Lack of marked cyto- and genotoxicity of cristobalite in devitrified (heated) alkaline earth silicate wools in short-term assays with cultured primary rat alveolar macrophages.

Alkaline earth silicate (AES) wools are low-biopersistence high-temperature insulation wools. Following prolonged periods at high temperatures they may devitrify, producing crystalline silica (CS) polymorphs, including cristobalite, classified as carcinogenic to humans. Here we investigated the cytotoxic and genotoxic significance of cristobalite present in heated AES wools. Primary rat alveolar macrophages were incubated in vitro for 2 h with 200 µg/cm² unheated/heated calcium magnesium silicate wools (CMS1, CMS2, CMS3; heat-treated for 1 week at, or 4 weeks 150 °C below, their respective classification temperatures) or magnesium silicate wool (MS; heated for 24 h at 1260 °C). Types and quantities of CS formed, and fiber size distribution and shape were determined by X-ray diffraction and electron microscopy. Lactate dehydrogenase release and alkaline and hOGG1-modified comet assays were used, ± aluminum lactate (known to quench CS effects), for cytotoxicity/genotoxicity screening. Cristobalite content of wools increased with heating temperature and duration, paralleled by decreases in fiber length and changes in fiber shape. No marked cytotoxicity, and nearly no (CMS) or only slight (MS) DNA-strand break induction was observed, compared to the CS-negative control Al2O3, whereas DQ12 as CS-positive control was highly active. Some samples induced slight oxidative DNA damage, but no biological endpoint significantly correlated with free CS, quartz, or cristobalite. In conclusion, heating of AES wools mediates changes in CS content and fiber length/shape. While changes in fiber morphology can impact biological activity, cristobalite content appears minor or of no relevance to the intrinsic toxicity of heated AES wools in short-term assays with rat alveolar macrophages.

Evaluation of the deposition, translocation and pathological response of brake dust with and without added chrysotile in comparison to crocidolite asbestos following short-term inhalation: interim results.

Chrysotile has been frequently used in the past in manufacturing brakes and continues to be used in brakes in many countries. This study was designed to provide an understanding of the biokinetics and potential toxicology following inhalation of brake dust following short term exposure in rats. The deposition, translocation and pathological response of brake dust derived from brake pads manufactured with chrysotile were evaluated in comparison to the amphibole, crocidolite asbestos. Rats were exposed by inhalation 6 h/day for 5 days to either brake dust obtained by sanding of brake-drums manufactured with chrysotile, a mixture of chrysotile and the brake dust or crocidolite asbestos. No significant pathological response was observed at any time point in either the brake dust or chrysotile/brake dust exposure groups. The long chrysotile fibers (>20 µm) cleared quickly with T(½) estimated as 30 and 33 days, respectively in the brake dust and the chrysotile/brake dust exposure groups. In contrast, the long crocidolite fibers had a T(½)>1000 days and initiated a rapid inflammatory response in the lung following exposure resulting in a 5-fold increase in fibrotic response within 91 days. These results provide support that brake dust derived from chrysotile containing brake drums would not initiate a pathological response in the lung following short term inhalation.

Pulmonary toxicity of printer toner following inhalation and intratracheal instillation.

The pulmonary effects of a finished toner were evaluated in intratracheal instillation and inhalation studies, using toners with external additives (titanium dioxide nanoparticles and amorphous silica nanoparticles). Rats received an intratracheal dose of 1 mg or 2 mg of toner and were sacrificed at 3 days, 1 week, 1 month, 3 months and 6 months. The toner induced pulmonary inflammation, as evidenced by a transient neutrophil response in the low-dose groups and persistent neutrophil infiltration in the high-dose groups. There were increased concentrations of heme oxygenase-1 (HO-1) as a marker of oxidative stress in the bronchoalveolar lavage fluid (BALF) and the lung. In a 90-day inhalation study, rats were exposed to well-dispersed toner (mean of MMAD: 3.76 µm). The three mass concentrations of toner were 1, 4 and 16 mg/m(3) for 13 weeks, and the rats were sacrificed at 6 days and 91 days after the end of the exposure period. The low and medium concentrations did not induce neutrophil infiltration in the lung of statistical significance, but the high concentration did, and, in addition, upon histopathological examination not only showed findings of inflammation but also of fibrosis in the lung. Taken together, the results of our studies suggest that toners with external additives lead to pulmonary inflammation and fibrosis at lung burdens suggest beyond the overload. The changes observed in the pulmonary responses in this inhalation study indicate that the high concentration (16 mg/m(3)) is an LOAEL and that the medium concentration (4 mg/m(3)) is an NOAEL.

Evaluation of immunohistochemical markers to detect the genotoxic mode of action of fine and ultrafine dusts in rat lungs.

Data on local genotoxicity after particle exposure are crucial to resolve mechanistic aspects such as the impact of chronic inflammation, types of DNA damage, and their role in lung carcinogenesis. We established immunohistochemical methods to quantify the DNA damage markers poly(ADP-ribose) (PAR), phosphorylated H2AX (γ-H2AX), 8-hydroxyguanosine (8-OH-dG), and 8-oxoguanine DNA glycosylase (OGG1) in paraffin-embedded tissue from particle-exposed rats. The study was based on lungs from a subchronic study that was part of an already published carcinogenicity study where rats had been intratracheally instilled with saline, quartz DQ12, amorphous silica (Aerosil(®) 150), or carbon black (Printex(®) 90) at monthly intervals for 3 months. Lung sections were stained immunohistochemically and markers were quantified in alveolar lining cells. Local genotoxicity was then correlated with already defined endpoints, i.e. mean inflammation score, bronchoalveolar lavage parameters, and carcinogenicity. Genotoxicity was most pronounced in quartz DQ12-treated rats, where all genotoxicity markers gave statistically significant positive results, indicating considerable genotoxic stress such as occurrence of DNA double-strand breaks (DSB), and oxidative damage with subsequent repair activity. Genotoxicity was less pronounced for Printex(®) 90, but significant increases in γ-H2AX- and 8-OH-dG-positive nuclei and OGG1-positive cytoplasm were nevertheless detected. In contrast, Aerosil(®) 150 significantly enhanced only 8-OH-dG-positive nuclei and oxidative damage-related repair activity (OGG1) in cytoplasm. In the present study, γ-H2AX was the most sensitive genotoxicity marker, differentiating best between the three types of particles. The mean number of 8-OH-dG-positive nuclei, however, correlated best with the mean inflammation score at the same time point. This methodological approach enables integration of local genotoxicity testing in subchronic inhalation studies and makes immunohistochemical detection, in particular of γ-H2AX and 8-hydroxyguanine, a very promising approach for local genotoxicity testing in lungs, with prognostic value for the long-term outcome of particle exposure.

Change in agglomeration status and toxicokinetic fate of various nanoparticles in vivo following lung exposure in rats.

The deposition characteristics in lungs following inhalation, the potential toxic effects induced and the toxicokinetic fate including a possible translocation to other sites of the body are predominantly determined by the agglomeration status of nanoscaled primary particles. Systemic particle effects, i.e. effects on remote organs besides the respiratory tract are considered to be of relevant impact only for de-agglomerated particles with a nanoscaled aspect. Rats were exposed to various types of nanoscaled particles, i.e. titanium dioxide, carbon black and constantan. These were dispersed in physiologically compatible media, e.g. phosphate buffer, sometimes including auxiliaries. Rats were treated with aqueous nanoparticle dispersions by intratracheal instillation or were exposed to well-characterized nanoparticle aerosols. Subsequently, alterations in the particle size distribution were studied using transmission electron microscopy (TEM) as well as the bronchoalveolar lavage (BAL) technique. Based on the results in various approaches, a tendency of nanoscaled particles to form larger size agglomerates following deposition and interaction with cells or the respiratory tract is predominant. The contrary trend, i.e. the increase of particle number due to a disintegration of agglomerates seems not to be of high relevance.

Impact of variations in the chemical composition of vitreous mineral fibers on biopersistence in rat lungs and consequences for regulation.

The chronic toxicity of vitreous fibers is substantially dependent on their biopersistence. Removal of fibers deposited in the respiratory tract is dependent on a combination of physiological clearance processes (like mechanical translocation) and physico-chemical processes like dissolution and leaching. This publication presents data of about 60 different fibers investigated in the biopersistence test which was standardized in the European Union. This test is based on in vivo investigation of biopersistence after intratracheal instillation in rats of a respirable fiber fraction, and it is a basis for the regulatory classification of vitreous fibers. Regression analysis is carried out employing the data of glass fiber compositions and the corresponding results of biopersistence tests (half-times). The study leads to a model that enables prediction of half-times for stone wool fibers as well as for glass wool fibers on the basis of their chemical composition. The aim of this paper was to investigate the stringency of the existing limits for the range of the chemical composition of glass and stone wools in view of the currently available data base. For regulatory purposes, however, this model is currently not sufficient to replace biopersistence tests completely.

Calibration study on subchronic inhalation toxicity of man-made vitreous fibers in rats.

This 3-mo inhalation study investigated the biological effects of a special-purpose glass microfiber (E-glass microfiber), the stone wool fiber MMVF21, and a new high-temperature application fiber (calcium-magnesium-silicate fiber, CMS) in Wistar rats. Rats were exposed 6 h/day, 5 days/wk for 3 mo to fiber aerosol concentrations of approximately 15, 50, and 150 fibers/ml (fiber length >20 microm) for E-glass microfiber and MMVF21. For the CMS fiber only the highest exposure concentration was used. During a 3-mo postexposure period, recovery effects were studied. In the highest exposure concentration groups, gravimetric concentrations were 17.2 mg/m3 for E-glass microfiber, 37 mg/m3 for MMVF21, and 49.5 mg/m3 for the CMS fiber. After 3 mo of exposure, lung retention of fibers longer than 20 micro m per lung was 17 x 10(6) for E-glass microfiber, 5.7 x 10(6) for MMVF21, and 0.88 x 10(6) for CMS. After 3 mo of recovery the concentration of the long fiber fraction was decreased to 38.4%, 63.9%, and 3.0% compared to original lung burden for the E-glass microfiber, MMVF21, and CMS, respectively. Biological effects measured included inflammatory and proliferative potential, histopathology lesions, and the persistence of these effects over a recovery period of 3 mo. Generally, observed effects were higher for E-glass microfiber when compared to MMVF21. The following clear dose-dependent effects on E-glass microfiber and MMVF21 exposure were observed as main findings of the study: increase in lung weight, in measured biochemical parameters and polymorphonuclear leukocytes (PMN) in the bronchoalveolar lavage fluid (BALF), in cell proliferation (BrdU-response) of terminal bronchiolar epithelium, and in interstitial fibrosis. The values observed in the proliferation assay on the carcinogenic E-glass microfiber indicate that this assay has an important predictive value with regards to potential carcinogenicity. Surprisingly, for the biosoluble CMS fiber, fibrogenic potential was detected in this study. The results of the CMS exposure group indicate that effects may be dominated by the presence of nonfibrous particles and that fibrosis may not be a predictor of carcinogenic activity of fiber samples, if the fiber preparation contains a significant fraction of nonfibrous particles. In summary, this study demonstrates the importance of fiber dust contamination by granular components. For future subchronic studies a longer posttreatment observation period would be advisable.