Trent R. Lewis

Establishing aerosol exposure concentrations for inhalation toxicity studies

Criteria for the selection of aerosol concentrations to be used in inhalation studies assessing the toxicity and carcinogenicity of chemical substances were discussed by the authors in a meeting sponsored by the National Toxicology Program. Concepts in the design of aerosol inhalation studies emerged from that meeting and are being communicated through this publication. Inhalation studies assessing the toxicity and carcinogenicity of aerosols have often used maximum exposure levels on the basis of technological feasibility. Evidence has now accumulated that the amount of pulmonary burden of deposited particles impacts on particle clearance above some as yet not well-defined exposure concentration. The sequelae are such that lung clearance decreases with increased particulate burden to the point of approaching complete cessation. This paper focuses on the major determinants in establishing maximal aerosol concentrations for use in inhalation toxicity studies with special emphasis on experimental design features to assess lung retention. The subject matter of this paper is a rapidly developing area in terms of knowledge. Accordingly, the contents of this article are intended as guidelines and not as absolute rules for the conduct and interpretation of inhalation exposure studies.

Long-term exposure to low levels of air pollutants: effects on pulmonary function in the beagle

One hundred and four beagles have been exposed for 18 months to natural and photochemically reacted auto exhaust, oxides of nitrogen and oxides of sulfur. No differences in singlebreath carbon monoxide diffusing capacity, dynamic pulmonary compliance, or total expiratory pulmonary resistance were found between exposed and control animals. Removal of reactive gases in the upper airway was studied during brief exposures in an additional small group of animals. Under these conditions, 100% removal of O3 and SO2, 90% removal of NO2, 73% removal of NO, and no removal of CO or hydrocarbon were found.

Influenza virus infection in mice after exposure to coal dust and diesel engine emissions

Influenza virus infection initiated after aerosol exposure of CD-1, white Swiss mice for durations of 1, 3, and 6 months to respirable particulates maintained at 2 mg/m3 of either coal dust (CD), diesel engine emissions (DEE), a combination of both (CD/DEE), or to filtered air (control) was studied. The course of infection in mice previously exposed for 1 month to various particulates did not differ appreciably among the four animal groups with respect to mortality, virus growth in lungs, interferon levels, or hemagglutinin antibody response. In mice exposed for 3 and 6 months to different particulates, the mortality response was similar among all animal groups. However, the percentage of animals showing lung consolidation was significantly higher in the 3-month groups exposed to DEE (96.5%) and CD/DEE (97%) than in the control (61.2%); in the 6-month groups, the percentages were twice that of the control for both DEE- and CD/DEE-exposed animals. Complementing these observations of both 3- and 6-month-exposed animals was the higher virus growth levels attained in the DEE and CD/DEE animals with concomitant depressed interferon levels which were the inverse of findings noted in the control group. Hemagglutinin-antibody levels in particulate-exposed animals, especially at the 6-month interval, were fourfold less than the control. Histopathologic examination of lungs revealed no qualitative differences in the inflammatory response at any one specified time interval of exposure to influenza virus among the control and particulate-exposed animal groups. However, there were differences in severity of reaction in relation to the particulate component of the exposures. Focal macular collections of pigment-laden macrophages were seen only in DEE and CD/DEE but not in CD animals after 3- and 6-month exposures. The findings of this study indicated that the severity of influenza virus infection is more pronounced in mice exposed to diesel engine emissions than in control animals and it is not appreciably accentuated by coal dust.

Sister-chromatid exchanges and chromosome aberrations in lymphocytes from monkeys exposed to ethylene oxide and propylene oxide by inhalation

The ability of long-term exposures to inhaled ethylene oxide (EO) and propylene oxide (PO) to induce sister-chromatid exchanges (SCEs) and chromosome aberrations in peripheral lymphocytes of monkeys was investigated. Five groups of adult male cynomolgus monkeys were exposed at 0 (shared control), 50, or 100 ppm EO, and at 100 or 300 ppm PO (7 hr/day, 5 days/week) for 2 years. EO exposures at 50 and 100 ppm resulted in statistically significant increases in sister-chromatid exchange rates and in the incidence of chromosome aberrations in monkey lymphocytes. Both EO-exposed groups had increased numbers of SCEs/metaphase compared to controls, with the SCEs/metaphase of the EO 100 ppm group also significantly elevated versus the EO 50 ppm group. Variability of SCEs/metaphase within each monkey increased even more than the increase in total SCEs/metaphase group with increasing EO exposure. Chromatid-type aberrations were also significantly increased for both EO 50 and EO 100 ppm groups compared to controls. Statistically significant increases in the number of chromosome-type aberrations (excluding gaps) were found only in the EO 100 ppm group. Combined chromatid- and chromosome-type aberrations were increased in both EO 50 and EO 100 ppm groups. No group differences in the number of gaps were found. In lymphocytes from monkeys exposed at 100 and 300 ppm PO, there were no group differences compared to controls for any variable-chromatid or chromosome-type aberrations, gaps, or SCEs/metaphase. These results indicate that EO is a more potent clastogen than PO and demonstrate, for the first time, statistically significant effects of EO on both SCEs and chromosome aberrations in lymphocytes of nonhuman primates.

A Chronic Inhalation Toxicity Study of Diesel Engine Emissions and Coal Dust, Alone and Combined:

To evaluate the potential health hazards of diesel engine emissions in underground coal mines, inhalation studies were performed using three species of animals. A wide range of toxicological responses was measured. Exhaust was provided by a 425 in.3 displacement four-cycle, water-cooled, naturally aspirated diesel engine (Caterpillar Model 3304) equipped with a water scrubber. Exposures were 7 h/day, 5 days/week, for periods up to 24 months. Micronized coal dust was generated using a Wright dust feeder. Four exposures were evaluated: (1) filtered ambient air, (2) 2 mg/m3 diesel particulate, (3) 2 mg/m3 respirable coal dust, and (4) 1 mg/m3 each of 2 and 3. Gaseous and vapor concentrations were similar in both exposures employing diesel exhaust. Male cynomolgus monkeys, Fischer-344 male and female rats, and female CD-1 mice were the experimental subjects. Monkeys were sacrificed at 24 months, rats at 3, 6, 12, and 24 months, and mice at 1, 3, and 6 months. Gross morphology and histopathology demonstrated that both diesel and coal dust particles are deposited in the lungs and retained in alveolar tissue. Alveolar type II cell hyperplasia and pulmonary lipidosis occurred in rats, being most evident in rats exposed to diesel exhaust alone. There was, however, no evidence of emphysema or chronic bronchitis, and only minimal fibrosis was seen in association with the retained particulate. Both particulates affected the defense mechanisms of the lung. Exposure to coal dust activated responses associated with phagocytosis, whereas exposure to diesel exhaust depressed them. Severity of influenza challenge increased concomitantly with decreased interferon production in diesel-exposed mice. Exposure to diesel emissions did not result in genotoxic effects as measured by increases in sister chromatid exchange, chromosomal aberrations, micronucleus testing, and urine genotoxic assays. Pulmonary function studies in monkeys showed mild obstructive airway disease in coal dust, diesel exhaust, and the combined exposed animals. This effect was most pronounced in monkeys exposed to diesel exhaust. Evidence of restrictive lung disease was not seen in any group. Clearance of F3O4 particles appeared to be stimulated by exposure to diesel exhaust in the first 3 months, but long-term clearance of diesel particulate appeared to be inhibited. No evidence was found for increases in tumorogenicity (rats) or induction of xenobiotic metabolizing enzymes in the lung or liver (rats). Humoral and cellular immunities were not significantly affected by exposure (rats). No adverse seminal effects were observed in monkeys exposed for 2 years. There was no frank evidence of chronic toxicity as demonstrated by changes in mortality, body weight gains, organ-body weight ratios, or clinical parameters in rats or monkeys. Synergistic effects between diesel exhaust and coal dust were not demonstrated.

Toxicity of long-term exposure to oxides of sulfur.

Thirty-two beagles, 16 previously impaired with 191 days exposure to 26 ppM NO/sub 2/ (bullous alveolar enlargement, increased peribronchial and perialveolar reticulum fibers and collagen), were exposed to (1) control, (2) 5.1 ppM SO/sub 2/, (3) approx. 900 ..mu..g/m/sup 3/ H/sub 2/SO/sub 4/ (90% < 0.5 ..mu..m), or (4) combination of 2 and 3, for 620 days, 21 hr/day. No effect of SO/sub x/ on hematology or lung measurements. SO/sub 2/ increased nitrogen washout in unimpaired animals but lowered washout in impaired animals. H/sub 2/SO/sub 4/ significantly reduced CO diffusing capacity, net inflated lung volume, net deflated lung volume, lung weight, and residual volume, and increased total expiratory resistance. Decreased heart weight was also noted. Impairment increased heart weight, trapped lung air. Lung previously altered was more difficult to alter more. H/sub 2/SO/sub 4/ at low levels can produce serious pulmonary impairment, is more dangerous than SO/sub 2/.

Effects of methyl n-butyl ketone on behavior and the nervous system

The effects of methyl n-butyl ketone (MBK) on nervous system function and operant behavior were investigated in monkeys and rats. Mean MBK exposure levels approximated 100 and 1000 ppm and lasted up to 10 months. Both exposures were 6 hours/day 5 days/week. Results showed that the 1000 ppm exposure effected 1) reduced motor conduction velocities in ulnar and sciatic-tibial nerves, 2) decreased the amplitude of evoked muscle action potentials, 3) lengthened implicit time of visual evoked potentials, 4) impaired operant behavioral performance, and 5) reduced body weight. Reductions in nerve conduction velocities and evoked muscle action potentials were found at 100 ppm. Recovery, as measured by sciatic-tibial nerve conduction velocity, was found to occur 6 months and 2 months after termination of the 1000 ppm and 100 ppm MBK exposures, respectively.

Fibrogenic Potential of Intratracheally Instilled Quartz, Ferric Oxide, Fibrous Glass, and Hydrated Alumina in Hamsters

As a first step in the development of an animal model for determining the role of pulmonary fibrosis in the etiology and pathogenesis of lung cancer, the fibrogenic potential of quartz, quartz and ferric oxide administered together, fibrous glass, and hydrated alumina were studied by multiple intratracheal instillation in groups of male Lak:LVG Syrian golden hamsters. Dose-related decreases in survival were evident for the groups instilled with the two highest doses of quartz or quartz and ferric oxide. Instillation of quartz or quartz and ferric oxide induced the greatest pulmonary fibrosis in response to the materials tested. However, the dense fibrous tissue present in the lungs in classical human silicosis and in experimental silicosis of rats was not observed in this study. The results of this study indicate that the Syrian golden hamster is not a suitable species for studying the role of quartz-induced pulmonary fibrosis in pulmonary carcinogenesis.

Pulmonary hyperreactivity in cynomolgus monkeys (Macaca fasicularis) from nose-only inhalation exposure to disodium hexachloroplatinate, Na2PtCl6☆

The pulmonary and dermal effects of exposure to Na2PtCl6 were investigated in cynomolgus monkeys (Macaca fascicularis) exposed by the nose-only inhalation and percutaneous routes. Separate inhalation exposures were performed in monkeys at 200 micrograms/m3 and 2 mg/m3 (4 hr/day, biweekly for 12 weeks), while another group of monkeys was percutaneously exposed biweekly by an open patch method. After a 2-week refractory period, serial Na2PtCl6 bronchoprovocation challenges and intradermal Na2PtCl6 sensitivity evaluations were performed. Na2PtCl6 bronchoprovocation in naive control monkeys yielded significant impairments in post-challenge pulmonary mechanics and ventilatory function. These results indicate a pharmacologic or irritant-mediated bronchoconstriction mechanism for acute exposure to this compound. When the post-challenge pulmonary function of animals exposed for the 12-week exposure regimen (across treatments) was compared to pulmonary deficits observed in control animals upon challenge, significantly greater pulmonary deficits were seen in animals exposed at the 200 micrograms/m3 concentration. Exposure at this concentration yielded significant changes in post-challenge average pulmonary flow resistance (RL) and forced expiratory volume in 0.5 sec corrected for vital capacity (FEV0.5/FVC) when compared to control monkey responses. Animals exposed by the percutaneous route or at 2 mg/m3 showed no significant post-challenge pulmonary deficits when compared to control animals. Intradermal Na2PtCl6 sensitivity was found not to be exposure related in the conditions of this experiment.

Genotoxicity studies of rodents exposed to coal dust and diesel emission particulates

Genotoxicity studies with mice and/or rats have been conducted to evaluate the potential mutagenic hazard associated with exposures of coal miners to diesel emission particulates (DEP) and/or coal dusts (CD). Rats and mice were exposed to filtered air, DEP, and/or CD for periods ranging from 3 months to 2 years. Levels of respirable particulates were maintained at 2 mg/m3 in all exposed groups. DEP and/or CD were collected in the inhalation chambers in which animals were exposed. Urine samples were collected for 5 consecutive days from rats exposed to DEP and/or CD for 3, 6, and 24 months. The particulate samples extracted with dichloromethane and the urine samples concentrated with XAD-2 columns were analyzed for mutagenic activity by the Ames Salmonella/microsome assay system. Peripheral blood lymphocytes from rats exposed for 3 months were analyzed for sister chromatid exchanges (SCE). The femur bone marrow cells from rats exposed for 24 months and mice exposed for 6 months were analyzed for micronuclei in both polychromatic and normochromatic erythrocytes. The results indicate that the solvent extract of DEP was mutagenic, while no mutagenic activity was found for the CD extract. Combination of CD and DEP did not show any synergistic effect. No mutagenic activity was found for urine samples from rats exposed to DEP and/or CD for up to 2 years. A slight increase in the micronucleated polychromatic erythrocytes over the control level was found in mice exposed to DEP and DEP plus CD for 6 months but the increase was not statistically significant. No increase in micronuclei was detected in rats exposed for 24 months. The frequencies of SCE in the peripheral lymphocytes of the 3-month-exposed rats were similar for control and DEP plus CD-exposed groups.