaust fails to get
into mammalian gonads, this would
lead to negative findings in the experi-
ments reported, and presumably also to
an absence of risk from genetic lesions
transmitted to future generations.
Reports of Individual
Experiments'
Project No. 1 I
Test for Heritable Point
Mutations in Male Mice
Objectives and relation to
other projects
The objective of this project was to
test for the induction of transmitted
point mutations (mtragemc changes
and small deficiencies) by means of the
specific-locus method. In planning the
experiment, the number of offspring to
be scored was calculated so as to be
sufficient either for showing a signif-
icantly positive effect, or for ruling out,
with a high degree of confidence, that
the induced mutation rate, atthe level of
human exposure (see calculation under
Results), could be higher than a small
fraction of the spontaneous rate In
order to meet the objectives of the latter
alternative, mice had to be exposed to a
quantity of diesel exhaust that was high
multiple of that accumulated by the
average American in one human gen-
eration.
For assessments of human risk, the
stage of prime importance in the male is
the spermatogonial stem cell, which
can harbor (and transmit) mutations for
the lifetime of the individual. The bulk ol
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our data were therefore derived from
diesel exposures to that stage. However,
we also obtained enough specific-locus
data .for the (transitory) postsperma-
togonial stages to rule outthe possibility
of a greatly elevated mutational sensi-
tivity during that period.
Since a very large (> 800,000) and
reliable historical control exists for
spontaneous mutations in specific-
locus experiments, all our available
resources were used for the study of
experimental groups only. A large
sample could therefore be accumulated.
Only four exceptional animals were
found, and none of these turned out to
be due to a mutation at one of the seven
loci
Conclusion
There is no evidence for the induction
of point mutations in spermatogonia or
m meiotic and postmeiotic stages. For
spermatogonia, the male stage of major
importance to risk assessment, the
experimentally observed zero frequency
rules out, with 97.5% confidence, that,
at the level of exposure encountered by
man, the induced mutation ratecould be
higher than 0.01 times the spontaneous
rate.
Project No. 2
Test for Induction of Dominant
Lethals in Male Mice
Objectives and relationship
to other projects
The effectiveness of diesel exhaust in
producing chromosomal aberration
effects in germ cells of the male mice
was studied using two procedures: the
dominant-lethal test (this project) and
the heritable translocation tests (Project
No. 3). Since males were exposed to
diesel exhaust for a prolonged period
and mated immediately afterwards (see
below), all spermatogenic cell stages
that are known to be sensitive to
dominant-lethal induction were pre-
sumably exposed. Dominant-lethal
effects were evaluated by analyzing
uterine content of unexposed females
mated to the exposed or control males
When a sperm carrying a lethal mutation
is used in fertilization, the resulting
embryo dies before or shortly after
implantation.
Conclusions
Results of the dominant-lethal test
indicate that the exposure of male mice
to diesel exhaust did not induce de-
tectable chromosomal aberration effects
in germ cells.
Project No. 3
Test for Induction of Heritable
Translocations in Male Mice
Objectives and relationship
to other projects
The effectiveness of diesel exhaust in
producing chromosomal aberration
effects in germ cells of male mice was
studied using two procedures: the
heritable-translocation test (this project)
and the dominant-lethal test (Project
No. 2).
The heritable-translocation method is
a sensitive and reliable procedure for
measuring the frequency of chromo-
some breakage and rearrangement
(exchange of parts) that is transmitted to
the next generation. When a sperm
carrying chromosome mterchange(s) is
used in fertilization, the resulting
progency is heterozygous for the trans-
location and produces two types of
gametes, balanced and unbalanced, in
approximately equal proportions. Both
types of gametes are capable of fertili-
zation, but the unbalanced gametes
result in embryonic lethality. For this
reason, most translocation heterozy-
gotes are only about half as productive
as normal mice. Heterozygotes for
certain types of translocations are
incapable of producing sperm In the
heritable-translocation procedure, pro-
geny of treated parents are therefore
tested for sterility and "partial sterility "
Confirmed sterile and partially sterile
progeny are then verified cytologically
for presence of a translocation Thus,
the heritable-translocation procedure
generates the most meaningful infor-
mation for evaluating hazards from
induced chromosome aberration to
human population, because it measures
transmissible genetic damage.
Conclusions
Results of the heritable-translocation
study indicate that the exposure of male
mice to diesel exhaust, as described
above, did not induce transmissible
chromosome exchange in male germ
cells. This result is consistent with that
of the dominant-lethal test (Project No.
2), which indicated that there was no
induction of chromosomal breakage in
male germ cells. Together, the two tests
fail to provide evidence of chromosomal
aberration effect induced in male mice
by the diesel exposure.
Project No. 4
Test for Induction of Genetic
Effects and Oocyte Killing
in Females
Objectives and relationship to
other projects
The effectiveness of diesel exhaust in
producing chromosomal and cytotoxic
damages to oocytes was studied by
means of total reproductive capacity.
The mouse ovary is known to be
sensitive to even relatively slight insults
from known mutagens. Genetic and/or
cytotoxic effects on oocytes in different
stages of follicular development were
measured by simply counting the
offspring produced by exposed and
control females over a period of time
Conclusions
The similarity in long-term repro-
ductive performance (pregnancy rate
and littersize) between control females
and females exposed to diesel exhaust
indicates that the exposure to diesel
described above did not induce detect-
able chromosomal or cytotoxic effects in
oocytes of the strain of mice employed
in this study. Comparisons with short-
term effects on female reproduction,
studied in another strain, are discussed
under Project No. 5
Project No. 5
Test for Dominant-Lethal
Induction in Female Mice
Objectives and relationship to
other projects
The primary objective of this experi-
ment was to test for induction of
chromosomal damages in mature and
maturing oocytes. Since the accessibility
of the ovary to diesel metabolites maybe
very different from that of the testis, this
project constituted an important com-
panion to Projects No. 1, 2, and 3, which
assayed for genetic damage in males.
An additional objective of this experi-
ment was to test for any short-term
effects of diesel exhaust on reproductive
physiology, long-term effects were
tested in Project No 4.
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Conclusions
A seven-week exposure to exhaust
fumes from a Nissan diesel engine
produced no transmissible chromo-
somal damage, as measured in a
dominant-lethal experiment, in mature
and maturing oocytes. However, sig-
nificant, though slight, effects were
produced on female reproductive func-
tions, as expressed in a decreased
number of ovulations (14% overall) and
a lengthening (by about 1.5 days) of the
time interval between mating oppor-
tunity and copulation.
The results of this study, which
concerns matings made mostly within a
week after the end of exposure, may be
compared with first-litter results for
Project No.'4 (births 18-24 days after
end of treatment). The females used in
these two projects, though of the same
age, were genetically different (SEC x
C57BL)Fi in Project 4 and (101 xCSHtF,
in Project No. 5. For the first-week
period, only 75.6% of mated control
females produced litters in Project No
4, but 100% of controls got pregnant in
Project No. 5. It is therefore difficult to
predict whether a 1.5-day (average)
diesel-induced lengthening in time
interval between mating opportunity
and copulation (shift in mean from 2.9 to
44 days), such as was determined
through day-by-day analysis in Project
No. 5, would have been detectable in
Project No. 4. It seems probable that the
method of Project No. 4 was not
sensitive enough to detect such a shift,
had it been induced in the (SEC x
C57BL)Fi females Project No 4 should,
however, have detected a 16% reduction
in average littersize, such as appears to
have been induced in Project No. 5. The
conclusion was reached that there is a
genetic difference in sensitivity of
females to the physiological damage(s)
responsible for reducing number of
ovulated eggs.
Project No. 6
Test for Effects of Spermatogonial
Survival in the Mouse
Objective
Differentiating spermatogonia of the
mouse are extremely sensitive to
cytotoxic agents such as radiation and
chemicals, and often show a positive
response even when no genetic damage
is observed. In the latter case, they
provide a sensitive test system to
determine if test materials reach the
gonads. The response observed, how-
ever, is a somatic one (i.e. cell death)
and is not necessarily associated with
heritable effects. Killing of sperma-
togonia can, of course, have an effect on
fertility.
Conclusions
Exposure to diesel exhaust had no
effects on the number of spermatogonia
or preleptotene spermatocytes of mice
after 5 or 10 weeks' exposure Likewise,
no irregularities in the normal dynamics
of the seminiferous epithelium were
observed; cell ular associations were the
same in controls, and division of the
different spermatogonial types occurred
at the normal times in the cycle of the
seminiferous epithelium.
L. B Russell, W M. Generoso, W. L. Russell, and E. F, Oakberg are with the
Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830.
Larry Claxton, John Orthoefer, and Michael Pereira are the EPA Project
Officers (see below/.
The complete report, entitled "Evaluation of Mutagenic Effects of Diesel Emis-
sions: I Tests for Heritable and Germ-Cell Effects in the Mouse," (Order No.
PB 81-235 814; Cost $6.50, 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 (Claxton) can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
The EPA Project Officers (Orthoefer and Pereira) can be contacted at:
Health Effects Research Laboratory
U S Environmental Protection Agency
Cincinnati, OH 45268
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
•it US GOVERNMENT PRINTING OFFICE, 1981 — 757-01 2/7317
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