PB81-235814
Evaluation of Mutagenic Effects of Diesel
Emissions: I. Tests for Heritable and
Germ-Cell Effects in the Mouse
(U.S.) Oak Ridge National Lab., TN
Prepared for
Health Effects Research Lab,
Research Triangle Park, NC
Aug 81
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
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EPA-600/1-81-056
August 1981
PB31-235814
EVALUATION OF MUTAGENIC EFFECTS OF DIESEL EMISSIONS
I. TESTS FOR HERITABLE AND GERM-CELL EFFECTS
IN THE MOUSE
by
L. B. Russell, W. M. Generoso, W. L. Russell, and E. F. Oakberg
Biology Division
Oak Ridge National Laboratory
P. 0. Box Y
Oak Ridge, Tennesse 37830
Contract No. EPA-D-X0710
EPA Project Manager and Technical Project Officer:
Larry Claxton
Genetic Toxicology Division
Health Effects Research Laboratory .
Research Triangle Park, North Carolina 27711
EPA Technical Project Officers for Exposure:
John Orthoefer and Michael Pereira
Health Effects Research Laboratory
Cincinnati, Ohio 45268
ORNL Project Manager and Technical Project Officer:
Laine B. Russell
Biology Division
Oak Ridge National Laboratory
This study was conducted in cooperation with
The Department of Energy at the
Oak Ridge National Laboratory
Interagency Agreement DOE No. 40-728-78
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U,S, ENVIRONMENTAL PROTECTION AGENCY
Research Triangle Park, North Carolina 27711
REPRODUCED 8T .
NATIONAL TECHNICAL
INFORMATION SERVICE
U.S. DEPARTMENT OF COMMERCE
SPRWGflElO, VA 22161
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
. REPORT NO.
EPA-600/1-81-056
2.
ORD Report
3. RECIPIENT'S ACCESSION NO.
PB81 2 3 3 8 1 A
4. TITLE AND SUBTITLE
Evaluation of Mutagenic Effects of Diesel Emissions:
J. Tests for Heritable and Germ-Cell Effects in
the Mouse _^____
5. REPORT DATE
August 1981
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
L. B. Russell, W. M. Generoso, W. L. Russell, and
E. F. Oakbero
3. PERFORMING ORGANIZATION NAME AND ADDRESS
Biology Division
Oak Ridge National Laboratory
Oak Ridge, Tennessee 37830
10. PROGRAM ELEMENT NO.
09035A105
11. CONTRACT/GRANT NO.
EPA-79-D-X0710
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory, RTF, NC
Office of Research and Development
U.S. Environmental Protection Agency
Research Trianole Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
PrniPrt Rpnnrt Final
14. SPONS
jprt ppnnpt
oRfNG AGE~NCY~
CODE
EPA/600/HERL. RTF
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The Environmental Protection Agency, under the Clean Air Act, is charged.
with the responsibility for regulating the emissions from new motor vehicles.
In order to assess potential heritable effects of diesel exhaust, mice were
exposed to whole diesel exhaust by.inhalation and a number of genetic end
points were studied. Exposure times varied from 5 to 10 weeks for the
different groups. The diesel particulate concentration averaged 6 mg/m3
during the exposure period of 8 hours per day and 7 days of the week. The
results of all genetic assays in both sexes were negative; however, small
but unequivocal effects on the reproductive performance of females of one
strain were observed.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
mutagenesis, heritable
effects, diesel, germ-
cell
18. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (Tills Report!
Unclassified
21. NO. OF PAGES
34
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
Triangle Park, NC, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily reflect
the views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
ii
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FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order.to promote the public
health and welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxi-
cology, epidemiology, and clinical studies using human volunteer subj-
ects. These studies address problems in air pollution, non-ionizing
radiation, environmental carcinogenesis and the toxicology of pesticides
as well as other chemical pollutants. The Laboratory participates in
the development and revision of air quality criteria documents on pollu-
tants for which national ambient air quality standards exist or are
proposed, provides the data for registration of new pesticides or
proposed suspension of those already in use, conducts research on
hazardous and toxic materials, and is primarily responsible for provi-
ding the health basis for non-ionizing radiation standards. Direct .
support to the regulatory function of the Agency is provided in the form
of expert testimony and preparation of.affidavits as well as expert
advice to the Administrator to assure the adequacy of health care and
surveillance of persons having suffered imminent and substantial endanger-
ment of their health.
The Genetic Toxicology Division supported this study in order to
determine whether or not whole diesel emissions produced a demonstrable
effect in a battery of in vivo mammalian assays for heritable effects.
The assays were chosen to detect several types of genetic end points
after exposure by inhalation to whole diesel exhaust. Such an
assessment helps to provide an adequate data base for future decisions
by the Administrator regarding requirements for emission standards from
mobile sources.
F. G. Hueter, Ph.D.
Director
Health Effects Research Laboratory
Research Triangle Park, NC
ill
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ABSTRACT
In order to assess potential risk from heritable effects in human popu-
lations, mice were exposed by inhalation to whole diesel exhaust,' and a num-
ber of genetic endpoints were studied. Exposure 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), chromosome
damage in males (dominant-lethal :and heritable-translocation tests), and
chromosome damage in females (dominant-lethal tests). Ancillary studies were
carried out to look for direct reproductive damage in both sexes; thus,
various parameters were used to assess reproductive performance in females,
and histological 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 perfor-
mance of females of one strain could be observed, consisting of a decrease in
....... <>;.".,; the/number ^pf pvulatiprvs and ;an increaseV.iri 'the 'interval--between .mating
... ; " r opportunity and"copulation. There was no detectable effect'of diesel expo--
sure 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 germcells have an efficient re-
pair system against induction of mutational events by such metabolites.
Thus, transmitted genetic effects appear not to be a major hazard from expo-
sure to diesel exhaust. The findings reported must, however, not be used to
draw any conclusions concerning possible risks to the exposed individual
' him/herself.
iv
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CONTENTS
Foreword ill
Abstract iv
Figures vi
Tables vii
1. Introduction ........ 1
2. Overall Conclusions 2
3. General Procedure 3
4. Reports of individual experiments 5
Test for heritable point mutations in male mice 5
Test for induction of dominant lethals in male mice ... 10
Test for induction of heritable translocations: in
male mice 13
Test for induction of genetic effects and oocyte
J.J killing in females 15
Test for dominant-lethal induction in female mice .... 18
Test for effects on spermatogonial survival in the
mouse 24
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FIGURES
Number Page
1 Distribution of fertile females with respect to number
of corpora lutea 21
2 Distribution of copulated females with respect to caging-
copulation interval 21
vi
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TABLES
Number Page
1 Results of specific-locus test, following exposure of
male mice to diesel exhaust 7
2 Specific-locus results grouped by germ-cell stage 8
3 Test for induction of dominant-lethal mutations in male mice . . 11
4 Induction of heritable translocations in male mice with
diesel exhaust 14
5 Reproductive performance of female mice exposed to
diesel exhaust 16
6 Mating performance and results of uterine dissections
for exposed females and controls 19
. 7 Distribution of corpora lutea counts in exposed females . .. .
and controls . . . ".-. ., . .. . . . .' ...-.-;. . . . ...... . 20
8 Distribution of mating intervals in exposed females and
controls . . . 22
9 Average number of corpora lutea at different mating intervals . . 23
10 Mean number of spermatogonia and preleptotene spermatocytes
observed after exposure to diesel exhaust for 5 and 10 weeks. . . 25
vii
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1. INTRODUCTION
Although non-mammalian and in vitro assays can provide evidence on pre-
sence or absence of mutagenicity, they have limitations in addressing the
complexities associated with reproductive-cell targets in intact organisms.
The assessment of risk from heritable damage must therefore utilize in vivo
mammalian germline mutagenicity 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 as-
says chosen were designed to detect a number of genetic endpoints, 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 experiments were
carried out there. Exposure to the diesel exhaust took place at the EPA
laboratory at Cincinnati. In the maximum-exposure group, the total intake
of exhaust per mouse was about 85 times the 30-year (generation-length) in-
take by a person in an average U.S. environment.
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2. OVERALL CONCLUSION
The results of all genetic tests in both sexes.were negative. Small
but unequivocal effects on the reproductive performance of females of one
strain could be observed, consisting of a decrease in the number of ovula-
tions and an increase in the interval between mating opportunity and copula-
tion. It is not 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 distri-
bution of cell types in the testis.
The absence of genetic effects could indicate either that no active
metabolites reached the gonads, or.that the germcells 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 germcells 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, ar.e. not a major hazard from
\exposure.".to .di^sei'1,exhaust. -'The 'findings reported''here must, ;howeyer, not .,..
be used to draw any conclusions concerning possible risks to the exposed
individual himself.. ... . .: . . : ; :
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3. 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 or air, and returned to Oak Ridge for the subsequent genetic experi-
ments. 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 de-
parture to Cincinnati and, again, after their return to the Oak Ridge
quarantine building. The first group to return to Oak Ridge was found to
*
Disease monitoring was under the supervision of the ORNL Biology Division
veterinarian, Dr. G. A. Bingham. Tests were performed for 21 organisms and
external parasites. These tests were designed to detect the presence of the
various organisms with a confidence, level of 95% or better, based upon esti-
mates of the prevalence of each organism within the colonies (if present at
all). Necropsies were performed on 80 representative mice [(101 x C3H)Fi, .
T, and SB] before any animals were taken to Cincinnati for exposure. Fol-
lowing return of the first batch, after 5 weeks at Cincinnati, 34 mice were
necropsied. Some of the organisms that were present before the mice were
sent to Cincinnati were not tested for on their return; i.e., the returning
mice were not tested for Polyoma virus, internal parasites, and external
parasites. Antibodies to the following three viruses were detected in this
batch of mice. Reovirus type 3, Theiler's GDVII and MVM, and Stapholococcus
aureus. On the basis of these findings, the animals were not returned to
the Oak Ridge Mammalian Genetics facility where they might have infected
the valuable colony. From the batches of mice that came back from
Cincinnati after about 8 and 11 weeks, respectively, 34 and 20 animals were
necropsied and tested as above. Test results revealed no organisms that
were not detected in some of the mice before being sent to Cincinnati; or
did these mice have the antibodies to Reovirus type 3, Theiler's GDVII, or
MVM that were detected in the first returning batch. These groups never-
theless joined the first batch in the separate building and all experiments
joined the first batch in the separate building and all experiments were
completed there. Disease monitoring was again carried out in December of
1979, when the collection of offspring in the specific-locus experiment was
completed and had yielded 3 (surviving) exceptional animals (see Project #1).
In order to make the genetic testing of these animals more convenient, we
desired to transfer them to the Mammalian Genetics facility. This transfer
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carry 3 viruses not present before they were shipped to Cincinnati. We
therefore 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 5x5 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
particulate concentration in the chamber averaged 6 mg/m^ during the exposure
period of 8 hours per day and 7 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
in 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).
These numbers are listed separately; for each experiment.]
We calculate that, during a 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 particulate concentration (mouse, 6 mg/m^ vs.
man 0.3 yg/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 , .
/ (dbminant-rlethal and heritable transldcation tests),. '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, substances
reached the gonads. If active material from diesel exhaust fails to get into
mammalian gonads, this would lead to negative findings in the experiments
reported, and presumably also to an absence of risk from genetic lesions
transmitted to future generations.
was effected subsequent to obtaining negative results in the disease
monitoring.
**
cages of different size
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.4.. REPORTS OF INDIVIDUAL EXPERIMENT
Project #1
TEST FOR HERITABLE POINT MUTATIONS IN MALE MICE
Objectives and relation to other projects
The objective of this project was1.to test for the induction of trans-
mitted point mutations (intragenic changes and small deficiencies) by means
of the specific-locus method currently the only practical and reliable
method in use in mammals that will detect and quantify this endpoint. This
method has recently proved the high sensitivity of in vivo mammalian germ-
cells to chemicals that are only weakly mutagenic (ethylnitrosourea) or non-
mutagenic (procarbazine) in bacterial tests or in in vitro systems. Where
chemicals that are mutagenic in other tests fail to produce mutations in
mammalian spermatogoniai this can be due to unstable metabolites not reach-
ing the target, to cell selection, or to spermatogonial repair systems.
Thus, one does not necessarily expect a ;good correlation between transmitted
damage induced in mammalian germcells in; vivo and results from other test
systems, However, it is the former result that is pertinent to transmission
of genetic lesions to future generations of human beings.
In planning the experiment, the number of offspring to be scored was
calculated so as to be sufficient either for showing a significantly posi-
tive effect, or for ruling out, with a high degree of confidence, that the
induced mutation rate, at the 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 ex-
posed to a quantity of Diesel exhaust that was a high multiple of that
accumulated by the average American in one human generation.
For assessments of human risk, the stage of prime importance in the
male is the spermatogonial stem cell, which can harbor (and transmit) muta-
tions for the lifetime of the individual. The bulk of our data were-there-
fore derived from Diesel exposures to that stage. However, we also obtained
enough specific-locus data for the (transitory) postspermatogonial stages to
rule out the possibility of a greatly elevated mutational sensitivity during
that period.
Since a very large (> 800,000) and reliable historical control exists
for spontaneous mutations in specific-locus experiments, we decided to use
all our available resources for the study of experimental groups only. A
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large sample could therefore be accumulated.
Exposure -.
Two groups of males were exposed: group A (114 mice) for 5 weeks
(3/21-4/24/79) and group B (138 mice) for 10 weeks (3/23-6/4/79). Exposure
to the Diesel exhaust was provided for 8 hours per day, 7 days per week, ex-
cept for an engine shut-down 4/24-4/26, inclusive.
Materials and methods
The exposed males in both groups were (101 x C3H)F^ or (C3H x 101)F,
hybrids who were returned to Oak Ridge the day after end of exposure.
Immediately upon their return, 86 males from each of groups A and B were
mated to 4 females, each, of the multiple-recessive T stock (ja/a; b/b_; cc^p/
cc^p; fi se/d se; s/s). Once a week for the next 4 weeks in group A, and
next 5 weeks in group B, the males were shifted to a new set of four fe-
males, while the females they left behind were separated one to a cage. In
group A, males were killed following these 5 weeks of serial mating, having
had the opportunity to mate with altogether 1720 females (20 for each male).
In group B, after the first 6 weeks of serial matings (2064 mating opportuni-
ties), the males were returned to their first-week mates to repeat the 6-
week cycle several more times until early November 1979.
All offspring were observed at about 3 weeks of age for any externally
visible changes from the normal phenotype. Exceptionals were saved for
further genetic testing, while, normals were discarded.
Results ' ' :'"'.' ' ..-. : '.'' '""' ;' :'.'" '''''-'.'.:'.'.''.' '''' ' ''"' '''''.''
. ,V! Except for. possible slight, effects, of: travel .(which-resulted in only.81
or 84% as many offspring from first-week than average later-week matings, see
Table 1), both groups of males appeared normal with regard to breeding per-
formance, producing average littersizes of 7.11 and 7.09 overall, respective-
ly, and impregnating 75.6% and 76.3% of the females during the first 5 weeks
of matings. While we had no concurrent controls, these figures are within
the range of the control experience with similar mating regimes (e.g., for
an unrelated specific-locus experiment conducted in 1979, average littersize
was 6.94 over a 21-week period). Nothing in the breeding performance
suggested even a short temporary sterile period which would have been indica-
tive of spermatogonial killing1
The number of offspring examined and exceptionals found are shown in
Table 1. . Only 4 exceptional animals were found*, and none of these
The 4 exceptionals were as follows: 2 animals with medium white belly spot,
probably resulting from heterozygous expression of s_ (Note: all offspring
of these animals are +/s); one slightly mottled with white, still under test,
but not a 7-locus mutant; one, very small and with various anomalies (fused
eyelids, scaly tail, shorter ears), died untested at 4 weeks of age.
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TABLE 1. RESULTS OF SPECIFIC-LOCUS TEST, FOLLOWING EXPOSURE OF MALE MICE
TO DIESEL EXHAUST
Week, after end
of exposure, in
which matings
occurred-
Group A
1st
2nd
3rd
4th
5th
Total
Group B
1st
2nd
.3rd
4th
5th
6th
7th
8th-20th
Total
Grand Total
No. of weeks of exposure in follow-
ingrgermcellnstages"'"
Meiotic and Differen- Sp'gonial
postmeiotic tiating stemcells
sp * gonia
5
4
3
2
1
5
V
"' "3
2
1
0
0
0
0 0
1 0
2 0
2 1
2 2
2 3
2 4
.- "-2 ; . ' s ' .
2 6
2 7
2 8
1 9
0 10
Numbers of
offspring excep
examined tionals
1556
1911
1753
1945
2075
9240
1643
1802
1970
1923
2181
1976
1866
19,971
33,332
42,572
0
0
0
0
0
0
0
0
0
0
0
0
0
4
4
4
mu-
tants
0
0
0
0
. . .0.'
0
0
. o
o
0
0
0
0
0
0
0
Germcell-stage information (mid-section of the table) applies to matings
made at the beginning of the week shown in this column.
^Analysis based on findings of Oakberg (1968) with regard to duration of
various germcell stages.
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turned out to be due to a mutation at one of the 7 loci. There is no sig-
nificant, difference between 4 exceptionals in 19,971 offspring (8th-20th
week) and-0 in 13,361 (lst-7th week).
It will be noted from Table 1 that, except for the 8th through 20th
weeks of group B, the exposure was divided differently among the germcell
stages for each week's production of offspring. For. purposes of analysis,
we have grouped the results (Table 2) into offspring derived from germcells
that were exposed 3 or more weeks during meiotic and postmeiotic stages
("postspermatogonial" group) and those derived from germcells exposed 8 or
more weeks in spermatogonial stages (stemcells or differentiating
"spermatogonial" group). This grouping omits the results for group A, 4th
and 5th weeks.
TABLE 2. SPECIFIC-LOCUS RESULTS. GROUPED BY GERM-CELL STAGE
Designation Weeks of exposure during following
.'".':.:..-. .".'.stages Number of
meiotic and spermatogonial offspring mutants
postmeiotic (stemcell & diff.)
"Postspermatogonial" 3-5 0-7 10,635 0
"Spermatogonial" 0-2 8-10 27,917 0
.: ;. Calculations have been made with each of:these groupings .for .the ratio .
of induced to spontaneous rate. Using the upper 95% Confidence limit of 0,
namely 3.3, we have computed the multiple of the spontaneous rate (43/801,406
or 0.000053655) that would not be exceeded in 97.5% of determinations made at
the experimental exposure level. For the "spermatogonial" group, for
example, the calculation is as follows
3'3 - 0.00053655
27,919
=1.20
0.000053655
Since there is some evidence, both from the absence of a sterile period
and the normal testis histology (Project #6), that no spermatogonial killing
has occurred, there is little likelihood for.a humped dose-response curve.
We therefore feel that it is conservative to extrapolate these results to the
level of the average human exposure, which is about 1/85 of the 10-week
experimental exposure [(6 x 103/3 x 10~1) (23.3/10,957) (2/1), see General
Procedure]. The experimentally observed zero mutation frequency rules out,
8
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with 97.5% confidence, that, at the level encountered by man, the induced
mutation rate could be higher than 0.014 times the spontaneous rate. In the
absence of information to the contrary, this extrapolation assumes that the
proportion of the total exposure that reaches target cells in the testis is
roughly similar in mouse and man.
For the "postgonial" group of 10,635 (as defined above and in Table 2)
the multiple calculated from the experiment was 4.8, and the weighted
average exposure was roughly 34x average human exposure. The multiple of
spontaneous rate, extrapolated to human dose, is thus 0.14.
Conclusion
There is no evidence for the induction of point mutations in spermato-
gonia or in 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 expo-
sure encountered by man, the induced mutation rate could be higher than 0.01
times the spontaneous rate.
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, _ Project #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 test
(see report on project #3). Since males were exposed to diesel exhaust for
a prolonged period and mated immediately afterwards (see below), all spermato
genic cell stages that are known to be. sensitive to dominant-lethal induction
were presumably 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 result-
ing embryo dies before or shortly after implantation.
Exposure . . - -
:.. .:. . .Mice were exposed for. 8..hours every day fr.om.3-22-7,9. to 5-14-79 (7.5 .
:week^X, /except .during':thefpe^^ :?'.
.Materials'.'-and.''Methods , ,-'...:-,. , . ; '..--. . ' ...'
A total of 216 male T-stock mice, 10-16 weeks old, were sent to
Cincinnati for exposure. Half of this number were exposed to diesel exhaust
and half served as controls. Immediately after the end of treatment, 102
and 100 mice in the experimental and control groups, respectively, were re-
turned to Oak Ridge. The reason why 14 mice disappeared is not known. Upon
their return to Oak Ridge, both control and experimental males were random-
ized into four groups of 24 each, with each group mated with one of four
different stocks of females: (C3H x 101^, (SEC x 05781^, (C3H x 05761)?^
or T-stock. Each male was caged with two females. Females were checked for
presence of vaginal plugs every morning for seven days posttreatment. Fe-
males that mated were replaced by new ones. Females that mated were killed
for uterine analysis 12-15 days after observation of the vaginal plug. The
use of four stocks of females was necessary in view of our finding that the
yield of dominant-lethal mutations in these stocks may vary markedly owing
to differences in the repair capability of .the fertilized eggs.
Results
Dominant-lethal data summarized in Table 3 represent the result for all
matings that occurred during the first 7 days after the end of exposure.
10
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TABLE 3 . TEST FOR INDUCTION OF .DOMINANT-LETHAL :MUTATIONS IN :MALE MICE*
Treatment^
Diesel
exhaust
Control
Stock of
female mice
(C3H x 101)F1
(C3H x C57BL)F1
(SEC x C57BL)F1
T-stock
(C3H x lODFj^
(C3H x C57BL)F1
(SEC x C57BL)F1
T-stock
Number of
mated
females+
85
83
94
89
94
85
83
82
Proportion
of pregnant
females (%)
81
87
80
82
77 .
84
77
87
Total implants
per fertile
female (avg)
7.8
10.5
9.9
8.8
7.6
10.1
9.6
8.1
Living embryos per
fertile female
(avg)
7.5
10.1
9.6
7.4
7.4
9.8
9.3
7.2
Dead implants/
total implants
(%)
4
4
3
16
1
; 3
3
23
T-stock males were used in the study.
Mice were exposed during a 7.5-week period.
+Males were mated during 7 days after the end of exposure. Tabulated data are from all matings during
this period.
-------
i These data do not show any indication of dominant-lethal effect. Breakdown
,j of data on a daily basis also did not reveal induced dominant lethality.
! ' . ' '
j Conclusions ;
j Results of the dominant-lethal test indicate that the exposure of male
.'j mice to diesel exhaust, as described above, did not induce detectable chromo-
I somal aberration effects in germ cells. ,,--
12
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Project #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
(see report on project #2).
The heritable-translocation method is a sensitive and reliable proce-
dure for measuring the frequency of chromosome breakage and rearrangement
(exchange of parts) that is transmitted to the next generation. When a
sperm carrying chromosome interchange(s) is used in fertilization, the re-
sulting progeny is heterozygous for the translocation and produces two types
of gametes, balanced and unbalanced, in approximately equal proportions.
Both types of gametes are capable of fertilization, but the unbalanced
gametes result in embryonic lethality. For this reason, most translocation
heterozygotes are only about half as productive.as normal mice. Heterozy-
gotes for certain' types of translocations'are incapable of producing sperm.
In the heritable-translocation procedure, progeny of treated parents are
therefore tested for sterility and "partial sterility." Confirmed sterile
and partially sterile progeny are then verified cyto'logically for presence
of a translocation. Thus, the heritable-translocation procedure generates
the most meaningful information for evaluating hazards from induced chromo-
some aberration to human population, because it measures transmissible
genetic damage.
Exposure
Mice were exposed for 8 hours every day from 3-22-79 to 4-22-79 (4.5
weeks), either to diesel exhaust or to air.
Materials and Methods
Altogether 160 T-stock males were used in the study. Each of these
males was caged with three (SEC x C57BL)F^ females just prior to shipment to
Cincinnati for the purpose of augmenting the number of progeny for estimating
the control frequency. This also served to rule out the possibility of pre-
existing translocations. The parental males were 15-18 weeks old when expo-
sure to diesel exhaust was started. They were randomized in equal numbers
into experimental and control groups.
13
-------
Soon after the last exposure, all animals were shipped back to Oak Ridge
(except for two mice from each group, lost at. Cincinnati). Each male was
caged with two (SEC x C57BL)Fi females immediately upon arrival. Males were
separated from females after one week. All male progeny were weaned and sub-
sequently tested for sterility and partial sterility. All sterile and par-
tially sterile animals were examined cytologically.
Results
Heritable-translocation data are shown in Table 4. We tested 1108 male
offspring of matings that occurred before parental males were sent to
Cincinnati. Among these 1108, one partially sterile translocation carrier
was found. In the contemporary control group (matings made after return from
Cincinnati), 358 male progeny were tested. No translocations were found in
this group. Thus, the control frequency (sum of the two groups) is 1 .
1466
TABLE 4. INDUCTION OF HERITABLE TRANSLOCATIONS IN MALE MICE WITH DIESEL
- EXHAUST
^ .. Number of ' Number of
Treatment progeny tested translocations
Control 1466 1
Diesel exhaust 358 0
.Male parents were exposed during a 4.5-:week period.
All offspring were conceived during the 7 days following the end of expo-
sure.
In the experimental group, 350 male progeny were tested - no transloca-
tions were found.
The test has an estimated power of better than 95% that it could detect
an induced rate of 0.015 x 10~2.
Conclusions
Results of the heritable-translocation study indicate that the exposure
of male mice to diesel exhaust, as described above, did not induce trans-
missible chromosome exchange in male germ cells. This result is consistent
with that of the dominant-lethal test (Project #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.
14
-------
_. , . Project #4
TEST FOR INDUCTION OF GENETIC EFFECTS AND OOCYTE KILLING IN FEMALES
' Objectives and relationship to other projects
I The effectiveness, of diesel exhaust In producing chromosomal and cyto-
i toxic damages to oocytes was studied by means of total reproductive capacity.
j The mouse ovary is known to be sensitive to even relatively slight insults
: from known mutagens. Genetic and/or cytotoxic effects on oocytes in differ-
ent stages of follicular development were measured by simply counting the
1 offspring produced by exposed and control females over a period of time.
Exposure
I
Mice were exposed to diesel exhaust for 8 hours every day from 3-22-79
i to 5-14-79 (7.5 weeks), except during the period of 4-22 to 4-27 due to
1 equipment failure.
J '
, Materials and Methods. , .
I Altogether 120 (SEC x C57BL)F1 females (about 12 weeks old) were
1 shipped to Cincinnati; one-half of these were, exposed to diesel exhaust, and.
, the remainder sham exposed'. The females were returned to Oak Ridge immedi-
ately after the last exposure and caged individually with (C3H x 101)F^
males upon arrival. Reproductive performance of the females was determined
by the size of the litters produced in successive matings during a 195-day
posttreatment observation period. Reduced litter sizes may indicate induced
dominant-lethal mutations or oocyte killing.
' Results
; Results are summarized in Table 5. During the observation period, no
I significant difference between experimental and control groups was observed
i with respect to the number of litters produced per female and with respect
to the size of litters produced at various posttreatment intervals - i.e.,
there was no significant difference in the total number of young produced
during the observation period.
j Conclusion
i
! The similarity in long-term reproductive performance (pregnancy rate
!* and littersize) between control females and females exposed to diesel ex-
j . haust indicates that the exposure to diesel described above did not induce
15
-------
TABLE 5. REPRODUCTIVE PERFORMANCE OF FEMALE MICE:EXPOSED!TO:DIESEL EXHAUST
Time after end
of treatment
(days)
;.;- ,;:' Proportion of females
, Number of females ; % .bearing litter (7,)
Diesel exhaust Control . ./-Diesel exhaust Control
18-24
25-43
44-62
63-81
: 82-100
101-119
120-138
139-157
158-176
177-195
45
45
45
45
45 .
45
45
45
45
45
45 ;'* ':'-
45 ':;.'' ';';
.45 :.:-: :^
45 ;-'.;; V;
. . . .45.. :,;' :/ :'?. .
45 ..;.-'.- vtrl
45 v 'S
43 /;::;.;:-.'
43 -..'.: -.:.'
43
80.0
95.6
93.3
95.6
88.9
84.4
.64.4
80.0
82.2
86.7
75.6
95.6
, 88.9
95.6
f
.91.1
1 82.2
: 84.4
, 86.0
86.0
86.0
Average littersize
Diesel exhaust
10.0
11.8 :
11.9 i
12.2 t.
11.7 - L -
i
12.2 |
12.4 |
11.2- | ;
10.9 !
10.0 :
Control
9.3 i
11.6 ;
12.0
11.8
12.5
12.3
11.9
11.9
11.1
9.6
-------
detectable 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 //5.
17
-------
Project #5_ _
TEST FOR DOMINANT-LETHAL INDUCTION IN FEMALE MICE
l Objective and relation to other projects
| . . .
i The primary objective of this experiment was to test for induction of
I . chromosomal damages in mature and maturing oocytes. Since the accessibility
| of the ovary to Diesel metabolites. may be very different from that of the
| testis, this project constituted an important companion to projects No. 1,
! 2, and 3, which assayed for genetic damage in males. An additional objective
| of this experiment was to test for any short-.term effects of Diesel exhaust
j on reproductive physiology; long-term effects were tested in project No. 4.
, t . ..
! ' ''.'' ' '
Exposure
j Females were exposed to Diesel exhaust at Cincinnati for 8 hours daily,
' 7 days per week from March 22 through May 14, 1979, except for a shutdown of
I the Nissan engine during the period of April 24-26, inclusive. The total
.!'.,. exposure. period, not,. counting shut-downs, was 7 weeks. The control group
j. ;'.' . > '': ' V;':. was" exposed ^ 'to'iafr ^ in .an identical chamber, during the ;. same period . There ;
i '': . were- 54 females in each group. ' Because of caging problems at Cincinnati,
., . . twelve 'mice escaped between ^removal. from .the chambers. and transport to. Oak . .
]':" :- '". Ridge. This left 45 in the experimental (Ex) and 51 in the control (Co) '
i group. ;
; Materials and Method
j , The exposed females were (101 x C3H)F1 virgins, 11.1/2 to 13 weeks old
j at the beginning of exposure. Immediately following their return to Oak
| Ridge. one day after end of exposure they were caged with (101 x C3H)F1
j males (2 ₯°-:l o) and checked for plugs every morning for the next 7 days.
i Females were sacrificed usually 14 (occasionally 13 or 15) days after the
j plug date, corpora lutea were counted, and the uterine contents examined.
The 6 females that had not copulated during the 7-day plug-check period were
! . left with their mate for an additional.15 days arid then sacrificed. For
; these females, a plug date was estimated from the stage of. development of
, their embryos.
Results
The top portion of Table 6 shows the mating performance, and the bottom
shows the dissection results. There is no significant difference in the
percentage of matings resulting in pregnancies. Of the ovulated eggs
. 18
-------
(represented by corpora lutea, c.l.)» approximately equal proportions re-
sulted in living fetuses, and the distribution among the remaining uterine-
content categories was also similar between the Ex and Co groups. There is
thus no evidence for the induction of a dominant-lethal effect.
TABLE 6. MATING PERFORMANCE AND RESULTS OF UTERINE DISSECTIONS FOR
. EXPOSED .FEMALES AND CONTROLS . .::.::.:.:'...::::.-:::':::::.
No. ?? exposed
No. 9$ caged with oo*
No. $°- failing to copulate
within 15 days
Non-fertile plugs
Fertile plugs
Corpora lutea
Live fetuses
Death after day 10 p.c.
Res orb ing
Preimplantation loss
Corpora lutea per female
No.
384
298
0
37
49
9.
Experimental
54
45
1
3
41
37 ± 0.
(2.2%)
(6.7%)
(91.1%)
% of c.l.
_^
77.6
0
9.6
12.8
21
Control
No.
521
418
1
37
65
10.85
54
51
0
3
48
7
± 0.15
(5.9%)
(94.1%)
: of c.l.
m
80.2
0.2
7.1
12.5
Difference between No. -rr exposed and No. -I-K caged with oo is due to -H- that.
escaped. ' . - .'. . , .: . . :; . . . . ;
In two parameters, however, clear effects of the Diesel exposure are
apparent, (a) There is a significiant difference between Ex and Co groups
in mean number of corpora lutea (Table 6, last line), and this difference
becomes even more striking when one compares the entire distributions (Table
7 and Fig. 1). (b) The average interval between caging a female with a male
and copulation (finding of a plug) is significantly longer, due to a shift
in the entire distribution (Table 8 and Fig. 2). In order to determine
whether the decrease in corpora lutea might be a function of the delay in
mating, we tabulated average number of corpora lutea for each mating interval
(Table 9) and found the experimental group to be consistently lower. This
indicates that the depression in number of ovulations is not merely the
result of mating later than normal.
Discussion and Conclusions
A seven-week exposure to exhaust fumes from a Nissan Diesel engine pro-
duced no transmissible chromosomal damage, as measured in a dominant-lethal
experiment, in mature and maturing oocytes. However, significant, though
slight, effects were produced on female reproductive functions, as expressed
19
-------
TABLE 7. DISTRIBUTION OF CORPORA LUTEA COUNTS IN EXPOSED FEMALES
CONTROLS
AND
No. of % (No.) of females
corpora lutea
Experimental Control
7 7.3 (3) 0
8 17.1 (7) 2
9 36.6 (15) 8
10 17.1 (7) 25
.11 ; 17.1 (7): 33
12 2.4 (1) 29
:','' ; ' ' >'".-.! ;: ; ". ' ':'.;*'''" - . ': ' " ' -'''; . ' >-,'.:'.!".''. ' . ...
.1
.3
.0
.3
.2.
:i.'
(1)
(4)
(12)
(16)
.(I*)..-.-,
(1)
Total (41). .'..'. . (48)
Mean No., corpora lutea, experimentals, 9.37 ± 0.21
Mean No. corpora lutea, controls, 10.85 ± 0.15
P < 0;.0001
20
-------
Figure
o*-
I
I
8 9 10 11
No. of corpora lutea
12
13
o»
_3
CL
O-l-
Figure 2
Control
Exposed
)
1
f 1
1
I ,,._ ,, _.,. .
1
2 " 3 ' 4 5,6,7
Days between caging with (/"and plug
8-14 >14
21
-------
TABLE 8. DISTRIBUTION OF MATING INTERVALS IN EXPOSED FEMALES AND
CONTROLS . ...I..:.::.::::...:.:::::::::::::::::::::::::
Interval: caging-
to-plug (days)
1
2
3
4
5 " ";
6
' 7
. 9 ''
-.^/'.i^r^tr
14 ..'..
20*
No.., of
Experimental
6
7
15 ,.
- ' 5-
' ' " ". .' "2 "" "V '"
2
2
..-..'' 1 .-,;'.
J-'^iii-fe?^.^
... _ 1
'-. - . ; , i v '.-
Total 45
females _ - ...
Control
3
22
11
8
"" 5
1
1
0
.:;,:"' 4-^^o^f:-
.0
0
51
Mean interval, experimental 4.42 ± 0.58
Mean interval, control 2.94 ± 0.18
P = 0.016
A 20-day interval was arbitrarily assigned for one female who showed no
implants 22 days after being caged with male. Minimum interval could
have been 16 days. If a smaller interval than 20 days is assigned,
significance increases slightly; e.g., for 15 days, P = 0.014.
22
-------
TABLE 9. AVERAGE NUMBER 07.CORPORA LUTEA.AT.DIFFERENT:MATING:INTERVALS
Interval: caging- Average No. of corpora lutea
to-plug (days) Experimental Control
5
8
1
2
3
4
, 6, 7
- 14
8
10
9
9
9
8
.8
.3
.6
.2
.0
.8
*
(5)
(6)
(14)
(5)
(6)
(5)
10
10
11
10
11
.0
.8
.2
.6
.0
(2)
(21)
(11)
(7)
(7)
No. of females on which the average is based is shown in parentheses.
in a decreased number of ovulations (14% overall) and a lengthening (by
about 1.5 days) of the time interval between mating opportunity and copula-
tion.
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 #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 C57BDF-L in. Project 4 and (101 x C3H)Fx in Project 5. 'For the'first-
week period, only 75.6% of mated control females produced litters in Project
#4, but 100% of controls got pregnant in Project #5. It is therefore diffi-
cult 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 4.4 days), such as was determined through day-by-day analysis in
Project 5, would have been detectable in Project #4. It seems probable that
the method of Project #4 was not sensitive enough to detect such a shift,
had it been induced in the (SEC x C57BL)F1 females. Project #4 should, how-
ever, have detected a 16% reduction in average littersize, such as appears
to have been induced in Project #5. We conclude that there is a genetic
difference in sensitivity of females to the physiological damage(s) respon-
sible for reducing number of ovulated eggs.
23
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. ' _ Project #6 ;.,.'_ . J _,
TEST FOR EFFECTS ON SPESMATOGONIAL SURVIVAL IN THE MOUSE
Objective
Differentiating spermatogonia of the mouse are extremely sensitive to
cytotoxic agents such as radiation .(1) and chemicals, and often show a posi-
tive response even when no genetic damage is observed (2). In the latter
'case, they provide a sensitive test, system to determine if test materials
reach the gonads. The response observed, however, is a somatic one (i.e.
cell death) and is not necessarily associated with heritable effects. Kill-
ing of spermatogonia can, of course, have an effect on fertility.,
Materials and Methods . . .
Hybrid male mice of the JH and H strains were exposed to diesel exhaust
8 hrs per day, seven days a week, for 5 and 10 weeks. The mice then were
returned to Oak Ridge and killed immediately. Contemporary controls were
transported to Cincinnati,handled.similarly. ,but exposed to air pnly,, re- . .
iturn'ed toiOak-Ridge^Vand :k^iledva^ mice..' (Controls
for the 10-week group, inadvertently, were returned
.; Mice:were killed by cervical' dislocation;. The testes were dissected '-
free of connective tissue and fat and.fixed, in Zenker-fonnol. Five-micron
paraffin sections were stained in periodic-acid-shiff and hematoxylin. One
hundred tubule cross sections, distributed among the stages of the semini-
ferous epithelium on the basis of the frequency distribution of stages of
the cycle in controls, were scored for each mouse. Eight spermatogonial
classes (As, Apr + Aai, Aj_, A£, Ag, A^, In, B) and preleptotene spermato-
cytes were enumerated. Three controls and 3 treated males were used for
each strain and exposure time.
Results .
No effect of exposure to diesel exhaust on number spermatogonia or
preleptotene spermatocytes was observed for either strain or exposure group.
Control counts were the same at 5 and.10 (actually, 9) weeks, so controls
were combined by strain. Data are given in Table 10. Differences in cell
counts all were within the limits normally observed for control mice.
24
-------
TABLE 10. MEAN NUMBER OF SPERMATOGONIA AND PRELEPTOTENE SPERMATOCYTES OBSERVED AFTER EXPOSURE TO
DIESEL EXHAUST FOR 5 AND 10 WEEKS
JH
Control
Cell Type
As
Apr + Aal
Al
A2
A3
ro
01 A
A4
In
B
Pre-lept
Sp ' gonia
Sp ' gonia
Sp ' gonia
Sp ' gonia
Sp ' gonia
Sp ' gonia
Sp ' gonia
Sp ' gonia
Sp'cytes
52. 8^
14.2
73.2
26.3
39.8
82.7
137.5
243.2
1129.2
5 wks.f
59.0
13.3
96.7
21.7
33.7
86.0
128.3
271.0
1184.0
mice
Treated
10 wks.'1"
45.6
9.0
80.0
19.7
41.3
78.7
138.0
.282.7
1117.7
H mice
Control*
5 & 10
weeks
52.3
11.2
88.3
20.7
37.5
82.3
133.2
276.8
1150.8
59.2
12.5
85.0
29.2
42.3
76.3
j 126.5
; 236.8
! 1106.8
5 wks.f
57.0
13.3
85.3
35.3
40.0
80.3
123.3
268.3
1089.7
Treated
10 wks.f
46.3
12.0
81.0
24.0
33.0
68.7
124.7
255.0
1079.0
5 & 10
weeks* '
51.7
12.7 .
83.2 :
29.7 '
36.5
74.5 ;
124.0 !
, i
261.7 s
!
1084.3 ';
Mean of 6 mice.
Mean of 3 mice.
+Number of cells scored in 100 tubule cross sections per mouse, distributed among the stages of the
cycle of the seminiferous epithelium on the basis of the frequency distribution in control mice.
-------
t. .
t.-..
Conclusion
Exposure to diesel exhaust had no effect on the number of spermatogonia
or preleptotene spermatocytes of-mice after 5 or 10 weeks' exposure. Like-
wise, no irregularities in the normal dynamics of the seminiferous epitheli-
um were observed; cellular associations were the same as in controls, and
division of the different spermatogonial types occurred at the normal times
in the cycle of the seminiferous epithelium.
1. Oakberg, E. F. Gamma-ray sensitivity of spermatogonia of the mouse.
J. Exp. Zool. 134:343-356, 1957.
2. Oakberg, E. F. Response of spermatogonia of the mouse to hycanthone:
A comparison with the effect of gamma rays. In, Physiology and Genetics
of Reproduction, Part A. E. M. Coutinho and F. Fuchs, eds. Plenum Pub.
Corp., N.Y. pp. 197-207..
26
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