Tumor induction in F344/N rats and B6C3F1 mice following inhalation exposure to ethylbenzene.

Carcinogenesis studies of ethylbenzene were conducted because of its extensive use as a solvent and because it is structurally similar to the known carcinogen benzene. Groups of 50 male and 50 female Fischer rats and B6C3F1 mice were exposed to ethylbenzene by inhalation at 0, 75, 250, and 750 ppm 6 h per day, 5 days per week, for 2 years. The dose levels were selected based on the results of 13-week studies. In the 750 ppm group of male and female rats, body weights were slightly lower and incidences of renal hyperplasia and tubular neoplasms were significantly increased compared with controls. Incidence of testicular tumors was also significantly increased in male rats. Survival and body weights of the exposed groups of male and female mice and controls were comparable. Incidences of alveolar epithelium metaplasia, alveolar/bronchiolar adenoma, and hepatocyte hypertrophy and necrosis were significantly increased in the 750 ppm male mice and incidences of liver eosinophilic foci and hepatocellular neoplasms were significantly increased in the 750 ppm female mice compared with controls. Ethylbenzene is carcinogenic inducing neoplasms in kidneys and testes in Fischer rats and in lungs in male and liver in female B6C3F1 mice.

13-week inhalation toxicity study of menthol cigarette smoke.

Menthol is a common pharmaceutical, food and tobacco flavouring ingredient used for its minty characteristics and cooling effects. A 13-wk comparative nose-only smoke inhalation toxicity study was conducted using an American-style, cellulose acetate-filtered, non-menthol reference cigarette and a similarly blended test cigarette containing 5000 ppm synthetic l-menthol tobacco. Male and female Fischer 344 rats were exposed for 1 hr/day, 5 days/wk for 13 wk at target mainstream smoke particulate concentrations of 200, 600 or 1200 mg/m3, while control rats were exposed to filtered air. Internal dose biomarkers (blood carboxyhaemoglobin, serum nicotine and serum continine) indicated equivalent exposures were obtained for the two cigarettes. Effects typically noted in rats exposed to high levels of mainstream tobacco smoke were similar for both cigarette types and included reduced body weights (males slightly more affected than females), increased heart-to-body weight ratios and lung weights, and histopathological changes in the respiratory tract. Rats exposed to reference cigarette smoke displayed a dose-related increase in nasal discharge that was not observed in menthol smoke-exposed rats. All smoke-related effects diminished significantly during a 6-wk non-exposure recovery period. The results of this 13-wk smoke inhalation study indicated that the addition of 5000 ppm menthol to tobacco had no substantial effect on the character or extent of the biological responses normally associated with inhalation of mainstream cigarette smoke in rats.

Ethylene dibromide: evidence of systemic and immunologic toxicity without impairment of in vivo host defenses.

Ethylene dibromide was administered intragastrically on 14 consecutive days to B6C3F1 female mice. Host resistance was not altered after challenge with B16F10 tumor cells, Listeria monocytogenes, influenza, or Herpes simplex viruses. In contrast, decreases were seen in relative thymus and spleen weights, red blood cells, hemoglobin, hematocrit, and in alveolar macrophage, natural killer cell, T-cell, and mixed lymphocyte culture responses. Increases occurred in relative kidney and liver weights, cholesterol, peripheral neutrophils, resident peritoneal exudate cells (with increased phagocytosis) and plaque-forming cells. There was little difference between the dose that caused immune modulation and that which produced significant toxicity.

Prechronic inhalation toxicity studies of isobutyl nitrite.

Isobutyl nitrite (IBN) is a volatile liquid that has become increasingly popular as an inhaled recreational drug. To investigate short-term toxic effects and establish exposure parameters for chronic inhalation studies, F344/N rats and B6C3F1 mice were exposed to IBN vapors on a 6 hr/day + t90, 5 days/week schedule. Twelve exposures were administered at concentrations of 0, 100, 200, 400, 600, and 800 ppm IBN. This exposure series resulted in mortality in rats exposed to greater than or equal to 600 ppm and mice exposed to 800 ppm. Animals exposed at the lower concentrations developed hyperplasia of the bronchiolar and nasal turbinate epithelium (rats and mice) and lymphocytic atrophy in the spleen and thymus (mice). Longer term, 13-week, subchronic exposures were conducted at concentrations of 0, 10, 25, 75, 150, and 300 ppm IBN. Exposure to 300 ppm IBN reduced the body weight gains in both sexes of rats and in female mice. IBN-related clinical pathology changes included reduced RBC counts accompanied by moderate increases in mean corpuscular volume and reticulocyte counts, increased WBC counts, and mildly increased methemoglobin concentration. Bone marrow hyperplasia was observed in all groups of IBN-exposed rats, while in mice only females at greater than or equal to 150 ppm IBN displayed this change. Excessive splenic pulp hematopoiesis was noted in mice at all IBN exposure levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental toxicity evaluation of inhaled citral in Sprague-Dawley rats.

Citral is a commonly used fragrance and flavour ingredient that has demonstrated a potential for teratogenicity in chick embryo screening studies. To investigate potential mammalian developmental toxicity, pregnant Sprague-Dawley rats were exposed to citral by inhalation for 6 hr/day on gestation days 6-15 at mean concentrations of 0, 10 or 34 ppm as vapour, or 68 ppm as an aerosol/vapour mixture. Dams were killed on gestation day 20 and the foetuses were removed and evaluated for gross, visceral and skeletal malformations. Exposure to 68 ppm was maternally toxic, with reduced body-weight gains, ocular opacity, breathing difficulty, nasal discharge and salivation noted in the dams. No maternal toxicity was seen at the lower vapour exposure levels. The number of corpora lutea, implantations, resorptions, foetal viability, litter size, and sex ratio were not adversely affected by citral at any exposure level tested, and no exposure-related malformations were observed. At a maternally toxic exposure level, a slight reduction in mean foetal body weight and a slight increase in the incidence of hypoplastic bones were noted. Results of this study indicate that citral does not produce developmental toxicity in the rat when administered by inhalation at concentrations up to a maternally toxic exposure level.

Prechronic inhalation toxicity studies of 2-mercaptobenzimidazole (2-MBI) in F344/N rats.

2-Mercaptobenzimidazole (2-MBI), used in rubber processing, is a suspect carcinogen structurally related to ethylene thiourea. The inhalation toxicity of 2-MBI was evaluated in male and female F344/N rats exposed 6 hr/day, 5 days/week to respirable aerosols generated by spray atomization of aqueous suspensions of the 2-MBI powder and subsequent drying of the resulting aerosols. Twelve exposures at target concentrations of 0, 6.3, 12.5, 25.0, 50.0, or 100 mg/m3 of 2-MBI produced a dose-related reduction in body weight gains, thyroid follicular cell hyperplasia, adrenal cortex fatty change, and pituitary atrophy. Sub-chronic exposures were conducted at target concentrations of 0, 3.1, 6.2, 12.5, 25.0, and 50.0 mg/m3 of 2-MBI. Rats at greater than or equal to 25 mg/m3 displayed hunched posture, hypoactivity, and reduced body weight gain, with compound related mortality at the highest exposure level. Anemia; increased SGPT, SGOT, alkaline phosphatase, sorbitol dehydrogenase, BUN, and cholesterol; and reduced free fatty acid were seen in rats at greater than or equal to 25 mg/m3. Increased thyroid weight and thyroid follicular cell hyperplasia were noted in both sexes at greater than or equal to 6.2 mg/m3, with reduced triiodothyronine and thyroxine levels in both sexes at greater than or equal to 12.5 mg/m3. Thyroid follicular cell hyperplasia was also seen in rats at 3.1 mg/m3. Thymus weights were significantly reduced in both sexes at all exposure levels with liver weight increases at greater than or equal to 6.2 mg/m3. Exposure-related histopathologic changes included pituitary cytoplasmic vacuolization, adrenal cortex necrosis, lymphoid depletion, thymic atrophy, liver cell hypertrophy, renal mineralization and tubular atrophy, and hypocellularity of the bone marrow.

Effects of inhaled hexachlorobenzene aerosols on rat pulmonary host defenses.

Pulmonary bactericidal activity, macrophage phagocytic activity, alveolar macrophage (AM) enzyme activity, and T- and B-cell mitogenesis of lymphocytes from lung associated lymph nodes (LALN) or mesenteric lymph nodes (MESLN) were assessed in Sprague-Dawley rats exposed 4 hr/d, 4 days/wk for 1, 4, or 16 days to hexachlorobenzene (HCB) aerosols. Pulmonary bactericidal activity was depressed after 1 or 4 but not 16 exposures to 35 mg/m3 of HCB. AM phagocytosis of 51Cr-RBC in vitro was increased after 4 but not 1 or 16 exposures to HCB, and no effect was observed in peritoneal macrophages. HCB significantly enhanced mitogenesis in MESLN to the B-cell mitogen Salmonella typhimurium lipopolysaccharide (STM) after 4 exposures; LALN STM mitogenesis and LALN and MESLN mitogenesis to phytohemagglutinin (PHA) were not affected. After 16 exposures, however, the PHA responses in LALN and MESLN were significantly increased and decreased, respectively.

Effects of inhalation of red phosphorus/butyl rubber combustion products on aveolar macrophage responses in rats.

The effects of inhalation of red phosphorus/butyl rubber (RP/BR) used as an obscurant smoke in tactical environments was examined. Sprague-Dawley male rats were exposed to 1000 mg/m3 of RP/BR for 3.5 h in single exposures, while in subsequent intermediate and subchronic studies the animals were exposed to concentrations ranging from 300 to 1200 mg/m3 for 2.25 h/day, 4 consecutive days/week for 4 and 13 weeks, respectively. Pulmonary bactericidal activity to inhaled [35S]-Klebsiella pneumoniae was depressed after the acute and the 13-week subchronic exposures but was unchanged after the 4-week exposures. The pulmonary free cells collected by lavage showed decreasing trends in total numbers, increased ATP levels and decreased ectoenzyme activity for 5'-nucleotidase after most of the exposures. Mild-to-moderate-to-severe terminal broncheolar fibrosis was observed in all rats after 4- and 13-week exposures to greater than or equal to 750 mg/m3 of RP/BR. The severity of the lesions increased with the severity of the exposure conditions. Except for the fibrosis, most changes were reversible.

Effects of inhalation of ethylene dichloride on pulmonary defenses of mice and rats.

The effects of single or multiple inhalation exposures to ethylene dichloride (DCE) on the pulmonary defense systems of mice and rats were evaluated. Single exposures of mice to the threshold limit value of DCE (10 ppm) resulted in decreased pulmonary bactericidal activity to inhaled Klebsiella pneumoniae and increased mortality from Streptococcus zooepidemicus respiratory infection. A single exposure to 5 ppm DCE caused increased mortality from streptococcal pneumonia although bactericidal activity was not affected. Neither of these two parameters changed following single or five consecutive daily exposures to 2.5 ppm DCE. Single exposures to 10 or 100 ppm DCE did not affect mouse alveolar macrophage (AM) inhibition of the proliferation of a tumor target cell in vitro or AM in vitro phagocytosis of red blood cells. In rats, no effects were observed on pulmonary bactericidal activity. AM in vitro phagocytosis, AM cytostasis and cytolysis of tumor target cells, AM ectoenzymes, or blastogenesis of mitogen-stimulated rat T- and B-lymphocytes from lung-associated, mesenteric, and popliteal lymph nodes following single exposure to 100 or 200 ppm DCE or after twelve 5-hr exposures to 10, 20, 50, or 100 ppm DCE.

Comparative study of various methods used for determining health effects of inhaled sulfates.

Various health effect parameters were compared to determine which tests were the most sensitive indicators of toxic effects of exposure to metallic sulfate aerosols. Inhalation studies were conducted involving either single 3-hr exposure to various concentrations of cupric sulfate (0.43-2.64 mg/m3 SO4), aluminum sulfate (1.65-2.75 mg/m3 SO4), and aluminum ammonium sulfate (1.47-3.81 mg/m3 SO4) or multiple (five and ten) daily 3-hr exposures to cupric sulfate (0.1 mg/m3 SO4). The test parameters studied in male and female CD1 mice were changes in mortality after respiratory infection with Group C Streptococcus zooepidemicus; pulmonary bactericidal activity; pulmonary cell number, type, viability, and ATP content; and pulmonary morphology by scanning electron microscopy. Tracheal ciliary beating frequency and morphology were also studied in both CD1 mice and Syrian golden hamsters. Differences in bacteria-induced mortality rate appeared to be the most sensitive and consistent indicators of pollutant damage. The other parameters produced evidence of damage but generally only at the higher pollutant concentrations. Cupric sulfate was the most toxic of the three sulfates, but the differences between the toxicity of the aluminum sulfate and aluminum ammonium sulfate were less clear.

Effects of acrolein on macrophage functions in rats.

Male Sprague-Dawley rats were exposed to 0.1, 1.0 or 3.0 ppm acrolein or filtered air 6 h/day, 5 days/week for 3 weeks. Rats were tested one day following the last exposure and exhibited no change in pulmonary clearance of inhaled 35S-labeled Klebsiella pneumoniae at any acrolein concentration. Decreased numbers of peritoneal cells were obtained from exposed rats while the number of cells lavaged from the lungs was unchanged. Macrophages of acrolein-exposed rats had altered phagocytic and enzymatic patterns as compared to macrophages from animals breathing filtered air. However, these changes had no apparent effect on macrophage killing of inhaled bacteria and were therefore probably not indicative of extreme chemical toxicity.

Absence of hydrocarbon-induced nephropathy in rats exposed subchronically to volatile hydrocarbon mixtures pertinent to gasoline.

Ninety-day inhalation studies were conducted on 50:50 weight percent (wt %) mixtures of n-butane:n-pentane and isobutane:isopentane, respectively, and on a distillation cut boiling below 145 degrees F of a reference unleaded gasoline blend to assess the nephrotoxicity of these volatile mixtures. The mixtures of butanes and pentanes were selected because these four hydrocarbons are the most prevalent components of gasoline vapors encountered under typical occupational exposures. The 0-145 degrees F gasoline distillation fraction was tested because it reasonably approximates the composition of gasoline vapors measured under occupational settings. Male and female F-344 rats were exposed to 2 levels of each mixture, 6 hours per day, 5 days per week, for 13 weeks. The target concentrations for the butane:pentane mixtures were 4500 and 1000 parts per million (ppm), while 5200 and 1200 ppm were set for the gasoline distillation fraction. An interim sacrifice was conducted after 28 days. The rats were not significantly affected by the exposures, and there was no evidence of hydrocarbon-induced nephropathy in either sex at the termination of each study. However, at the 28-day interim sacrifice period for both butane:pentane mixtures, mild, transient treatment-related but not exposure-related kidney effects were observed in the male rats. These perturbations were absent at the interim sacrifice period for the gasoline distillation fraction.

The effects of inhalation of organic chemical air contaminants on murine lung host defenses.

The potential health hazards of exposure to threshold limit value (TLV) concentrations of acetaldehyde, acrolein, propylene oxide, chloroform, methyl chloroform, carbon tetrachloride, allyl chloride, methylene chloride, ethylene trichloride, perchloroethylene, benzene, phenol, monochlorobenzene, and benzyl chloride, compounds which may be present in the ambient or work room atmosphere were investigated. The effects of single and multiple 3-hr inhalation exposures were evaluated in mice by monitoring changes in their susceptibility to experimentally induced streptococcus aerosol infection and pulmonary bactericidal activity to inhaled Klebsiella pneumoniae. When significant changes in these parameters were found, further exposures were performed at reduced vapor concentrations until the no-measurable-effect level was reached. Multiple exposures on 5 consecutive days were then performed at this concentration. Significant increases in susceptibility to respiratory streptococcus infection were observed after single 3-hr exposure to TLV concentrations of methylene chloride, perchloroethylene, and ethylene trichloride. For methylene chloride and perchloroethylene, these exposure conditions also resulted in significantly decreased pulmonary bactericidal activity.

Evaluation of host resistance and immune function in cadmium-exposed mice.

Adult female B6C3F1 mice received distilled water only or water containing 10, 50, or 250 ppm of cadmium chloride (CdCl2) for 90 days. Body weights were measured weekly. On selected days during exposure and on Day 91, Cd tissue concentrations were measured along with changes in primary antibody responses. On Day 91 mice also received a primary challenge with various infectious agents. T- and B-cell mitogenesis, natural killer (NK) cell function, delayed type hypersensitivity (DTH) as well as macrophage bactericidal activity, and phagocytosis were measured. There was no change in body weight gain, organ weights, or in humoral immunity during treatment even though cadmium had accumulated in significant quantities in the tissues. Compared with controls, exposure to cadmium had no statistically significant effect on mortality and mean survival time following primary or secondary challenge with any of the infectious agents. However, there was a dose-related, increased susceptibility to Herpes simplex type 2 virus. T- and B-lymphocyte proliferation was significantly reduced, and macrophage phagocytosis was significantly increased following cadmium exposure. NK cell activity was augmented, but not significantly. Macrophage bactericidal activity and DTH were not significantly altered.

Effects of toluene inhalation on pulmonary host defenses of mice.

The potential hazards of exposure to vapor-phase toluene on pulmonary host defenses were evaluated. Mice exposed to concentrations ranging from 2.5-500 ppm, including the threshold limit value level of 100 ppm, exhibited increased susceptibility to respiratory infection with Streptococcus zooepidemicus. The no-measurable-effect level for single, as well as for 5 exposures was 1 ppm. Significantly decreased pulmonary bactericidal activity was observed after single exposures to 500, 250, 100 and 2.5 ppm toluene, and after 5 daily 3-h exposures to 1.0 ppm of toluene. A 20-exposure study with toluene at 1 ppm produced no changes in either of the 2 assays.

The effect of dimethylnitrosamine on host resistance and immunity.

Adult female B6C3F1 mice were injected ip with 0.2 ml phosphate-buffered saline (PBS) only or PBS containing 1.5, 3, or 5 mg dimethylnitrosamine (DMN)/kg body wt daily for 14 days. On Day 16, mice were evaluated for changes in immune status as measured by the antibody response to sheep red blood cells (SRBCs), blastogenesis to T- and B-cell mitogens, natural killer (NK) cell function, delayed hypersensitivity, and alveolar macrophage (AM) bactericidal activity; and for changes in host resistance following challenge with various microorganisms or tumor cells. DMN-exposed animals exhibited reduced humoral antibody responses, T-cell mitogenesis, and AM bactericidal activity. B-cell mitogenesis, NK cell activity, and delayed hypersensitivity were increased. Resistance to challenge with Listeria monocytogenes, Trichinella spiralis, or Herpes simplex types 1 or 2 virus (HSV-1, HSV-2) was not significantly impaired, while that to Streptococcus zooepidemicus and influenza virus was significantly reduced. Resistance to B16-F10 tumor challenge was enhanced following DMN exposure. The data show that DMN treatment altered humoral immunity and antibody-mediated host defense mechanisms. Increased NK cell activity may account for the increased resistance to challenge with Herpes virus and B16-F10 tumor cells.

Effects of arsenic trioxide inhalation exposure on pulmonary antibacterial defenses in mice.

The effects of single and multiple (5 and 20) 3-h inhalation exposures to aerosols of arsenic trioxide on the pulmonary defense system of mice were investigated. Arsenic trioxide mist was generated from an aqueous solution and dried to produce particulate aerosols of 0.4 micron mass median aerodynamic diameter. Aerosol mass concentration ranged from 125 to 1000 micrograms As/m3. Effects of the exposures were evaluated by determination of changes in susceptibility to experimentally induced streptococcal aerosol infection and in pulmonary bactericidal activity to 35S-labeled Klebsiella pneumoniae. Significant increases in mortality due to the infectious challenge and decreases in bactericidal activity were seen after single 3-h exposures to 270, 500, and 940 micrograms As/m3. Similarly, 5 or 20 multiple 3-h exposures to 500 micrograms As/m3 produced consistently significant increases in mortality and decreases in pulmonary bactericidal activity. At 125 or 250 micrograms As/m3, a decrease in bactericidal activity was seen only after 20 exposures to 250 micrograms/m3. Results from earlier studies with an arsenic-containing copper smelter dust were compared to these data. The possibility of the development of adaptation during multiple exposures to arsenic trioxide is also considered.

The effects of intratracheally administered coarse mode particles on respiratory tract infection in mice.

Because coarse mode particles are rarely studied in their existing size ranges (greatest mass about 5-7 microns, aerodynamic diameter), we investigated the effects of four such particles, quartz, ferric oxide, calcium carbonate, and sodium feldspar, on host defenses against bacterial pulmonary infection. Mice which received intratracheal instillations of 10, 33, and 100 micrograms/mouse were exposed within an hour to aerosols of viable Streptococcus, and pneumonia-induced mortality was measured. At 33 and 100 micrograms/mouse, all particles significantly increased mortality. At the lower dose, only Fe2O3 caused a significant increase in mortality. To evaluate potential delayed effects, mice were challenged with the bacteria 24 h after exposure to 100 micrograms particles/mouse. Delaying the challenge did not significantly alter the response, except for the sodium feldspar group for which a partial recovery was observed. When mice exposed to 100 micrograms particles/mouse received aerosols of Klebsiella pneumoniae 24 h later, there was no significant effect on pulmonary bactericidal activity. For the model system used, it appears that Fe2O3, CaCO3, and sodium feldspar have effects roughly equivalent to quartz.

Effects of subchronic exposure to a mixture of O3, SO2, and (NH4)2SO4 on host defenses of mice.

Mice exposed 5 h/d, 5 d/wk up to 103 d, to 0.2 mg O3/m3 or to a mixture of O3, 13.2 mg SO2/m3, and 1.04 mg (NH4)2SO4 aerosol/m3 showed significantly greater susceptibility to group C streptococcal aerosol infection relative to filtered air controls. Pulmonary bactericidal activity by alveolar macrophages was significantly enhanced in the lungs of mice exposed to the mixture relative to those inhaling filtered air or O3 alone. The total number and distribution of the free cells lavaged from the lungs, as well as cellular ATP levels, did not change due to the pollutant exposures. In vitro cytostasis in tumor target cells cocultured with peritoneal macrophages from the exposed mice was significantly enhanced in the O3-exposed and in the mixture-exposed treatment groups relative to controls and also in the mixture-exposed relative to the O3-exposed group when a target-to-effector-cell ratio of 1:10 was used; no such effects were observed when this ratio was 1:20. Splenic T-lymphocyte function, as measured by blastogenesis to mitogens and alloantigens, was affected by exposure to O3 and/or the mixture, although the patterns of effects were qualitatively different. Splenic B-cell function and macrophage antigen processing, as measured by the generation of antibody plaque-forming cells, was unaffected by exposure.

The effect of diethylstilbestrol as measured by host resistance and tumor susceptibility assays in mice.

As part of a program to develop and validate methodology to measure chemically induced immunotoxicity, the effect of DES on resistance of adult B6C3F1 female mice to various microorganisms and to challenge with syngeneic tumor cells was evaluated. The mice received sc injections of 50 microliter corn oil alone or of corn oil containing the equivalent of 0.2, 1, and 4 mg DES/kg X d for 14 d. Three days later they were challenged with Listeria monocytogenes, Streptococcus sp. influenza virus, herpes virus, Trichinella spiralis, or B16-F10 tumor cells. Host resistance parameters were mortality for the bacterial and viral systems, expulsion of adult parasites from the gut for T. spiralis, and lung weights for the B16-F10 tumor-cell model. Host resistance to L. monocytogenes, herpes virus, and T. spiralis was significantly decreased following DES exposure. Resistance to Streptococcus sp. was decreased, but not at a statistically significant level following these doses of DES. However a dose of DES at 8 mg/kg X d resulted in a highly significant decrease in resistance to the organism. Resistance to influenza virus was unaffected by the DES. In contrast to the above, host resistance to iv-administered B16-F10 tumor cells was significantly increased as a consequence of DES exposure. These model systems for measuring alterations in host resistance have been indicated to hold potential for the routine screening of drugs, chemicals, and environmental agents for their possible immune effects, both adverse and potentiating. The results indicate the importance of selecting the appropriate assay for evaluating a particular agent. They also stress the necessity for including host resistance assays along with assays to measure specific immune aspects, in order to assess in the intact animal the overall effect of complex immune interactions following exposure to a test agent.