Carcinogenicity of diesel exhaust as tested in strain a mice

Groups of strain A mice were exposed to diesel exhaust by inhalation and diesel particulate by intraperitoneal injection. The animals were exposed from seven to eight weeks and then sacrificed 26–30 weeks postexposure. Other animals were exposed for up to seven months by the inhalation route. Some animals were promoted using urethane at a dose below which tumors would occur. There was no increase in incidence of pulmonary adenomas in the animals exposed to either diesel exhaust or diesel particulate over the control animals. In the animals which were promoted using urethane at a low dose, there was a significant increase in pulmonary adenomas. Diesel particulate was found in the lungs and bronchial lymph nodes of animals exposed to diesel exhaust 26–30 weeks after cessation of exposure.     

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.     

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.     

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).     

Behavioral alterations due to diesel exhaust exposure

Several experiments examining the effects of diesel exhaust on the behavior of rats are reported. Animals were exposed either as adults or neonates. The spontaneous locomotor activity (SLA), measured in standard running wheel cages, of adult rats exposed for 8 h/day, 7 days/week was significantly less than that of controls. Experiments involving diesel exhaust exposure to neonatal rats indicated that adult rats, exposed to diesel exhaust during their neonatal lives, were significantly less active as measured by SLA. Adult rats, exposed to 20 h diesel per day as neonates, were placed in skinner boxes after the SLA experiment described above had been completed. The exhaust exposed animals showed significantly decreased acquisition of a food reinforced bar pressing task. All animals that learned this task extinguished at the same rate. The results of the neonatal diesel exhaust experiments support the hypothesis that diesel exhaust exposure during development of an organism can lead to behavioral differences in adulthood.     

Test of diesel exhaust emissions in the rat liver foci assay

In the initiation/promotion assay in rat liver, partial hepatectomy is used to enhance initiation, and a choline-devoid diet as promoter. The induction of carcinogenesis is determined by the focal appearance of gamma glutamyl transpeptidase (GGT) positive hepatocytes. We adopted this assay to diesel exhaust emission by performing a partial hepatectomy, and exposing the rats to either clean air or diesel exhaust emission. The rats were fed either a choline-devoid or a choline-supplemented diet for three or six months. The animals were sacrificed and liver sections stained for GGT were examined for the presence of foci of GGT(+) hepatocytes. The results indicate that diesel exhaust exposure does not result in a systemic dose of carcinogens sufficient to be detected in the liver foci assay.     

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.     

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.     

Neurophysiological alterations due to diesel exhaust exposure during the neonatal life of the rat

This study was designed to assess the effects of diesel exhaust on the development of the nervous system in rats as measured by somatosensory and visual evoked potentials (SEPs and VEPs, respectively). SEPs, elicited by 1 mamp, 0.5 msec pulses delivered to the tibial nerve at the tibale, were recorded from the skull overlying the somatosensory projection area on days 28, 35, 42, and 49 postconception (pc). Analysis of the data for day 35 pc double pulse SEPs revealed significantly longer latencies for all peaks of the SEP in pups exposed to diesel exhaust. There were also differences indicating that the electrophysiological recoverability of the nervous system had been compromised in the diesel exhaust exposed group. On day 35 pc, the latencies of all VEP peaks occurring before 300 msec were greater in the exposed group. The differences, however, reached statistical significance only for the P2 peak of the response to the first of the paired stimuli. These data support the hypothesis that diesel exhaust influences the development of the nervous system.     

Pulmonary function testing of animals chronically exposed to diluted diesel exhaust for 267 days

Thw purpose of this work was to assess the potential effect that chronic inhalation of diesel exhaust may have on lung mechanics and lung volumes. Noninvasive pulmonary function tests have been conducted repeatedly on 25 rats exposed to diesel exhaust at a particulate concentration of 1500 μg/m³, for 20 h/day, days/week for 267 days. The same tests were conducted on 25 clean air control animals. When the data is normalized, there are no apparent functionally significant changes occurring in the lungs that may be attributed to the chronic inhalation of diesel exhaust.     

Pulmonary function evaluation of cats after one year of exposure to diesel exhaust

Adult male, inbred, disease-free cats of uniform age and size were exposed eight hours per day, seven days per week to a 1 : 18 dilution of diesel exhaust emissions. After one year of exposure, the animals were removed from the chambers for measurement of lung volumes, forced expiratory flow rates, dynamic compliance and resistance, diffusing capacity, and nitrogen washout. No important changes in pulmonary function were detected with the exception of a decrease in closing volume (P < 0.05). The inability to detect decrements in pulmonary function may have been due to insufficient cocentration of exhaust, insufficient exposure length, or to the use of a species resistant to diesel exhaust. To test these possibilities, the cats are being exposed for an additional year, and another species, hamsters, are being exposed for future testing at exhaust dilutions of 1 : 18 and 1 : 9.     

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.     

Deposition and biological availability of diesel particles and their associated mutagenic chemicals

To estimate the human health risk of inhaled diesel particles, it is necessary to know their deposition and retention in the respiratory tract and the rate of dissociation of mutagenic compounds associated with the particles. The deposition of a chain aggregate aerosol of ⁶⁷Ga₂O₃ with size and shape characteristics similar to diesel exhaust particles has been evaluated using Beagle dogs. Approximately one-third of the inhaled activity is deposited in the respiratory tract with most of the particles deposited in the lung. The mutagenic activity present in dichloromethane, dog serum, dog lung lavage fluid, saline, dipalmitoyl lecithin (DPL) and albumin following incubation of these fluids with diesel exhaust particles was determined in the Ames Salmonella system. As observed by other investigators, large quantities of mutagenic activity were removed by dichloromethane. A very small amount of mutagenic activity was removed by the serum and lavage fluid over a 3-day incubation period. No activity was detected following elution with the other solvents. The finding that minimal mutagenic activity could be demonstrated in the biological media following incubation with diesel exhaust particles may be due to a lack of removal of mutagens from the particles or an inactivation of removed mutagens by protein binding or other processes.     

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%).     

Particle size measurements of automotive diesel emissions

The automative diesel engine has long been acknowledged as being “dirtier” than the spark ignition engine and its particulate emissions may be carcinogenic. Possible solutions to the diesel emission problem are combustion modification or aftertreatment devices. Selection of candidate aftertreatment devices requires knowledge of the physical and chemical properties of the particles, including particle morphology, size distribution, mass concentration and emission rates in the exhaust gas stream. The study reported here represents the first of a series of experiments designed to characterize the exhaust emissions and test various aftertreatment devices. This paper deals only with the particulate characterization phase of the program. Results of size distribution, particle concentration and mass emission rate measurements for a 5.71 displacement Oldsmobile diesel engine are given for a variety of engine operating conditions.     

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.     

Facilities for diesel exhaust studies

It is the major intent of this report to examine the animal inhalation facilities at the EPA Health Effects Research Laboratory in Cincinnati, Ohio, associated with the exposure to the exhaust of diesel-powered equipment. The facility serves as a center for toxicologic evaluation, in relevant animal models, of potentially hazardous environmental pollutants from diesel engine exhaust. Briefly, the entire engine exhaust is mixed with filtered and conditioned air in a dilution tube. That diluted exhaust enters a large volume mixing chamber and a portion passes through dynamic flow irradiation chambers (to simulate sunlight) and is then conducted to animal exposure chambers. The system provides nonirradiated exhaust in the same concentration (directly from the mixing chamber) and also filtered, conditioned ambient air for control animal exposure. The major physical components of the diesel emission system include: air purifiers, animal exposure chambers, engine-dynamometers, irradiation chambers, monitoring instrumentation and data acquisition systems.     

Introduction to and experimental conditions during the first year of the GMR chronic inhalation studies of diesel exhaust

A chronic exposure study was initiated to determine the effects of diesel exhaust on the health of experimental animals. For this purpose, test atmospheres of clean air (control) or freshly diluted diesel exhaust at concentrations of 250, 750, and 1500 μg/m³ were supplied to four 12.6 m³ inhalation chambers which housed rats and guinea pigs. Diesel aerosol size and concentration, as well as chamber temperature and relative humidity, were continually monitored and controlled to maintain the exposure dose levels and an environment of 22±2°C and 50%±20% relative humidity. The concentrations of CO and NO_x were found to be 5.8±1.0 mg/m³ and 7.9±1.0 mg/m³ above ambient in the chamber containing 1500 μg/m³ of particulate. Animals were supplied from the chambers, on a random basis, for both intramural and extramural studies throughout the exposure period. The experiment ran uninterrupted for over twelve months with mean diesel particle mass concentrations within 2% of the target values.     

Control strategies for particulate emissions from vehicular diesel exhaust

The advantages and disadvantages of several possible control strategies for particulate emissions from vehicular diesel exhaust are discussed. The evaluation of the potential usefulness of the various control strategies is based on available data concerning the mass loading and particle size distribution and on anticipated control standards. Several studies have been made on devices for removing particulate emissions from vehicular diesel exhaust. These studies, which include the techniques of filtration, wet scrubbers, and electrostatic precipitation, are summarized. A comparison of the various control devices is made based on such factors as size, efficiency, and maintenance requirements.     

Mutagenic and carcinogenic potency of extracts of diesel and related environmental emissions: Two-stage carcinogenesis in skin tumor sensitive mice (SENCAR)

Skin tumors can be induced by the sequential application of a subthreshold dose of a carcinogen (initiation phase), followed by repetitive treatment with a noncarcinogenic tumor promoter. There is a very good dose-response relationship between the induction of the number of papillomas per mouse at early times (10 to 20 weeks) by either tumor initiators and promoters and the final carcinoma incidence after a longer latency (20 to 50 weeks) in SENCAR mice. This system not only can be used to determine the tumor initiating and promoting activities of a compound but if the agent is given repeatedly by itself one can also determine if it is a complete carcinogen, i.e., if it has both tumor initiating and promoting activity. In addition, if the agent is given concurrently with a known complete carcinogen or a tumor initiator one can also determine if the agent has cocarcinogenic or cotumor initiating activity or even possibly anticarcinogenic activity. Likewise, if the agent is given concurrently with a known tumor promoter one can determine if the agent has copromoting or antipromoting activity. Using the SENCAR skin carcinogenesis system we have undertaken the determination of the skin carcinogenic, cocarcinogenic, tumor initiating and promoting activities of various diesel emission particle extracts as well as for comparative purposes, standards such as benzo(a)pyrene and 12-0-tetradecanoylphorbol-13-acetate and extracts of emissions from a gasoline engine, roofing tar, coke oven and cigarette smoke condensate. Most of the studies are still in progress but some preliminary data is available on the comparative tumor initiating activities of the various samples at 14 weeks. Caterpillar diesel and cigarette smoke condensate were essentially without activity, whereas the Mustang gasoline catalyst and Olds diesel gave extremely low values although they were slightly above background. Roofing tar, coke oven and Nissan Diesel all gave moderate activity at high doses (1 to 10 mg) but when compared to benzo(a)pyrene were relatively low values.