Diesel particulate extracts in bacterial test systems

The Ames bacterial mutagenicity test system was used to evaluate parameters which may affect the mutagenic activity of diesel particulate extracts. The optimal extraction conditions, extractability of mutagens by simulated biological fluids and the effect of collection method were investigated. The role of solvent was examined by extracting diesel particles with methanol, acetone, cyclohexane, ethyl acetate, n-hexane, dichloromethane, benzene and a benzene-ethanol mixture. Of these, the dichloromethane extract exhibited the highest activity in the Ames test, although methanol yielded the largest extractable mass. Diesel particles were also extracted by dimethyl sulfoxide (DMSO) and four other simulated biological fluids for 48 h at 25, 37, and 45°C to study the effects of temperature. The mutagenic activity of the DMSO extract began to decline at temperatures higher than 37°C after 8 h of incubation. Fetal calf serum was the only simulated biological fluid which eluted mutagenic activity from the particles. No activity was detected in the 0.5% bovine serum albumin, simulated lung surfactant and saline extracts. Diesel particles collected by electrostatic precipitation (ESP) and filtration were studied. The mutagenic activities of both extracts were comparable when expressed as revertants per mg of particle. After the extracts were separated into nine fractions by a solvent partitioning scheme, the majority of the activity was found in the neutral-nonpolar II, neutral polar, strong acid and weak acid fractions. The acid salt fraction from the ESP sample was inactive. These results demonstrate that differences in the extraction conditions can result in differences in the mutagenic activity of diesel particulate extract. Since the mutagens in the extracts are not readily extractable by simulated biological fluids, the question of bioavailability of mutagens in diesel particles must be considered in the final assessment of their potential effects in biological systems and organisms.     

Lymphatic transport of inhaled diesel particles in the lungs of rats and guinea pigs exposed to diluted diesel exhaust

Factors influencing the disposition of the inhaled diesel particles were studied by analyzing the deposition of radioactively labelled diesel particles in the respiratory system, by determining the specific function of alveolar cellular mechanisms in the primary defense against inhaled particles and by identifying the important role of the lymphatic system in the lung clearance of experimental animals exposed to diluted emissions from a diesel engine. Radioactive ¹³¹Barium was used as a tracer of diesel particles and the deposition efficiency was determined to be 15%±6% of the inhaled dose in the Fischer 344 rat strain. The number of cells obtained by bronchial lavage increased significantly after a prolonged exposure to a concentration of 1500 μg/m³ of diesel particles. The increased cell number was more than twofold, contained two distinct cell populations (alveolar macrophages and neutrophils) and represented a reactive mobilization of the defense mechanisms in the organism. Light microscopy studies investigated the role of lymphatic transport of the particulate matter and revealed that the peribrochial and perivascular aggregates of lymphoid tissue contained diesel particles even after short exposure periods at low dose levels. With the increasing burden of particles in the respiratory system, the coloration of hilar and mediastinal lymph nodes continuously changed to gray and finally to dark black, depending upon the dose level and exposure. However, at all exposure levels, most of the diesel particles in the alveoli were phagocytized by an increased alveolar cellular defence and particle-containing macrophages were actively moving towards the mucociliary escalator or towards lymphatic channels leading to peribronchial lymphoid aggregates and bronchial or mediastinal lymph nodes. In the lymph nodes, alveolar macrophages containing diesel particles were found mostly in the afferent subcapsular lymphatic vessels and marginal sinuses. In the later stages, cellular structure disintegrated and large aggregates of particulate matter were dispersed throughout the medullary cords with increasing accumulation towards the hilus. It is concluded that the lymphoid aggregates and lymphatic nodes play an important role in sequestering diesel particles or particle-containing phagocytizing cells and provide a pathway, in addition to the mocociliary clearance for particulate removal from the deep pulmonary region.     

DNA-binding studies with diesel exhaust particle extract

This study examines whether chemical components from diesel exhaust particulates react with DNA to form covalently bound adducts. Experiments in this report describe the in vitro reaction of purified DNA with a dichloromethane extract of diesel exhaust particulates in the absence or presence of enzyme activation by rat liver microsomes. The reactivity of the particle extract was compared to that of benzo[a]pyrene metabolites using low temperature fluorescence techniques which detect small quantities of polycyclic aromatic compounds bound to DNA. Incubation of DNA with the particle extract in the presence of microsomal enzymes produced no detectable fluorescent adducts in contrast to model experiments using benzo[a]pyrene. However, addition of the particle extract to incubation mixtures containing benzo[a]pyrene markedly decreased formation of benzo[a]pyrene-DNA adducts because the particle extract inhibits microsomal enzymes which activate benzo[a]pyrene and other polycyclic aromatic hydrocarbons. In the absence of microsomal enzymes, fluorescent material was detected in DNA exposed to high concentrations of the particle extract, but probably not as a result of covalent binding because the mutagenic activity of the particle extract remained unchanged during prolonged incubation with DNA. This stability is in contrast to the rapid decrease in mutagenic activity of benzo[a]pyrene-4,5-oxide during incubation with DNA. Thus, direct mutation of bacteria by the particle extract may require activation by bacterial enzymes as is known to occur with nitroaromatic compounds.     

Physical characterization of diesel exhaust particles in exposure chambers

Since the deposition of particulate in the respiratory system is strongly influenced by particle size, a correct assessment of this parameter is important for any inhalation experiment studying the potential health effects of air pollutants. Measuring the distribution of particles according to their aerodynamic diameter and mechanical mobility diameter is crucial in analyzing the deposition of submicron particles in the lower respiratory system. Cascade impactor measurements of diluted diesel exhaust in 12.6 m³ animal exposure chambers in the GMR Biomedical Science Department showed that the mass median aerodynamic diameter of the aerosol was 0.2 μm with 88% of the mass in particles smaller than 1 μm. Diffusion battery measurements showed that the mass median mechanical mobility diameter was about 0.11 μm. Transmission electron micrographs of particles deposited on chamber surfaces revealed both agglomerates and nearly spherical particles. The particles in these chambers are similar in size and shape to diesel particles described elsewhere. The flux of diesel particles to food surfaces was measured. Calculations of the expected daily dose by inhalation and by feeding showed that the “worst case” dose by feeding was only about one-tenth the dose by breathing.     

Evaluation of techniques used in the preparation of diesel extract samples for mutagenicity studies

Diesel exhaust particles were used to compare methods and techniques used in the preparation of particulate matter for microbial mutagenesis testing. Investigated in this study were extraction, concentration, and solvent exchange methodologies as they affected recovery of mutagenic material from diesel samples using a Salmonella typhimurium plate incorporation assay. Solvent removal methods applicable for use in determining the mass concentration of extracts were also evaluated. Results indicated that particle samples Soxhlet extracted with dichloromethane yielded higher levels of mutagenic activity than did comparative samples utilizing sonication. No difference was seen between rotary evaporation or Kuderna-Danish macro concentration of extracts to volumes > 50 mL. In comparison of micro concentration techniques to volumes < 10 mL, vortex evaporation was found to be more efficient than a modified micro Kuderna-Danish method in recovery of mass and mutagenicity. Solvent exchanged samples were found to yield higher recoveries of mutagenic activity than samples taken to dryness and then reconstituted in the bioassay solvent. A dry mass weighing procedure utilizing desiccation was found to be more acceptable than either the use of an infrared heat lamp or nitrogen blowdown for solvent removal.     

Detection and average content levels of carcinogenic and mutagenic compounds from the particulates on diesel and gasoline engine mufflers

Carcinogenic and mutagenic compounds, which were extracted from the particulates that adhered to inner surfaces of diesel and gasoline engine mufflers, were quantified by the series method of Soxhlet extraction, liquid-liquid partition, thin-layer chromatography, and spectrofluorometry. Mutagenic activity of their neutral and acidic fractions was tested in the improved Ames assay by the preincubation method with Salmonella typhimurium TA98 in the presence and absence of metabolic activation system (S-9 mix). The average content levels (μg/g tar) of polycyclic aromatic hydrocarbons from gasoline engine cars were greater than those from diesel engine vehicles. However, the levels of nitro derivatives of PAHs and polycyclic quinones from the diesel engines were greater than from the gasoline engines. Mutagenic activity of the diesel acidic fraction was the highest among the diesel and gasoline fractions, and was significantly higher in the absence of the S-9 mix. Furthermore, the relative value (R_{c = 0}) of infrared absorption of carbonyl stretching vibration to that of methylene asymmetric stretching vibration of the diesel acidic fraction was the highest among the diesel and gasoline fractions. These results strongly suggest that highly direct-acting mutagens in the acidic fraction are at higher levels in diesel emission particulates than those from gasoline, and that these mutagens are carboxylic acid, aldehyde, and alcohol derivatives of PAHs and NPAHs.     

Salmonella/microsome mutagenicity assays of exhaust from diesel and gasoline powered motor vehicles

Motor vehicle exhaust from prechamber injection diesel and gasoline powered passenger cars, sampled during US FTP 1973 test cycles and comprising both particulate matter and compounds condensable at ambient temperature, has been assayed for mutagenicity in the Salmonella/microsome test. Mutagenic components were to a large extent active in the absence of the mammalian microsomal preparation. The mutagenicity of both particulate matter and condensate from diesel exhaust and condensate from gasoline exhaust was decreased in the presence of the microsomal preparation whereas the mutagenicity of particulate matter from gasoline exhaust was enhanced by microsomal activation. A comparison between the investigated diesel and gasoline exhaust samples shows that the mutagenic effect in the Salmonella test of the diesel exhaust is more than ten times higher than that of the gasoline exhaust. Fractionation with respect to polarity indicates that the mutagenic components mainly are distributed in neutral aliphatic, aromatic, and oxygenated fractions. Tests for mutagenic monofunctional nitroarenes by an anaerobic assay indicate that such compounds at most are marginally present in the exhaust samples as compared with their presence in airborne particulate matter collected in an urban environment.     

Mutagenic activity of diesel emission particulate extracts and isolation of the mutagenic fractions

Mutagenic activity in diesel emission particulate extracts was detected by the Salmonella typhimurium/microsome assay. Direct-acting mutagens as well as promutagens requiring metabolic activation were detected. The extracts were fractionated into acidic, basic, and neutral fractions, and the neutral fraction was chromatographed into seven subfractions. Differences in the mutagenic potency of these fractions and subfractions were determined by the Salmonella assay. Fractions containing as yet unidentified compounds, but not polynuclear aromatic hydrocarbons, were found to make a major contribution to mutagenic activity of the extracts.     

Mutagenic and carcinogenic potency of diesel and related environmental emissions: Salmonella bioassay

Due to the expected increase in the percentage of diesel vehicles in the United States, the Environmental Protection Agency must evaluate the health effects associated with exposure to diesel emissions. Respirable particles from a variety of combustion sources have the potential of being carcinogenic and mutagenic. The objective of these studies was to determine the relative biological activity of the organic material adsorbed on these particles in in vitro mutagenesis bioassays. The organic extracts from the following series of emission sources were bioassayed in the Salmonella assay for mutagenic activity: (1) a light-duty Oldsmobile diesel 350 engine; (2) a heavy-duty Caterpillar diesel engine; (3) a light-duty Nissan engine; (4) a Volkswagen Rabbit diesel engine; (5) cigarette smoke; (6) roofing tar; (7) coke oven; and (8) a gasoline catalyst Mustang. This paper provides a comparison of these sources within the Salmonella bioassay and also demonstrates how bacterial systems can be used as a quality assurance measure in in vivo testing.     

Genotoxicity of syrian hamster lung cells treated in vivo with diesel exhaust particulates

Polycyclic aromatic hydrocarbons extracted and concentrated from diesel exhaust particulates have been shown to be mutagenic and carcinogenic, but attempts to induce pulmonary tumors through chronic inhalation of diesel exhaust by experimental animals have failed. We have attempted to resolve this incongruity by measuring chromosomal damage in lung tissue of chronically exposed hamsters, using the highly sensitive test for genotoxic chemical agents, sister chromatid exchange (SCE) analysis. To determine the degree of responsiveness of the test system to both diesel exhaust particulates and benzo(a)pyrene (BaP), these agents were instilled intratracheally into anesthetized hamsters as suspensions in 0.25 ml volumes of Hank's balanced salt solution (HBSS). Lung tissues from these animals were subsequently cultured in vitro and chromosomes from the resulting cell divisions were scored for exchanges of chromatin between sister chromatids. Control animals, treated weekly with 0.25 ml of BSS for 10 weeks, showed an average value of 12 SCE's per cell, while animals treated weekly with 200 ng BaP over a 10-week period showed an average of 17 SCE's per cell. HBSS, given as a single treatment, also produced an average of 12 SCE's per cell in control animals, but animals treated with a single instillation of 12.5 μg BaP showed an average SCE value of 19. These data confirmed that the procarcinogen BaP can be metabolically activated by lung cells in vivo and also demonstrated the efficacy of using this technical approach to study the effect of chemical mutagens that enter the lungs. Diesel exhaust particulates, administered in a range from 0 to 20 mg per hamster over a 24 h exposure period, produced a linear SCE dose-response ranging from 12 to 26 SCE's per metaphase. This curve suggested that a concentration of 3 mg of diesel particulates per hamster would not produce a statistically significant increase in SCE's above control values. One group of 8 hamsters, chronically exposed to diesel exhaust particulates for 3 months showed an average of 12 SCE's per cell. This was equivalent to a set of 5 control animals which also showed an average of 12 SCE's per cell. Although the scope of this study was limited, the data demonstrated that diesel exhaust particulates can induce genotoxic damage but a 3-month exposure to 6 mg/m³ of diesel exhaust particulates was insufficient to produce measurable mutagenic changes in lung cells. This negative response is consistent with the results from other studies in which similar exposures failed to produce pulmonary tumors.     

Mutagenic and carcinogenic potency of extracts of diesel and related environmental emissions: Summary and discussion of the results

The proposed conversion from gasoline powered automobiles to diesel powered vehicels has prompted the Environmental Protection Agency to evaluate the potential health effects associated with exposure to diesel emissions. At present, there is no direct epidemiological link between this exposure and human health. Therefore, a research program was constructed to compare the health effects associated with diesel emissions with those from other emission sources for which epidemiological information was available. The emission sources chosen were cigarette smoke, roofing tar, and coke oven. An additional comparative emission source which was a gasoline catalyst engine. Respirable particles from a variety of combustion sources have the potential of being carcinogenic and mutagenic. The objective of these studies was to determine the relative biological activity of the organic material adsorbed on these particles in both in vitro mutagenesis and in vitro and in vivo bioassays. The organic extracts from the following series of emission sources were quantitatively bioassayed in a matrix of tests for their carcinogenic and mutagenic activity: (1) a light-duty Oldsmobile diesel 350 engine; (2) a heavy-duty Caterpillar diesel engine; (3) a light-duty Nissan engine; (4) a Volkswagen Rabbit diesel engine; (5) cigarette smoke; (6) roofing tar; (7) coke oven; and (8) a gasoline catalyst Mustang. The test matrix consisted of the following bioassay: reverse mutation in Salmonella typhimurium; mitotic recombination in Saccharomyces cerevisiae; DNA damage in Syrian hamster embryo cells (SHE); sister chromatid exchange in CHO cells; gene mutation in L5178Y mouse lymphoma cells, Balb/c 3T3 mouse embryo fibroblasts and CHO cells; viral enhancement of SHE cells; oncogenic transformation in Balb/c 3T3 cells; and skin tumor initiation in SENCAR and C57 black mice. The results of this test matrix are discussed.     

Preparation and characterization of diesel exhaust particles for biological experiments

Bioassays of diesel engine exhaust components are being conducted at IITRI to determine toxic and carcinogenic potentials of the exhaust. The bioassay method, intracheal instillation of saline suspensions of test materials in hamsters, requires preparation of stable suspensions of test materials. A method to prepare suspensions of whole particle diesel exhaust in saline has been developed. The diesel exhaust particle material was supplied to IITRI as a dry, loose powder by the U.S. EPA from a light duty diesel test engine. Preliminary characterizations of the powders indicated aggregation of exhaust particles had occurred both before and during capture on collection substrates. Flake-like sheets and hollow spheres of aggregated particles up to 150 μm in sie present in the powders. Therefore, the powder were ball-milled to geometric particle sizes more amenable to the animal administration technique to be employed. Grinding, suspension preparation and particle concentration assaying methods have been developed. Particle (geometric) size and morphological characterizations have also been performed on the as-received powders and prepared suspensions. A method to prepare emulsions (liquid-liquid suspensions) of the dichloromethane extracts of whole particle diesel exhaust has also been developed.     

In vivo detection of mutagenic effects of diesel exhaust by short-term mammalian bioassays

Male Chinese hamsters were exposed to diesel exhaust and clean air for six months at the Center Hill Facility of the U.S. Environmental Protection Agency in Cincinnati, Ohio. The animals were kept in specially constructed inhalation chambers and exposed to clean air or diesel exhaust for eight hours daily. The animals were sacrificed and slides prepared to study the mutagenic effects of diesel exhaust by four in vivo short term mammalian bioassays. Sperm morphology bioassay revealed a 2.67-fold increase in sperm abnormalities in the animals exposed to diesel exhaust as compared to those exposed to fresh air. Micronucleus bioassay revealed a 50% increase in the number of micronuclei in polychromatic erythrocytes obtained from animals exposed to diesel exhuast. However, no increase in sister chromatid exchange or chromosomal abnormalities was observed in bone marrow cells of animals treated with diesel exhaust. During these studies a decrease in mitotic index was observed in animals treated with diesel exhaust.     

Enhanced susceptibility to infection in mice after exposure to dilute exhaust from light duty diesel engines

A series of experiments was conducted in which groups of mice were first exposed for various durations to diluted exhaust from light duty diesel engines and then briefly to an infectious aerosol generated by nebulizing cultures of a bacterial pathogen (Streptococcus). Typically, postinfection mortality was significantly greater in groups exposed to exhaust than in their corresponding control groups exposed to purified air only. Data of recent diesel and of past diesel- and catalyst-treated gasoline engine exhaust experiments suggest a somewhat greater excess mortality from (enhanced susceptibility to) bacterial infection in mice exposed to diesel exhaust than in those exposed to catalytic gasoline exhaust. Limited data on acute tests of NO₂ and acrolein vapor alone suggest that the infectivity-enhancing effect of diesel exhaust could be accounted for in large part by these components. Exposures to diesel exhaust, NO₂, or acrolein did not enhance the mortality response to a viral pathogen (A/PR8-34).     

Mutagenic and carcinogenic potency of extracts of diesel and related environmental emissions: In vitro mutagenesis and DNA damage

The Saccharomyces cerevisiae D3 recombinogenic assay, the assay for forward mutagenesis in L5178Y mouse lymphoma cells, and the sister chromatid exchange (SCE) assay using Chinese hamster ovary cells were used to evaluate the in vitro mutagenic and DNA-damaging effects of eight samples of diesel engine emissions and related environmental emissions. The recombinogenic assay was not sufficiently sensitive for this evaluation, but mutagenicity was detected in the L5178Y mutagenesis assay following exposures of the cells to all of the emission samples, and DNA damage in the SCE assay was induced by most of the emission samples in the presence and absence of metabolic activation. The observation of positive results in the absence of activation indicated that the samples contained substances that were direct-acting mutagens and DNA-damaging agents.     

Mutagenic and carcinogenic potency of extracts from diesel related environmental emissions: Simultaneous morphological transformation and mutagenesis in BALB/c 3t3 cells

Particulate extracts from six different environmental emission sources were assayed for genotoxic activity in mouse BALB/c 3T3 clone A31-1 cells. All compounds were tested simultaneously for both transforming and mutagenic (induction of ouabain-resistance) potential with and without exogenous metabolic activation in the form of a 9000 × g postmitochondrial hepatic supernatant fraction from Aroclor-1254 induced Fischer 344 rats. Dichloromethane particulate extracts from the exhaust of two light duty diesel engines (Oldsmobile and Nissan), one heavy duty diesel engine (Caterpillar) and one late model gasoline engine (Mustang II) were assayed in an identical manner to particulate extracts from the emissions of a roofing tar pot and a coke oven. No clear dose-dependent responses were observed, but several of the samples showed significant transforming and mutagenic activity. A qualitative ranking system showed the activity of these particulate extracts for either mutagenesis or transformation was: coke oven = Mustang II gasoline engine > Nissan diesel engine > roofing tar. Particulate extracts from the Oldsmobile diesel engine and the Caterpillar diesel engine showed essentially no activity.     

The mutagenic activity of thirty polycyclic aromatic hydrocarbons (PAH) and oxides in urban airborne particulates

Data are presented on the mutagenicity of an organic extract of a composite sample of urban air particulates and of thirty PAH compounds in such samples, including four quinone derivatives, isolated quantitatively by thin-layer chromatography and identified by fluorescence or other spectral techniques. Mutagenic activity was determined by the Ames assay, using histidine auxotrophs of Salmonella typhimurium strains TA98, TA100, TA1537 and TA1538. All compounds were dissolved in dimethyl sulfoxide (DMSO) which was the least toxic of eight organic solvents tested.The mutagenic activity of a benzene extract of suspended particulates from the air of Hamilton, Ontario was significantly greater with strain TA98 than with strain TA100, suggesting the presence of more frame-shift acting mutagens. The mutagenic response of this extract was similar with and without S-9 activation.Mutagenic tests on the 30 PAH compounds indicated that only the benzo(a)pyrene quinones were direct acting mutagens. All of the chemical compounds were tested with and without S-9 activation. The following PAH showed unequivocal mutagenic activity, with S-9 activation: benz(a)anthracene, benzo-(a)pyrene, benzo(ghi)perylene, benzo(rst)pentaphene, benzophenanthrene, chrysene, 1, 2, 3, 4-dibenzanthracene, 2, 3, 6, 7-dibenzanthracene, and 3-methyl cholanthrene. The quinones of 1, 6-; 3, 6-; and 6, 12-benzo(a)pyrenes showed weak mutagenic activity but 3, 6-benzo(a)pyrene elicited also a photodynamic response.     

Metaphase analysis, micronuclei assay, and urinary mutagenicity assay of mice exposed to diesel emissions

Female Swiss mice were exposed 8 h/day to diesel exhaust for 1, 3, and 7 weeks. Urine was collected overnight for 4 days prior to sacrifice while the mice continued to be exposed for eight hours during the day. The presence of mutagens was determined by the Ames Salmonella test. One hour prior to sacrifice each mouse received 1 mg/kg colcemide. After sacrifice, the marrow from each femur was obtained. The marrow from one femur was used to prepare slides for metaphase analysis and the other for micronuclei assay. Other mice received IP 50 mg/kg cyclophosphamide 24 h prior to sacrifice or 1 μmole/kg benzo(a)pyrene in each of four daily doses prior to sacrifice and served as positive controls. The Ames Salmonella assay of the unconcentrated urine after 1, 3, and 7 weeks and concentrated urine after 7 weeks exposure to diesel exhaust did not significantly vary from clean air controls. In the micronucleus test, and metaphase analysis, cyclophosphamide produced a strong positive response and the 7 week diesel exposure was not different from clean air controls.     

Diesel soot: Mutation measurements in bacterial and human cells

Both hot pipe and dilution chamber samples of the exhaust from a diesel (Oldsmobile 350) engine have been collected, extracted with methylene chloride and those extracts have been tested for mutagenicity in forward mutation assays in human lymphoblasts and S. typhimurium. In the absence of a metabolic activation system, the extract was significantly mutagenic to the bacteria in the range of 0 to 30 μg/ml, but introduced no mutations in human cells at concentrations up to 200 μg/ml under the same conditions of assay medium. However, when assayed in the presence of a postmitochondrial supernatant derived from rat liver, the the soot extracts were significantly mutagenic to both bacteria and human cells in the range of 50–100 μg/ml. Fractionation of the soot extract on the basis of polarity by sequential elution from a silicic acid column permitted concentration of the mutagenic activity in the alkane/toluene eluate, as determined by bacterial assays. Preliminary characterization of this fraction and preliminary studies of pure compounds leads us to suspect the alkyl substituted phenanthrenes as representing at least a significant fraction of the mutagenic activity of this alkane/toluene eluate.     

A study of diesel emissions on Drosophila

A sex-linked recessive lethal test was performed on male fruit flies of the species Drosophila melanogaster, (Oregon-R strain), exposed to an approximate five-fold dilution of exhaust from a diesel engine. The eight hour exposure was achieved by drawing diluted diesel exhaust from a three cubic meter stainless steel exposure chamber housing laboratory animals through a two liter reaction flask modified for use with Drosophila. A preconditioned sampling bag was used to collect the emissions after passing through the exposure chamber containing the flies. Results of analyses performed on the diesel exhaust mixture showed the following: carbon dioxide—0.17%, carbon monoxide—12.2 ppm, hydrocarbons—11.6 ppm, nitrogen oxide—3.8 ppm, nitrogen dioxide—2.9 ppm, sulphur dioxide—1.0 ppm, and particulates—2.18 mg/m³. Two broods of the F₂ generation were investigated for the occurrence of recessive lethal events. These broods approximated the developing gametogenic stages of mature sperm (P₁ matings on days 2 and 3 postexposure) and spermatocytes (P₁ matings on days 8 and 9). Additionally, the F₃ generation was evaluated for the occurrence of mosaic recessive lethal events which might escape detection in the F₂ generation. An equal number of F₂ and F₃ flies for both broods served as concurrent controls. Results indicate that, under the conditions tested, the diesel exhaust did not increase the mutation frequency of the exposed flies (F₂ rate = 0.30%, F₃ rate = 0%) when compared to the concurrent controls (F₂ rate = 0.37%, F₃ rate = 0.15%).