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 ------- 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 ------- 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. ------- 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 Postage and Fees Paid Environmental Protection Agency EPA 335 Official Business Penalty for Private Use $300 0000 32 r L ,-, • CHICAGO ------- |