Subchronic inhalation exposures to aerosols of three petroleum lubricants.

Subchronic inhalation studies were performed with three petroleum lubricants: generic cutting oil (GCO), generic gear oil (GO), and generic commercial engine oil (CEO). Each formulation had a mineral oil base. Sprague-Dawley rats were exposed 6 hours/day, 5 days/week for 13 weeks to aerosol concentrations of 0 (untreated controls), 0 (sham-exposed controls), approximately 50, 150, or 400-520 mg/m3. At necropsy, 15 rats/sex/group were sampled for serum chemistry (18 parameters), hematology, and weights of 13 organs. Testis and epididymis of males in the control and high-dose group were used for number of spermatids and morphology of epididymal sperm. Histopathological slides were evaluated for 22 or more organs. Pulmonary function tests were done on 10 additional males/group. Pulmonary hydroxyproline was measured in these rats for GCO and GO. Residual oil in the lungs was determined for GCO. The primary organ affected by exposures to these three formulations was the lung; the main observed effects were accumulation of foamy macrophages in pulmonary alveoli and alveolar walls, very mild thickening of alveolar walls due to foamy macrophages and a mixed cell infiltrate, and subtle epithelial hyperplasia. The foamy macrophages tended to group together in aggregates, and the aggregates seemed responsible for plaques seen visibly on the surface of the lung. These histological changes were accompanied by concentration-related increases in lung weight and pulmonary hydroxyproline, whereas pulmonary function tests were generally unaffected. Effects distal to the lung were more limited. These results indicated low toxicity of these aerosols in this model.

Comparison of synthetic zeolite catalysts and alumina binders administered intratracheally to rats.

An intratracheal (IT) screening assay was performed in rats on a series of aluminosilicate catalysts (synthetic zeolites) and alumina binders to compare their relative ability to cause pulmonary fibrosis and related changes. Before initiation of IT screens, both the uniformity of deposition and residence time of a prototype catalyst in the lung were determined. Subsequently, the test materials were instilled and animals were evaluated 6 mo later for lung volumes, pulmonary pressure-volume curves, pulmonary hydroxyproline (OHPro) content, lung weights, and histopathology. Negative controls were saline and glass beads; the positive control was quartz. The test materials were organic-free ZSM-5 crystals, organic form of ZSM-5 crystals, alumina-bound ZSM-5, nickel/ZSM-5/ Al2O3 binder, used nickel/ZSM-5/Al2O3 binder, nickel-tungsten/ZSM-5/Al2O3 binder, and Bayer pseudoboehmite. For each, groups of 12 male rats were dosed once with either 25 or 50 mg of ground particles (> or =95% less than 2.4 microm). Second to quartz, the sample of alumina caused the most pronounced pulmonary reactions at 6 mo after dosing. Therefore, the effects of four aluminas were subsequently compared (Bayer pseudoboehmite, Bayer gammaAl2O3, Ziegler pseudoboehmite, and Ziegler gammaAl2O3). The results support the idea of a lack of long-term effects from exposure to normal concentrations of these materials in the workplace. Also, the IT assay proved to be a very useful tool for ranking the relative effects of this series of zeolites and aluminas.

Short-term exposures of rats to airborne hydrogen fluoride.

A series of acute inhalation exposures was performed with airborne hydrogen fluoride (HF) to establish the concentration response for nonlethal effects in the rat. Exposures were either 2 or 10 min long; concentrations ranged from 135 to 8621 ppm. Three additional exposures (20 to 48 ppm) were performed for 60 min. A mouth-breathing (MB) model with a tracheal cannula was used in most of the exposures to maximize delivery of the HF to the lower respiratory tract. Endpoints on the day after exposure included hematology, serum chemistry, bronchoalveolar lavage, pulmonary function, organ weights, and histopathology. Nasal resistance was measured in nose-breathing (NB) groups. Effects of exposure were generally limited to the respiratory tract and included alveolitis, bronchial lesions, altered parameters of pulmonary function and bronchoalveolar lavage, and mucosal necrosis, inflammation, and fibrinopurulent exudate in airways. Observed changes were concentration related and appeared more pronounced in major airways near the point of entry (trachea in MB animals and nose in NB animals). One group of MB animals exposed for 10 min to 1454 ppm was evaluated at 3 and 14 wk after exposure; the acute effects had resolved by those times. The effects of 2-min exposures were consistently more severe than those from 10-min exposures to the same product of concentration x time. Exposures of MB animals for 60 min to 20 or 48 ppm HF did not result in observable adverse effects, although quasistatic pressure-volume curves were shifted upward slightly after 48 ppm. These data provide an integrated picture of the concentration-related effects of short nonlethal exposures to HF.

Acute effects of 10-minute exposure to hydrogen fluoride in rats and derivation of a short-term exposure limit for humans.

A series of acute inhalation exposures of female rats was conducted with hydrogen fluoride (HF) to establish a concentration-response curve for nonlethal exposures. Durations of 2 and 10 min were used to simulate possible short-term exposures. Concentrations of HF ranged from 593 to 8621 ppm for 2-min exposures and from 135 to 1764 ppm for 10-min exposures. Additional exposures were performed for 60 min at 20 and 48 ppm HF for comparison to existing Emergency Response Planning Guidelines. Animals were evaluated on the day after exposure for changes in parameters of bronchoalveolar lavage, pulmonary function, hematology, serum chemistry, body weight, organ weights, and histopathology. Most exposures were performed with orally cannulated animals to bypass absorption of HF in the nose and achieve maximum delivery of HF to the lower airways. One of the primary uses of the resulting data was to estimate a concentration to which most people could be exposed for 10 min without severe of irreversible health effects. This level was 130 ppm. It was predicted that irritation would occur at this concentration, but the effects on t he respiratory tract would not be "serious" and would be expected to be reversible. The results of this experiment and the subsequent analysis of the data provide an important aid in he planning of responses to an accidental release of HF.

Use of a surrogate aerosol in a preliminary screening for the potential carcinogenicity of coal coated with No. 6 fuel oil.

Coal, which contains significant amounts of water, can be ground and dried to produce an efficient fuel for electric power plants; however, spontaneous combustion can occur in the dried coal. Liquid petroleum hydrocarbons inhibit this combustion, but not all petroleum streams are effective. No. 6 fuel oil, a readily available and inexpensive stream, provides an effective coating, but the carcinogenic potential of coal particles treated with No. 6 fuel oil, which contains polynuclear aromatic hydrocarbons (PNAs), was undefined. As part of the assessment process, a series of studies was conducted to compare this treated coal with similar particles (petroleum coke) that had been tested by chronic inhalation in monkeys and rats. The amounts of PNAs in petroleum coke and treated coal were compared in extraction studies; the treated coal had only two-thirds of the organics extractable with benzene compared with coke and only 7% as much of the 3-7 ring PNAs, the likely tumorigenic compounds. In addition, the analytical profile of 3-7 ring PNAs was of lower molecular weights in the coal treated with fuel oil. The mutagenicity of extracts from treated coal was much less than with petroleum coke and markedly less than that of No. 6 fuel oil itself. The percutaneous absorption of 3H-benzo(a)pyrene from both particles and from their benzene extracts, as measured in vitro, was approximately eight times greater with petroleum coke than with treated coal. Based on these preliminary results, there is no evidence suggesting that the treated coal would pose any greater carcinogenic risk than petroleum coke.

Toxicity evaluation of petroleum blending streams: reproductive and developmental effects of a distillate from light alkylate naphtha.

A distillate of light alkylate naphtha (CAS number 64741-66-8; LAN distillate) was administered via inhalation, 6 h/d, 7 d/wk to 4 groups of Sprague-Dawley rats (10/sex/dose) at target concentrations of 0 (filtered air control), 5, 12.5, or 25 g/m3 with the highest dose exceeding 60% of the lower explosive limit of LAND. Exposure began 2 wk prior to mating and continued throughout gestation until postnatal d 4 for females or for 8 consecutive weeks for males. No apparent clinical signs indicative of systemic toxicity were observed in the F0 and F1 animals of either sex. Inhalation exposure to LAND up to and including the 25 g/m3 dose level had no effect on parental food consumption, body weights, absolute and relative organ weights, and reproductive indices. All groups had comparable delivery data and a fertility index > or 80%. Pups in all groups showed comparable birth weights, weight gain, a viability index (postnatal d 4) for all groups of > or = 97%, and no histopathological changes. In the dams, there were no significant differences in the mean numbers of corpora lutea, implantation sites, and resorptions recorded at necropsy. In the males, the only remarkable findings at necropsy were a small right epididymis and testis seen in one mid-dose male and an abscess on the right epididymis of a high-dose male. In both cases, the dams that had been bred to these males produced normal litters. There were no test material-related microscopic changes observed in the testes and epididymis of the F0 male rats or ovaries of the F0 female rats exposed to LAND. Under the conditions of this experiment, the no-observed-adverse-effect level (NOAEL) for LAND via inhalation in rats is established at greater than 24.7 g/m3 (analytical concentration).

Subchronic and developmental toxicity studies of vaporized diisopropyl ether in rats.

Two inhalation studies were performed with a vaporized sample of commercial-grade diisopropyl ether (DIPE). In the subchronic study, Sprague-Dawley rats (14/sex) were exposed to 0 (both untreated and sham-exposed controls), 480, 3300, or 7100 ppm DIPE for 6 h/d, 5 d/wk, for approximately 90 d. DIPE itself accounted for 91-95% of the vapors, with the remainder being a mixture of 27-29 compounds. Exposure to DIPE did not adversely affect clinical signs, body weight, serum chemistry, hematology, or the number of sperm or spermatids. Exposure of males to 7100 ppm resulted in hypertrophy of liver cells associated with increased liver weight and in increased kidney weight with an increased incidence of hyaline droplets in proximal tubules of the kidney. Females had increased weight of both liver and kidney, although kidney increased only in relation to sham-exposed controls and no morphological changes were observed in either organ. At 3300 ppm, weights of liver and kidney were again increased in males; the liver weights were increased in females only compared to sham-exposed controls and not untreated controls. No abnormalities were observed morphologically. No changes were observed with 480 ppm. In the developmental toxicity study, pregnant Sprague-Dawley rats (22/group) were exposed to 0 (both untreated and sham-exposed controls), 430, 3095, or 6745 ppm for 6 h/d on gestation d 6-15. Animals were sacrificed on gestation d 20. With 6745 ppm, dams had a slight reduction in body weight gain and a significant decrease in food consumption. A concentration-related increase in the incidence of rudimentary 14th ribs was observed, but its significance was uncertain. There was no apparent toxicity, either maternal or fetal, at the lowest exposure concentration. Both studies indicated a low order of toxicity for DIPE.

Cell proliferation in rat forestomach following oral administration of styrene oxide.

A series of range-finding studies was conducted in a limited number of male F344 rats on the relation between cell proliferation and styrene oxide (SO) given as gavage doses in corn oil ranging from 550 to 1500 mg SO/kg. In each study, rats were injected with [3H]thymidine (0.50 mCi/g, ip) at intervals from 1 to 48 hr after dosing with SO. One hour later, stomachs were removed and fixed in formalin. Autoradiograms were prepared and labeling index (LI) was determined as the percentage of epithelial cells with 3H-labeled nuclei. Mean LI increased with a peak at approximately 15 hr after one or nine doses of SO. The increases were multifocal and not restricted to the area near the limiting ridge. The magnitude of the response in LI at 24 hr after dosing tended to decrease with progressive multiple doses (3/week). Dose-related morphologic lesions from SO (particularly submucosal) were multifocal and variable across the forestomach; they appeared unrelated to LI in a given area. In a final study, groups of 10 rats were given a single dose of 0, 20, 50, 125, 250, 500, or 800 mg/kg and LI was determined 15 hr later. Mean LI was dose-related with increases up to 250 mg/kg. A maximum response had apparently been reached and higher doses did not cause any further increase in LI. The degree of involvement of cell proliferation in the tumorigenicity of SO remains uncertain; additional studies are suggested to help in the understanding of such a possible relation.

Light catalytically cracked naphtha: subchronic toxicity of vapors in rats and mice and developmental toxicity screen in rats.

Both a subchronic inhalation study and a developmental toxicity screen were performed with vapors of light catalytically cracked naphtha (LCCN). In the subchronic study, four groups of mice and rats (10 animals per sex per species) were exposed for approximately 13 wk (6 h/d, 5 d/wk) to concentrations of LCCN vapors of 0, 530, 2060, or 7690 mg/m3. An untreated control group was also included. Animals were observed daily and body weights were taken weekly. No significant treatment-related changes were found in clinical signs, body weight, serum chemistry, hematology, histopathology of 24 tissues, or weights of 12 organs. A marginal decrease was noted in the number of sperm per gram of epididymis. In the developmental toxicity screen, presumed-pregnant Sprague-Dawley rats were exposed to 0, 2150, or 7660 mg/m3 of LCCN vapors, 6 h/d on d 0-19 of gestation. Females were sacrificed on d 20; dams and fetuses were examined grossly and fetuses were later evaluated for skeletal and visceral effects. The number of resorptions was increased by approximately 140% in the group receiving 7660 mg/m3; no other definite treatment-related changes were observed. Overall, the effects of exposure to partially vaporized LCCN were minimal.

Four-week inhalation exposures of rats to aerosols of three lubricant base oils.

Sprague-Dawley rats were exposed to aerosols of one of three base stocks used to formulate lubricating oils. These stocks were a solvent-refined oil (SRO), a hydrotreated and acid-washed white oil (WTO) and a severely hydrotreated and hydrocracked oil (HBO). Exposures were for 6 h per day, 5 days per week for ca. 4 weeks. There were four groups of rats for each study (10 per sex per group). Aerosol concentrations were ca. 0, 50, 210 and 1000 mg m-3 for each material; the mass median aerodynamic diameter was ca. 1 microns. Following the last exposure, all animals were sacrificed and necropsied. Samples were taken for serum chemistry, hematology, sperm morphology, weights of seven organs and histopathology on at least nine organs. Body weights and clinical signs were not affected by exposures. The only treatment-related changes were in the lung and associated lymph nodes. Both the wet weight of the lung and the dry/wet weight ratio increased in a concentration-related manner. Associated with the increased weight were accumulations of foamy alveolar macrophages, particularly in alveoli close to alveolar ducts. Mild infiltration by neutrophils was observed with WTO and SRO; thickened alveolar walls were noted with the highest concentration of HBO. These mild responses to exposures at very high concentrations indicate a low degree of toxicity for these aerosols.

Role of exposure parameters in toxicity of aerosolized diesel fuel in the rat.

Sprague-Dawley rats were exposed to aerosolized diesel fuel (ADF) to determine the potential health effects in military troops during exposure to this visual obscurrant. Acute range finding studies were performed to estimate exposure conditions at which minimal (less than 1%) mortality would occur. This information was used in the design of a repeated exposure study to test the relative significance of the frequency of exposures, the duration of exposure, and aerosol concentration in any toxicity which might occur. Animals were exposed once or three times per week for a total of nine times to concentrations ranging from 1.3 mg ADFl-1 to 6 mg ADFl-1 and exposure durations of 2 or 6 h. Body weight and food consumption were recorded at weekly intervals during the exposure period and in animals that were retained for 2 weeks after the last exposure. Animals were used for a variety of endpoints, including neurotoxicological tests, pulmonary function tests, hematology, clinical chemistry, organ weights, and histopathology, either within 2 days of the last exposure or after the 2-week recovery period. Animals exposed to ADF showed an initial depression in body weight followed by a slower rate of growth than the sham exposed controls. No exposure related changes were observed in any of the neurotoxicology assays. The lung was the primary site of toxicity. Focal accumulations of pulmonary free cells were observed histologically, with thickening and hypercellularity of nearby alveolar walls. The number of lavaged pulmonary free cells increased similarly. Both pulmonary wet and dry weights increased. Lung volumes were altered by exposure, including increased FRC and decreased TLC. Carbon monoxide diffusing capacity was decreased. Histologic abnormalities were not observed in any other organs. Of the exposure parameters tested, the frequency of exposure appeared to be the most significant in relation to degree of toxicity.

A composite model for multiple assays of skin irritation.

A model for skin irritation was developed for simultaneous evaluation of the influence on irritation of abrasion, occlusion, and duration of treatment and for fulfillment of requirements for labeling considerations under DOT, CPSC-FHSA, OSHA, and EEC. This model greatly reduces the number of animals required to address submissions under multiple agencies compared to performing each test separately. In this model, which we have called a Composite Skin Irritation test, a test material is placed on three pairs of intact and abraded sites on each rabbit; one pair of sites is occluded for 4 hours, one for 24 hours, and the other left unoccluded for 24 hours. Results are presented from 88 composite tests with 80 petroleum-related materials. For the materials tested, abrasion of the skin had no effect on the irritation response. Occlusion of the test site generally did not result in dramatic increases in response, except for petroleum refinery streams with a boiling range below 500 degrees F. Exposure for 4 hours rather than 24 hours generally resulted in less irritation; however, for individual compounds, the irritation from the 4-hour exposure could not be predicted from the response to the 24-hour exposure. Of the 80 materials tested, 12 would be labeled as skin irritants under CPSC guidelines, three under OSHA, and 20 under EEC. Of the 20 that would be labeled under EEC criteria, only seven would be labeled under CPSC criteria. At least for petroleum-related materials, results from skin irritation studies performed under one set of conditions cannot be used to predict the degree of irritation that would be produced under a different set of exposure conditions.

Potentiating effects of oxygen in lungs damaged by methylcyclopentadienyl manganese tricarbonyl, cadmium chloride, oleic acid, and antitumor drugs.

The intraperitoneal administration of methylcyclopentadienyl manganese tricarbonyl (MMT) and cyclophosphamide, exposure to an aerosol of cadmium chloride, intravenous administration of oleic acid, and intratracheal instillation of bleomycin to young female BALB/c mice or CD/CR rats result in acute lung injury. Pulmonary morphology and lung collagen content were examined in animals treated with these chemicals alone or in combination with an elevated oxygen concentration (80%) in the inspired air. In mice, the development of fibrosis could be significantly enhanced if animals treated with MMT, cadmium chloride, cyclophosphamide, or bleomycin were exposed to 80% oxygen immediately following exposure to these agents. In rats only cyclophosphamide- and bleomycin-induced acute lung injury was potentiated by hyperoxia, resulting in significant enhancement of lung collagen content. The pathogenesis responsible for this differential species response of pulmonary injury to hyperoxia remains to be investigated.

Pathogenesis of lesions induced in rat lung by chronic tobacco smoke inhalation.

Lesions were induced in the lungs of specific-pathogen-free F344 rats by chronic tobacco smoke exposure. Animals exposed to 7 cigarettes/day were killed after 1, 1.5, or 2 years of exposure. Parallel lifetime exposures induced pulmonary tumors in 9% of the animals. In serially killed animals, four types of lesions were found: 1) perivascular or peribronchiolar accumulation of lymphoreticular cells. 2) fibrotic and cellular enlargement of peribronchiolar septa, 3) type II cell hyperplasia with septal fibrosis, and 4) air-space enlargement (emphysema). However, emphysema occurred only in animals exposed to a higher (10 cigarettes) dose of tobacco smoke. Ultrastructural studies showed all of the focal lesions to be infiltrated by cells typical of the inflammatory response. The type II hyperplastic and peribronchiolar alveolar lesions involved larger portions of the parenchyma in fibrotic changes but differed in structure, location, and frequency. The incidence of the peribronchiolar alveolar lesions was temporally related to tumor incidence.

Formaldehyde and tumors in hamster respiratory tract.

Male Syrian golden hamsters were exposed to 10 ppm of formaldehyde (H2CO) 5 times/week for lifetime. Survival of the treated animals was reduced relative to unexposed controls. No tumors were observed in histologic sections of respiratory tract tissues from either unexposed or treated animals. Only a minimal increase in hyperplastic and metaplastic areas was observed in nasal epithelium of exposed animals. However, there was evidence that H2CO could serve as a cofactor in the incidence of respiratory tumors induced by diethylnitrosamine (DEN). Animals were exposed once per week to 30 ppm H2CO (5 h/day) for lifetime. No tumors were observed in the respiratory tract of the H2CO-only control group exposed to this regimen. Hamsters receiving exposures to H2CO at 2 days prior to each of 10 weekly DEN injections had a higher number of tracheal tumors/tumor-bearing animal at autopsy than those receiving DEN alone.

Effect of sulfur dioxide on the morphology and mucin biosynthesis by the rat trachea.

Specific-pathogen-free rats were exposed to 400 ppm sulfur dioxide daily for up to 7 weeks. At intervals during exposure, tracheas were removed and incubated in vitro in culture medium containing radioactive glycoprotein precursors. The most prominent histological changes due to SO2 were progressive hypertrophy and hyperplasia of the submucosal mucous glands accompanied by a flattening of the epithelium with eventual recovery. Uptake of radioactive precursors into a highly purified mucin fraction correlated with these histological changes in the submucosal mucous glands, increasing progressively up to 4 times that of control. Uptake of precursors into specific mucins purified by DEAE-Sephacel showed that uptake into the 0.2 and 0.3 M NaCl fractions was stimulated several fold by SO2, and uptake into more highly acidic fractions, which was nearly absent in the control, was also greatly increased. Two weeks following the last exposure of the tracheas to SO2, their morphological and mucus-secreting properties showed signs of returning to that of the control.

Chronic inhalation of cigarette smoke by F344 rats.

Specific-pathogen-free female F344 rats were exposed by inhalation to what was considered a maximal tolerated dose of cigarette smoke. Total pulmonary deposition of smoke particulates from a single cigarette was 0.25 mg in young rats. Rats were exposed to smoke from 7 cigarettes/day for as long as 2.5 years, at which time 30% of the rats remained alive. Mortality of smoke-exposed animals was not different from that of untreated or sham-exposed controls. Hyperplastic and metaplastic areas in the epithelium of the nasal turbinates, larynges, and tracheae of exposed animals were observed at death. The lungs of exposed rats contained areas of focal alveolitis consisting of accumulated pigmented macrophages, epithelial hyperplasia, fibrosis, and disrupted alveolar structure. Smoke exposure did not change the total number of tumor-bearing animals relative to controls; however, exposed rats had significantly fewer tumors in the hypophyses, hematopoietic-lymphoid systems, uteri, and ovaries but an increased number of tumors in the respiratory tracts and dermes. Only 1 of 93 (1%) control rats had a tumor (an alveologenic carcinoma) in the respiratory tract as opposed to 7 of 80 (9%) exposed animals (nasal tumors: 1 adenocarcinoma and 1 squamous cell carcinoma; pulmonary tumors: 5 adenomas, 2 alveologenic carcinomas, and 1 squamous carcinoma).

A method for rapid scoring of respiratory tumors in hamsters.

The larynx, trachea, and lung of Syrian golden hamsters treated with diethylnitrosamine were stained with modified Wright's stain and rendered semitransparent by a clearing technique. Areas of dense cell aggregation (tumors) were readily observable under a dissecting microscope. Tumor scoring at this subgross level correlated with subsequent microscopic evaluation, except that several tumors were found on the subgross level which would not have been observed during routine histologic procedures. The clearing method was used successfully to discern differences in tumor incidence in a dose-response study with diethylnitrosamine.