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
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