T. R. Webb

Pulmonary toxicity studies in rats with triethoxyoctylsilane (OTES)-coated, pigment-grade titanium dioxide particles: Bridging studies to predict inhalation hazard

The aim of this study was to assess and compare the acute lung toxicities of intratracheally instilled hydrophobic relative to hydrophilic surface-coated titanium dioxide (TiO 2) particles using a pulmonary bridging methodology. In addition, the results of these instillation studies were bridged with data previously generated from inhalation studies with hydrophilic, pigment-grade (base) TiO 2 particles, using the base, pigment-grade TiO 2 particles as the inhalation/instillation bridgematerial. To conduct toxicity comparisons, the surface coatings of base pigment-grade TiO 2 particles were made hydrophobic by application of triethoxyoctylsilane (OTES), a commercial product used in plastics applications. For the bioassay experimental design, rats were intratracheally instilled with 2 or 10 mg/kg of the following TiO 2 particle-types: (1) base (hydrophilic) TiO 2 particles; (2) TiO 2 with OTES surface coating; (3) base TiO 2 with Tween 80; or (4) OTES TiO 2 with Tween 80. Saline instilled rats served as controls. Following exposures, the lungs of sham- and TiO 2 -exposed rats were assessed both using bronchoalveolar lavage (BAL) biomarkers and by histopathology of lung tissue at 24 hours, 1 week, 1 month, and 3 months post exposure. The results demonstrated that only the base, high-dose (10 mg/kg) pigment-grade TiO 2 particles and those with particle-types containing Tween 80 produced a transient pulmonary inflammatory response, and this was reversible within 1 week postexposure. The authors conclude that the OTES hydrophobic coating on the pigment-grade TiO 2 particle does not cause significant pulmonary toxicity.

Pulmonary toxicity screening studies in male rats with TiO2 particulates substantially encapsulated with pyrogenically deposited, amorphous silica

The aim of this study was to evaluate the acute lung toxicity in rats of intratracheally instilled TiO2 particles that have been substantially encapsulated with pyrogenically deposited, amorphous silica. Groups of rats were intratracheally instilled either with doses of 1 or 5 mg/kg of hydrophilic Pigment A TiO2 particles or doses of 1 or 5 mg/kg of the following control or particle-types: 1) R-100 TiO2 particles (hydrophilic in nature); 2) quartz particles, 3) carbonyl iron particles. Phosphate-buffered saline (PBS) instilled rats served as additional controls. Following exposures, the lungs of PBS and particle-exposed rats were evaluated for bronchoalveolar lavage (BAL) fluid inflammatory markers, cell proliferation, and by histopathology at post-instillation time points of 24 hrs, 1 week, 1 month and 3 months.The bronchoalveolar lavage results demonstrated that lung exposures to quartz particles, at both concentrations but particularly at the higher dose, produced significant increases vs. controls in pulmonary inflammation and cytotoxicity indices. Exposures to Pigment A or R-100 TiO2 particles produced transient inflammatory and cell injury effects at 24 hours postexposure (pe), but these effects were not sustained when compared to quartz-related effects. Exposures to carbonyl iron particles or PBS resulted only in minor, short-term and reversible lung inflammation, likely related to the effects of the instillation procedure.Histopathological analyses of lung tissues revealed that pulmonary exposures to Pigment A TiO2 particles produced minor inflammation at 24 hours postexposure and these effects were not significantly different from exposures to R-100 or carbonyl iron particles. Pigment A-exposed lung tissue sections appeared normal at 1 and 3 months postexposure. In contrast, pulmonary exposures to quartz particles in rats produced a dose-dependent lung inflammatory response characterized by neutrophils and foamy (lipid-containing) alveolar macrophage accumulation as well as evidence of early lung tissue thickening consistent with the development of pulmonary fibrosis.Based on our results, we conclude the following: 1) Pulmonary instillation exposures to Pigment A TiO2 particles at 5 mg/kg produced a transient lung inflammatory response which was not different from the lung response to R-100 TiO2 particles or carbonyl iron particles; 2) the response to Pigment A was substantially less active in terms of inflammation, cytotoxicity, and fibrogenic effects than the positive control particle-type, quartz particles. Thus, based on the findings of this study, we would expect that inhaled Pigment A TiO2 particles would have a low risk potential for producing adverse pulmonary health effects.

Four-week inhalation toxicity study in rats with nylon respirable fibers: rapid lung clearance.

This inhalation toxicity study in rats was conducted to assess the hazard potential for workers inhaling Nylon respirable fibers. Groups of 48 male rats each were exposed, nose-only, 6h per day, 5 days per week, for 4 weeks to aerosols of uncoated, finish-free Nylon respirable-sized, fiber-shaped particulates (RFP) at concentrations of 0, 4, 15 and 57 fibers (f)/cm3. Nylon RFPs were prepared using flock rotary cutters followed by vigorous opening procedures. After exposures, the lungs of sham and Nylon-exposed rats were assessed at 1 and 8 days, and 1, 3, 6 and 12 months postexposure. The results showed that the retained mean lung burdens at 1 day postexposure were 1.75E+07 RFP/lung (high level). Mean lengths and diameters of the Nylon aerosol were 9.8 and 1.6 microm, respectively. Lung clearance of Nylon RFPs was rapid over the 12-month period. There were no significant increases in lung weights, indications of pulmonary inflammation, or alveolar macrophage functional deficits in Nylon-exposed animals versus controls based on cell differentials, bronchoalveolar lavage (BAL) fluid analyses, and macrophage phagocytosis or chemotaxis activity. Histopathology revealed no adverse lower pulmonary or upper respiratory effects. In summary, the no-observed-effect level (NOEL) for inhaled Nylon RFP was 57f/cm3 (20mg/m3), the highest concentration tested.

Pulmonary toxicity screening studies in male rats with M5 respirable fibers and particulates

M5 fiber is a high-strength, high-performance organic fiber type that is a rigid rod material and composed of heterocyclic polymer fibers of type PIPD. The aim of this study was to evaluate the acute lung toxicity of intratracheally instilled M5 respirable fibers and particulates in rats. Using a pulmonary bioassay and bridging methodology, the acute lung toxicity of intratracheally instilled M5 particulates and that of its fibers were compared with a positive control particle type, quartz, as well as a negative control particle type, carbonyl iron particles. Moreover, the results of these instillation studies were bridged with data previously generated from inhalation studies with quartz and carbonyl iron particles, using the quartz and iron particles as the inhalation/instillation bridge material. For the bioassay experimental design, in the bronchoalveolar lavage studies, the lungs of rats were intratracheally instilled with 0.5 or 0.75 mg/kg of M5 particulate or 1 or 5 mg/kg of the following control or particle types: (1) M5 long fiber preparation, (2) silica–quartz particles, and (3) carbonyl iron particles. Phosphate-buffered saline (PBS)-instilled rats served as additional controls. Following exposures, the lungs of PBS and particle-exposed rats were assessed using bronchoalveolar lavage (BAL) fluid biomarkers, cell proliferation methods, and histopathological evaluation of lung tissue at 24 h, 1 wk, 1 mo and 3 mo post instillation exposure. The bronchoalveolar lavage results demonstrated that lung exposures to quartz particles, at both concentrations but particularly at the higher dose, produced significant increases vs. controls in pulmonary inflammation and cytotoxicity indices. Exposures to M5 particulate and M5 long fiber preparation produced transient inflammatory and cell injury effects at 24 h postexposure (pe) as well as at 24 h and 1 wk pe, respectively, but these effects were not sustained when compared to quartz-silica effects. Exposures to carbonyl iron particles and PBS resulted in only minor short-term and reversible lung inflammation, likely related to the effects of the instillation procedure. Histopathological analyses of lung tissues revealed that pulmonary exposures to M5 particulate and in particular, the M5 long fiber preparation in rats produced some inflammatory responses, observed up to 1 wk postexposure. These responses were often associated with the presence of M5 long fiber in the airways or in the proximal alveolar regions but appeared to be reversible at 1 and 3 mo postexposure. In contrast, pulmonary exposures to silica–quartz particles in rats produced a dose-dependent lung inflammatory response characterized by neutrophils and foamy (lipid-containing) alveolar macrophage accumulation and evidence of early lung tissue thickening consistent with the development of pulmonary fibrosis. Based on our results, we conclude the following: (1) It was very difficult to produce M5 fibers into a respirable fibrous form; these findings suggest that aerosol exposure concentrations of respirable fibrous M5 in the workplace are likely to be rather low. (2) The particulate and long fiber preparations of M5 that were tested produced a moderate amount of pulmonary inflammatory activity, more active than our negative control, carbonyl iron particles, but substantially less active in terms of inflammation, cytotoxicity, and fibrogenic effects than the positive control particle type, silica–quartz particles. Thus, based on the results of this study, we would expect that inhaled M5 respirable fibers have a low risk potential for producing adverse pulmonary effects.

Assessing the role of neutrophil apoptosis in the resolution of particle-induced pulmonary inflammation

Following inflammatory-cell recruitment in the lung, neutrophil apoptosis and subsequent engulfment by macrophages are regarded as important components in the resolution of pulmonary inflammation. The goal of this study was to further investigate the role of apoptosis and its influence, if any, on the pulmonary inflammatory process following exposures to the following particulate types: amorphous (AMO) or crystalline silica (Si), lipopolysaccharide (LPS), or pigment-grade titanium dioxide (TiO 2) . Rats were intratracheally instilled either with TiO 2, AMO, or Si particles at doses of 1 or 5 mg/kg or 6 µg LPS. Following exposures, bronchoalveolar lavage fluids and lung tissues were collected and evaluated at 12, 24, 48, or 168 h (i.e., 1 wk). At the 1 mg/kg dose, AMO instillation produced the highest pulmonary inflammatory response, concomitant with a rise in apoptotic cells that mirrored temporally the transient nature of the inflammatory response. At 5 mg/kg, amorphous silica and crystalline silica particles induced substantial pulmonary inflammation [~50-60% neutrophils (PMNs)] at 12 h postexposure (pe). A fundamental difference between the two inflammatory patterns, however, was the subsequent reversibility of inflammation in the AMO-exposed rats at 168 h postexposure and the sustained inflammatory effect in the Si-exposed animals measured through 168 h pe (~40% PMNs). Pulmonary apoptotic responses in AMO-exposed rats mirrored temporally and correlated with the time-course reduction of inflammatory responses, leading to resolution. In the Si-exposed rats, apoptotic levels remained elevated, concomitant with sustained inflammation measured through 168 h pe. High doses of TiO 2 particles produced transient lung inflammation, but with low levels of apoptosis. In addition, instillation of LPS produced a transient inflammatory response which mirrored the time course of apoptosis levels and was resolved by 168 h pe. cDNA microarray methods demonstrated that gene expression was altered for several apoptosis-related genes in AMO-, Si-, and LPS-exposed animals at 24 h pe. The results of these studies demonstrate that, following exposures, the resolution of lung inflammation correlated temporally with apoptotic levels of neutrophils in AMO- and LPS-exposed rats. Alternatively, instillation of crystalline silica resulted in sustained pulmonary inflammation and measurable apoptosis at 1 wk postexposure, but the apoptotic cell processes were not effective in resolving the inflammatory response. The findings suggest that the coordination between the resolution of inflammation and inflammatory cell apoptosis in the lung is dependent on the particle-type and that other factors, such as particle cytotoxicity, may also be important.

Biodegradability of inhaled p-aramid respirable fiber-shaped particulates (RFP): mechanisms of RFP shortening and evidence of reversibility of pulmonary lesions

These studies elucidated mechanisms of inhaled p-aramid respirable fiber-shaped particulates (RFP) biodegradation in the lungs of exposed rats and hamsters. We postulate that lung fluids coat/activate inhaled p-aramid RFP which deposits in the lung and promote enzymatic attack and consequent shortening. p-Aramid or cellulose (biopersistent control) RFP were instilled into the lungs of rats and the lungs digested 24 h later using two different (KOH or enzymatic) digestion techniques. In vivo, the enzyme but not the KOH solution produced shortening of p-aramid but not cellulose RFP recovered from the lungs. For in vitro studies, the two RFP-types were incubated with BAL fluids and underwent simulated digestions; also rat lung epithelial cells, macrophages or co-cultures were incubated with p-aramid and digested at 1, 24, or 168 h postexposure. The results of in vitro acellular studies demonstrated that only p-aramid RFP incubated in BAL fluids and digested by the enzyme method were shortened. In vitro cellular studies demonstrated a shortening of p-aramid RFP in macrophages and co-cultures but not in lung epithelial cells at 24 h and 1 week postexposure. These results demonstrate that lung fluids coat and catalyze the p-aramid RFP as a prelude for shortening and describe a likely mechanism for the biodegradability of inhaled p-aramid RFP in the lungs of exposed animals.

TIME COURSE OF EOSINOPHILIC RECRUITMENT AND PULMONARY TOXICITY BIOMARKERS IN AN ALLERGIC ASTHMA MODEL IN BROWN NORWAY RATS

Allergic asthma is a pulmonary disease characterized by antigen-induced pulmonary eosinophilia, airway hyperresponsiveness, antigen-specific IgE antibody responses, and broncho-constriction. In attempting to elucidate mechanisms associated with the pathogenesis of this disease, a number of animal models have been developed. The current studies were undertaken to develop a model of allergic asthma model in Brown Norway rats. Unlike the neutrophilic inflammatory response to inhaled particles in most strains of rats, inhalation of antigens in sensitized Brown Norway rats results in a complex cellular response which is characterized by a variety of inflammatory cell types, and is dependent on the time course of inflammatory cell recruitment. In characterizing this ovalbumin-challenge model of allergic asthma, it was important to assess the time course of pulmonary inflammation, cell proliferation, and apoptosis. Male Brown Norway rats were sensitized and boosted with intraperitoneal injections of ovalbumin in aluminum hydroxide on experimental days 1 and 8. On days 15-17, rats were challenged by an inhalation exposure to 5% ovalbumin and were evaluated by bronchoalveolar lavage (BAL) at 24 or 48 h postexposure (PE). Control rats were similarly treated to ovalbumin aerosol exposures; however, these animals had been sensitized and boosted with aluminum hydroxide (minus the ovalbumin). Cell differential evaluations demonstrated that the rats exposed for 3 days/24 h postexposure and for 2 days/ 48 h postexposure produced the greatest numbers of BAL eosinophils and corresponding indicators of pulmonary toxicity. It was interesting to note that earlier exposure time periods (i.e., 1 day/24 h PE) generated a predominantly neutrophilic inflammatory response, while longer exposure/postexposure time periods (i.e., 3 days/48 h) produced a predominant mononuclear inflammatory response. Subsequent studies demonstrated that the 2-day/ 48-h protocol produced the optimum eosinophilic, cytotoxic, cell proliferative, and apoptotic response. Histopathological evaluations demonstrated a chronically active alveolitis and bronchiolitis, characterized by epithelial cell proliferation in the airways and inflammatory cell proliferation in the alveoli. Studies are ongoing to assess the cell types undergoing apoptosis in both the airway and parenchymal regions to fully characterize this model in order to assess its relevance and utility for studying asthma in humans.