Michael C. Carakostas

Development of a short-term inhalation bioassay to assess pulmonary toxicity of inhaled particles: Comparisons of pulmonary responses to carbonyl iron and silica

This paper describes a short-term inhalation bioassay for evaluating the lung toxicity of inhaled particulate materials. To validate the method, rats were exposed for 6 hr or 3 days to various concentrations of either aerosolized alpha-quartz silica or carbonyl iron particles. Cells and fluids from groups of sham- and dust-exposed animals were recovered by bronchoalveolar lavage (BAL). Alkaline phosphatase, lactate dehydrogenase (LDH), and protein values were measured in BAL fluids at several time points postexposure. Cells were identified, counted, and evaluated for viability. Pulmonary macrophages (PM) were cultured and studied for morphology, chemotaxis, and phagocytosis by scanning electron microscopy. The lungs of additional exposed animals were processed for histopathology and transmission electron microscopy. Brief exposures to silica elicited a sustained granulocytic inflammatory response (primarily neutrophils) with concomitant increases in alkaline phosphatase, LDH, and protein in the lavage fluids (p less than 0.05). In addition, PM functional capacity was depressed (p less than 0.05) and histopathologic lesions were observed within 1 month after exposure. In contrast, 6-hr or 3-day exposures to CI produced no cellular, cytotoxic, or alveolar/capillary membrane permeability changes at any time postexposure. PM function was either enhanced or unchanged from controls. These data demonstrate that short-term, high-dose inhalation exposures of silica produce effects similar to those previously observed using intratracheal instillation or chronic inhalation models, and lend support to this method as a reliable short-term bioassay for evaluating the pulmonary toxicity and mechanisms associated with exposures to new and untested materials.

Attenuation of perfluoropolymer fume pulmonary toxicity: Effect of filters, combustion method, and aerosol age

Thermal decomposition products of some perfluorinated polymers are toxic to experimental animals in small-scale combustion toxicity tests; the toxicity is dependent upon the heating procedure, combustion temperature, and other experimental conditions. In the current studies we investigated the time course of fume generation and exposure on pulmonary effects in rats following a 30-min exposure to perfluoropolymer decomposition products (i.e., fume concentration = 0.2 mg/m3 of tetrafluoroethylene/hexafluoropropylene copolymer (FEP)) pyrolyzed with either static or dynamic airflows. In the first set of experiments, five different groups of rats were exposed to FEP fumes in a static combustion toxicity test system. Three groups were exposed to unfiltered FEP fumes during 0- to 15-, 15- to 30-, and 0- to 30-min intervals, respectively, and one to a filtered (particle-free) atmosphere of combusted FEP for 30 min. Sham-exposed rats constituted the control group. Immediately after exposure, the rats were sacrificed and their lungs weighed and lavaged or perfused to assess indices of cytotoxicity. Our results showed that lung weights, markers of inflammation, and pulmonary hemorrhage and alkaline phosphatase, beta-glucuronidase, lactate dehydrogenase, and protein levels in bronchoalveolar lavage fluids were significantly elevated in all unfiltered FEP-exposed groups compared to those in either the rates exposed through filters or controls (P less than 0.01). In a second set of experiments using a dynamic pyrolysis toxicity test system, rats were exposed for 30 min to FEP-pyrolyzed fumes which were either freshly generated or aged for 1 or 5 min prior to delivery to the animals breathing zone. Subsequently, lung cytotoxicity parameters were measured. Rats exposed directly to the fresh fumes demonstrated toxic effects consistent with those described above (P less than 0.01), but the pulmonary toxicity of aged (i.e., 1 or 5 min delay) FEP fumes was diminished in a time-dependent manner, suggesting that the toxicant was unstable. Histopathological studies correlated with biochemical results and revealed that inhalation of unfiltered or freshly generated FEP fumes produced a severe lung injury characterized by the development of alveolar and interstitial edema, intraalveolar hemorrhage, congestion, and fibrin deposition. Electron microscopy studies demonstrated severe damage to terminal bronchiolar cells and detachment of Type I epithelial and endothelial cells in pulmonary regions. The severity of pathology observed in lungs of rats exposed to 1-min aged fumes was intermediate between unfiltered/unaltered fume-exposed animals and sham controls. The results of these studies demonstrate that the lung toxicity of perfluoropolymer fumes is associated with the aerosol phase generated in perfluoropolymer pyrolysis.

Interpreting rodent clinical laboratory data in safety assessment studies: Biological and analytical components of variation☆

Evaluation of the biological and toxicological significance of clinical laboratory results obtained in safety assessment studies requires an understanding of factors unrelated to the treatment that may affect test results. Since the magnitude of the components of variation is usually unknown, the toxicologic significance of small, but statistically significant, differences between control and treatment group results can be difficult to assess. Over a 12-week period, components of variation were determined for a wide range of hematologic and clinical biochemical assays in clinically normal Sprague-Dawley-derived rats. Estimates of variance components and ratios were obtained for each test. While the intra-animal/inter-animal ratios (r ratios) revealed some important tests with a high or low degree of individuality, many hematologic and clinical biochemical tests had r ratios within the 95% confidence interval for equal variances. Analytical variance ratios revealed tests that are sensitive to the effects of experimental error and experimental design. Due to the diversity of the variance component patterns among clinical laboratory tests, complex experimental designs may be required to reduce the effects of analytical and biological variation on the statistical analysis of clinical laboratory data. The results of this study suggest that statistically significant clinical laboratory findings that are not biologically or toxicologically important will be present in many rodent safety assessment studies with a standard design. Overreliance on the result of standard prepackaged statistical analyses for determining the presence of toxicologically significant findings can lead to misinterpretation of clinical laboratory data. Sound medical judgment must be applied to clinical laboratory findings using appropriate statistical analyses as a tool for pattern recognition.

Effect of circulating neutrophil depletion on lung injury induced by inhaled silica particles

Polymorphonuclear leukocytes (PMNs) recruited into the alveolar region during inflammation may injure the lung parenchyma by releasing cytotoxic oxygen radicals and proteases. Because brief exposures to crystalline silica elicit recruitment of PMNs into the alveolar region, which is strongly correlated with parameters of cytotoxicity, increased alveolar epithelial permeability, and lysosomal enzyme release, we sought to evaluate the potential role of PMNs in silica‐induced lung injury. Rats were depleted of PMNs by administration of an anti‐rat PMN antiserum prior to exposure to silica. Pulmonary inflammatory responses to silica in this group were compared to responses in normal silica‐exposed rats as well as sham‐exposed normal or PMN‐depleted rats. Bronchoalveolar lavage fluids from normal, silica‐exposed rats contained 9.7 × 106 PMNs immediately after exposure for 3 days, compared to 0.01 × 106 PMNs for both normal or PMN‐depleted, sham‐exposed rats. Bronchoalveolar lavage fluids from successfully PMN‐depleted, exposed rats contained significantly fewer (0.7 × 106) PMNs compared to normal silica‐exposed rats. In both groups of silica‐exposed rats, a variety of biochemical indicators of lung injury were increased significantly compared to measurements from both sham‐exposed groups, but there were no differences between PMN‐depleted and normal silica‐exposed groups. The results suggest that recruitment of PMNs into the alveolar region is not a necessary prerequisite for the observed increases in biochemical indicators of silica‐induced acute lung injury.

Four-week inhalation toxicity study with ludox colloidal silica in rats : pulmonary cellular responses

This study was designed to complement a traditional subchronic inhalation toxicity study with Ludox colloidal silica. CD rats were exposed nose-only for 2 or 4 weeks at concentrations of 0, 10, 50, and 150 mg/m3 Ludox (dried SiO2). Additional groups of rats exposed for 4 weeks were given a 3-month recovery period. Following exposure and/or recovery, fluids and cells were recovered from the lungs by bronchoalveolar lavage (BAL) and measured for cellular and biochemical parameters. Additional groups of animals were processed for cell labeling studies or lung deposition studies. Inhaled doses of Ludox colloidal silica were measured after 4-week exposures and were found to be 489 micrograms/lung (10 mg/m3 group), 2418 micrograms/lung (50 mg/m3), and 7378 micrograms/lung (150 mg/m3), respectively. Results showed that exposures to 150 mg/m3 Ludox for 2 or 4 weeks produced pulmonary inflammation along with increases in BAL protein, LDH, and alkaline phosphatase values (p less than 0.05) and reduced macrophage phagocytosis. Inflammatory responses, evidenced by increased numbers of neutrophils, were also measured in the lungs of the 50 mg/m3 group following 2 and/or 4 weeks of exposure. Most biochemical parameters for all groups returned to control values following a 3-month recovery period. Autoradiographic studies demonstrated that the labeling indices of terminal bronchiolar and lung parenchymal cells were generally increased in the 50 and 150 mg/m3 groups after 2 and 4 weeks of exposure but, with one exception, returned to normal levels following a 3-month postexposure period. No significant alterations in any measured parameters were detected in rats exposed to 10 mg/m3 Ludox at any time postexposure. The determination of a no-observable-effect level (NOEL) of 10 mg/m3 was consistent with results obtained by conventional toxicology methods and affirms the utility of these biochemical, cellular, and autoradiographic techniques for providing a predictive screen to assess the toxicity of inhaled particles.

A 90-Day Inhalation Toxicity Study with Benomyl in Rats

Benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate, CAS Registry No. 17804-35-2] is a fungicide and the possibility for inhalation exposure exists for field workers. To assess the toxicity of benomyl, groups of 20 male and 20 female CD rats were exposed nose-only 6 hr a day, 5 days a week, to concentrations of 0, 10, 50 or 200 mg/m3 of a benomyl atmosphere. At the midpoint (approximately 45 days on test) and at the end of the exposure period, blood and urine samples for clinical evaluation were collected from 10 rats/group/sex, and these animals were sacrificed for pathological examination. Similar evaluations were performed on all remaining rats at the end of the 90-day test period. After approximately 45 days on test, compound-related degeneration of the olfactory epithelium was observed in all males and in 8 of 10 female rats exposed to 200 mg/m3 benomyl. Two male rats exposed to 50 mg/m3 had similar, although less severe, areas of olfactory epithelial degeneration. After approximately 90 days of exposure, the remaining 10 rats/group/sex were sacrificed and examined. Of these rats, all of the males and females exposed to 200 mg/m3 had olfactory degeneration, along with 3 males exposed to 50 mg/m3 of benomyl. No other observed lesions were interpreted to have been caused by the benomyl exposure. In addition, male rats exposed to 200 mg/m3 benomyl had depressed mean body weights compared to controls and this finding correlated with a reduction in food consumption. Based on pathological observations, 10 mg/m3 represents the no-observable-effect level (NOEL) for the male rats, and 50 mg/m3 is the NOEL for the female rats.

Assessments of Lung Toxicity to Acrawax® C Following Acute Inhalation Exposure

Acrawax is a trademark for a series of synthetic waxes which are used as flatteners in paint, and lubricants in plastics, and these materials have been routinely regarded as nuisance dusts. Due to a paucity of information regarding the pulmonary toxicity of this material, we investigated the effects of acute inhalation of Acrawax C in rats. CD rats were exposed to aerosols of Acrawax C for 6 hours at 112 mg/m3. Fluids and cells from sham and exposed animals were recovered by bronchoalveolar lavage (BAL) and measured for cellular and biochemical parameters at 0, 24, 48, 172 hrs (8 days), and 1 month postexposure. Pulmonary macrophages (PM) were cultured and studied for in vitro and in vivo phagocytosis, as well as surface morphology. The lungs of additional animals exposed to Acrawax were fixed for assessment by histopathology, and transmission electron microscopy. Our results showed that Acrawax C exposure produced a mild inflammatory response at 24 hours postexposure, but cell differentials were not significantly different from controls at 48 hrs after exposure. BAL levels of lactate dehydrogenase, alkaline phosphatase and protein were slightly different from controls only at 8 days postexposure, and had returned to control values by 1 month of recovery. Acrawax exposure had no adverse effects on either morphology or the phagocytic capacity of pulmonary macrophages recovered from exposed animals. Histopathologic analysis of lung tissue from Acrawax C-exposed rats revealed normal lung architecture. Based on acute studies, our results suggest that the response to inhaled Acrawax C is not substantially different from the response to other nuisance dusts such as carbonyl iron and titanium dioxide.

Complement facilitates macrophage phagocytosis of inhaled iron particles but has little effect in mediating silica-induced lung inflammatory and clearance responses

Complement-mediated mechanisms are known to play a role in pulmonary inflammation and clearance responses to some types of inhaled particles. The present studies were undertaken to investigate the role of complement in mediating pulmonary inflammation and/or phagocytosis as a function of particle clearance in rats exposed to silica or carbonyl iron (CI) particles. Both particle types were shown to be weak activators of serum complement in vitro. In these studies, normal and complement-depressed (CVF-treated) rats were exposed to aerosols of CI or silica particles for 6 hr at 100 mg/m3. Following exposure, alveolar fluids and cells from sham and dust-exposed animals were recovered by bronchoalveolar lavage (BAL) at several time periods postexposure and measured for a variety of biochemical and cellular indices. In addition, pulmonary macrophages were cultured and studied for morphology and phagocytosis. Our results showed that CI exposure did not produce cellular or biochemical indices of pulmonary inflammation, either in normal or complement-depleted rats. However, fewer phagocytic macrophages were recovered from the lungs of CVF-treated, CI-exposed rats than from normal exposed animals. In contrast, silica inhalation produced a sustained PMN inflammatory response in the lungs of exposed rats, measured up through 1 month postexposure, along with significant increases in BAL fluid levels of LDH, protein, and alkaline phosphatase (P less than 0.05) and deficits in pulmonary macrophage phagocytic functions. Cobra venom factor (CVF) treatment prior to exposure in rats had no significant effect upon the silica-induced parameters, suggesting that complement may not play an important role in the acute pulmonary response to silica. The results indicate that complement may play a role in mediating CI-related macrophage clearance responses but has little effect upon sustained silica-induced pulmonary inflammatory parameters.

Inhalation Toxicity of an Isomeric Mixture of Hydrochlorofluorocarbon (HCFC) 225 in Male Rats

A blend of the hydrochlorofluorocarbon isomers HCFC-225ca and HCFC-225cb has been proposed as a potential substitute for CFC-113, an important solvent and cleaning agent. The toxicity following repeated inhalation of an HCFC-225 isomer mixture was assessed in male Crl:CDBR rats. Three groups of 10 male rats were exposed to the test compound in air at design concentrations of 500, 5000, and 13,000 ppm. Rats were exposed 6 hr/day, 5 days/week for 2 weeks. A control group of 10 male rats was exposed to air only. Decreased serum cholesterol, triglycerides, and glucose; dose-related increased mean absolute and relative liver weights; and microscopic hepatocellular hypertrophy were present at all exposure concentrations. Hepatocellular hypertrophy correlated ultrastructurally to proliferation of peroxisomes. Clinical chemical parameters and organ weight and morphologic changes in the liver were reversible following 14 days of recovery.

Subchronic Inhalation Study with Vinyl Fluoride: Effects on Hepatic Cell Proliferation and Urinary Fluoride Excretion

Vinyl fluoride is used widely in the manufacture of fluoropolymers. Based in part on the structural similarity of vinyl fluoride to the hepatocarcinogens vinyl chloride and vinyl bromide, a TSCA Section 4 test rule mandated the testing of vinyl fluoride for oncogenicity. This report presents the results of a 90-day inhalation study in rats and mice with vinyl fluoride designed to set test concentrations for a subsequent oncogenicity study. Groups of 15 male and female rats and mice were exposed 6 hr per day, 5 days per week for approximately 90 days to target concentrations of 0, 200, 2000, or 20,000 ppm vinyl fluoride. Clinical chemical, hematological, and urine analyses were performed on rats after 45 and 90 days of exposure. A hematological evaluation was performed on mice following 45 and 90 days of exposure. A complete gross and microscopic evaluation was conducted at the end of the study. After 93 days on test, groups of five rats and five mice per sex were implanted with osmotic minipumps containing [3H]thymidine and were exposed for an additional 5 days to measure cell proliferation in liver, kidney, lung, and nasal cavity tissues. Results of the histopathological, clinical chemical, and hematological evaluations showed no significant effects of vinyl fluoride exposure at any concentration following either 45 or 90 days of exposure. A concentration-related increase in fluoride ion in urine was observed in rats at 45 and 90 days of exposure. A plateau in urinary fluoride excretion was observed at approximately 2000 ppm, suggesting saturation of vinyl fluoride metabolism. Vinyl fluoride-related cell proliferation effects were largely restricted to liver. Hepatic cell proliferation in male and female rats and mice was elevated at all concentrations. The response was similar at concentrations of either 2000 or 20,000 ppm and was consistent with concentration-response relationships for other haloethylenes. Taken together, the urinary fluoride excretion and hepatic cell proliferation data suggest a mechanistic link between the two effects. On the basis of these findings and experience with other haloethylenes, concentrations of vinyl fluoride to be tested for oncogenicity should be chosen such that the full linear range of the concentration-response curve is evaluated. The present study demonstrates through example the value of incorporating cell proliferation studies in standard testing protocols.