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The article discusses a study to evaluate the acute lung toxicity in rats of intratracheally instilled TiO2 particles that were substantially encapsulated with pyrogenically deposited, amorphous silica. It aims to determine if Pigment A TiO2 particles impart significant toxicity in the lungs of rats and how its activity compares with other reference particulate materials. It suggests that the inhalation of Pigment A TiO2 particles have a low risk potential for producing detrimental pulmonary health effects.

Titanium dioxide (TiO2) is manufactured worldwide in large quantities for use in a wide range of applications and is normally considered to be toxicologically inert. Findings of tumours in the lungs of rats exposed chronically to high concentrations of TiO2, but not in similarly exposed mice or hamsters, suggest that the tumorigenic response may be a rat-specific phenomenon but nonetheless raises concerns for potential human health effects. With the limited toxicological understanding of species differences in response to inhaled TiO2 and a similarly limited amount of epidemiological information with respect to TiO2 exposure in the workplace, a consortium of TiO2 manufacturers in Europe (under the European Chemistry Industry Council; CEFIC) and in North America (under the American Chemistry Council; ACC) initiated a programme of research to investigate inter-species differences as a result of exposure to TiO2 and to conduct detailed epidemiological surveys of the major manufacturing sites. The toxicology studies exposed rats, mice and hamsters to pigment-grade TiO2 (PG-TiO2, 0, 10, 50 and 250 mg m-3) or ultrafine TiO2 (UF-TiO2, 0, 0.5, 2 and 10 mg m-3) for 90 days and the lung burdens and tissue responses were evaluated at the end of the exposure period and for up to 1 year after exposure. Results demonstrated clear species differences. Rats and mice had similar lung burdens and clearance rates while hamsters showed high clearance rates. At high lung particle burdens, rats showed a marked progression of histopathological lesions throughout the post-exposure period while mice and hamsters showed minimal initial lesions with recovery apparent during the post-exposure period. Lung neutrophil responses, a sensitive marker of inflammatory changes, reflected the development or recovery of the histopathological lesions. The use of surface area rather than gravimetric lung burden provided closer correlates of the burden to the biological effect across both TiO2 types. The epidemiological investigations evaluated the mortality statistics at 11 European and 4 US TiO2 manufacturing plants. They concluded that there was no suggestion of any carcinogenic effect associated with workplace exposure to TiO2. [ABSTRACT FROM AUTHOR]

The article presents a study that compares the acute pulmonary responses inhaled silica in mouse strains using Min-U-Sil crystalline silica particles. It notes the cells and fluid from sham and silica-exposed animals that were recovered bronchoalveolar lavage (BAL) which also measures granulocytes, lactate dehydrogenase (LDH) and N-acetylglucomsaminidase (NAG). The study revealed that a five-day silica exposure produced a low-level inflammatory response.

The article presents a study which investigates the role of inhaled overload particle concentrations on high particle burden and inflammation of alveolar macrophage (AM). Male rats were exposed to TiO2 or CI particles and the lungs of sham and dust-exposed animals were lavaged in the study. The development of a persistent pulmonary inflammatory response and high TiO2 was determined in the study by neutrophils recovered in bronchoalveolar lavage fluids from exposed rats.

The article presents a study which investigates the mechanisms of particle-induced pulmonary inflammation in rats after exposures to crystalline and amorphous silica, or titanium dioxide particles. Crystalline and amorphous silica, or titanium chemotaxis were intratracheally instilled in groups of male rats and bronchoalveolar lavage (BAL) as well as chemotaxis assay were performed in the study. Results in the study reveal that all three dusts induced neutrophilic inflammation.

The article compares the pulmonary effects of size-separated p-aramid and chrysotile asbestos inhalation exposure in rats. It also examines the effect of p-aramid inhalation in rats and in hamsters after exposures to aerosolised, size-separated fibrils. Moreover, it introduces a new term in characterizing para-fibrils.

The article presents a study which examines the reversibility of fibrotic lesions in rats with exposure and inhalation of chrysotile asbestos fibres. The researchers have evaluated the tissue changes during short-term exposure of rat lung models to aerosolized chrysotile asbestos fibres. The study addresses the issue on initial changes detected early or on recovery from continued exposure to the hazardous asbetos fibres.

The aim of these studies is to develop a respiratory allergy model in Brown Norway (BN) rats using ovalbumin (Ova) as the antigenic stimulus. Specifically, we are interested in studying the mechanisms underlying the development of pulmonary T helper type 2 (Th2) responses following Ova sensitization and inhalation challenge. We have previously reported on the development of an active pulmonary inflammatory response characterized by the presence of eosinophils and measurement of serum immunoglobulin E levels in Ova-sensitized but not in alum-treated, Ova-challenged or sham control rats. In the current study, we have used cDNA microarray technology to assess the gene expression in tracheobronchial, mesenteric and submaxillary lymph nodes, as well as in pulmonary cells and tissues of Ova-sensitized, alum-sensitized or sham control BN rats. Preliminary cDNA microarray results of lung tissue from BN rats exposed to ova-ova (Ova-sensitized, Ova-challenged) 4 or 24 h after ova challenge did not show up-regulation (relative to sham controls) of mRNA for interleukin-4 (IL-4) or IL-5 but did show up-regulation of eotaxin (4 h), intercellular adhesion molecules (ICAM) and vascular cell adhesion molecules (VCAMs), which are likely to be prerequisites for eosinophilic pulmonary inflammatory responses measured at 24 and 48 h post Ova inhalation exposure. In addition, reverse transcriptase-polymerase chain reaction analysis of lung tissue from BN rats exposed to ova-ova 4 and 24 h after challenge demonstrated up-regulation of mRNA for IL-10 (Th2 response). Studies are ongoing to identify the time course and anatomic location of Th2 responses related to the development of Ova-induced respiratory allergy responses in BN rats. [ABSTRACT FROM AUTHOR]

The aims of this study were to assess in rats, using a well-developed, short-term pulmonary bioassay, the acute pulmonary toxicity of intratracheally instilled triethoxyoctylsilane (OTES)-coated (i.e. hydrophobic) pigment-grade TiO2 particles relative to uncoated, hydrophilic TiO2 particle control samples and to bridge the results of these instillation studies with data previously generated from inhalation studies with uncoated, pigment-grade TiO2 particles, using these uncoated pigment-grade TiO2 particles as the inhalation/instillation bridge material. Rats were intratracheally instilled with 2 or 10 mg/kg of the following TiO2 particle types: TiO2 with OTES, TiO2 with Tween 80, or TiO2 with OTES and Tween 80. Saline-instilled rats served as controls. The lungs of sham and exposed rats were evaluated by bronchoalveolar lavage and histopathology at 24 h, 1 week, 1 month and 3 months post-exposure. The results demonstrated that only the high dose (10 mg/kg) pigment-grade TiO2 particles and those with Tween 80 produced a transient pulmonary inflammatory response, and this reached control levels within 1 month post-exposure. We conclude that the OTES coating on the pigment-grade TiO2 particle does not cause significant pulmonary toxicity. [ABSTRACT FROM AUTHOR]

Four groups of 48 male rats each were exposed, nose-only, 6 h/day, 5 days/week, for a total of 4 weeks (i.e. 20 exposures) to aerosols of Nylon respirable-sized, fiber-shaped particulates (RFPs) at concentrations of 0, 4, 15 and 57 f/cm³ (ratio of RFPs:particles = 1:10-20). The samples containing Nylon RFPs were prepared using flock rotary cutters followed by vigorous opening procedures. Following 4 week exposures, the lungs of sham and Nylon-exposed rats were evaluated at 1 day, 1 week, as well as 1, 3, 6 and 12 months after exposure. At each of the post-exposure time points, the lungs of rats were either lavaged (for 3 months after exposure) or infusion fixed for cell proliferation, histopathology and fiber clearance/retention studies. This report contains data obtained during a 6 month post-exposure period. Preliminary results show that the retained mean lung burdens at 1 day after exposure were 1.75 × 107 (high level), 3.4 × 106 (mid level) and 4.8 × 105 (low level) RFPs/lung. Mean lengths and diameters of the Nylon aerosol were 9.8 and 1.6 µm, respectively. There were no significant increases in lung weights or indications of pulmonary inflammation in Nylon-exposed animals versus controls--based on cell differentials, bronchoalveolar lavage (BAL) fluid analyses and chemotaxis activity. More than 90% of the alveolar macrophages recovered by BAL (high level) contained Nylon RFPs or particles within their cytoplasm. Interim histopathological analyses have thus far revealed no adverse lower pulmonary or upper respiratory effects. [ABSTRACT FROM AUTHOR]

These studies were designed to investigate mechanisms through which inhaled p-aramid respirable- sized fiber-shaped particulates (RFP) are biodegraded in the lungs of exposed rats and hamsters. We have postulated that lung fluids coat/activate inhaled p-aramid RFP which deposit in the lung and promote enzymatic attack and consequent shortening. Aliquots of 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 methods. For in vitro studies the two RFP types were incubated with lavage fluid and processed via simulated digestion; in addition, rat lung epithelial cells, macrophages or co-cultures were incubated with p-aramid and digested 1, 24 or 168 h post-exposure. In vivo the enzyme but not the KOH digestion method resulted in shortening of p-aramid but not cellulose RFP recovered from rat lungs. The results of in vitro studies showed that mean lengths of p-aramid RFP incubated with saline and processed by either digestion method were not found to be altered. Indeed, only the preparation of p-aramid RFP that had been incubated with BAL fluid and processed with the enzyme solution resulted in cleavage of p-aramid RFP. In contrast to the in vitro acellular studies with p-aramid RFP, the combination of BAL fluid incubation and enzyme digestion method had no measurable effect on shortening of cellulose RFP, indicating that the results with p-aramid were specific. In vitro cellular studies demonstrated a shortening of p-aramid RFP in exposed macrophages and co-cultures but not in lung epithelial cells 24 h and 1 week post-exposure. These findings demonstrate that lung fluids coat and activate the p-aramid RFP as a prerequisite for cleavage and describe a likely mechanism for the biodegradability of inhaled p-aramid RFP in the lungs of exposed animals. [ABSTRACT FROM AUTHOR]

The aims of this study were to assess in rats, using a well-developed, short-term pulmonary bioassay, the acute pulmonary toxicity of intratracheally instilled triethoxyoctylsilane (OTES)-coated (i.e. hydrophobic) pigment-grade TiO2 particles relative to uncoated, hydrophilic TiO2 particle control samples and to bridge the results of these instillation studies with data previously generated from inhalation studies with uncoated, pigment-grade TiO2 particles, using these uncoated pigment-grade TiO2 particles as the inhalation/instillation bridge material. Rats were intratracheally instilled with 2 or 10 mg/kg of the following TiO2 particle types: TiO2 with OTES, TiO2 with Tween 80, or TiO2 with OTES and Tween 80. Saline-instilled rats served as controls. The lungs of sham and exposed rats were evaluated by bronchoalveolar lavage and histopathology at 24 h, 1 week, 1 month and 3 months post-exposure. The results demonstrated that only the high dose (10 mg/kg) pigment-grade TiO2 particles and those with Tween 80 produced a transient pulmonary inflammatory response, and this reached control levels within 1 month post-exposure. We conclude that the OTES coating on the pigment-grade TiO2 particle does not cause significant pulmonary toxicity. [ABSTRACT FROM AUTHOR]

Data generated using standardized testing protocols for toxicity studies generally provide reproducible and reliable results for establishing safe levels and formulating risk assessments. The findings of three OECD guideline-type oral toxicity studies of different duration in rats are summarized in this publication; each study evaluated different titanium dioxide (TiO2) particles of varying sizes and surface coatings. Moreover, each study finding demonstrated an absence of any TiO2 -related hazards. To briefly summarize the findings: 1) In a subchronic 90-day study (OECD TG 408), groups of young adult male and female rats were dosed with rutile-type, surface-coated pigment-grade TiO2 test particles (d50 = 145 nm - 21% nanoparticles by particle number criteria) by oral gavage for 90 days. The no-adverse-effect level (NOAEL) for both male and female rats in this study was 1000 mg/kg bw/day, the highest dose tested. The NOAEL was determined based on a lack of TiO2 particle-related adverse effects on any in-life, clinical pathology, or anatomic/microscopic pathology parameters; 2) In a 28-day repeated-dose oral toxicity study (OECD TG 407), groups of young adult male rats were administered daily doses of two rutile-type, uncoated, pigment-grade TiO2 test particles (d50 = 173 nm by number) by daily oral gavage at a dose of 24,000 mg/kg bw/day. There were no adverse effects measured during or following the end of the exposure period; and the NOAEL was determined to be 24,000 mg/kg bw/day; 3) In an acute oral toxicity study (OECD TG 425), female rats were administered a single oral exposure of surface-treated rutile/anatase nanoscale TiO2 particles (d50 = 73 nm by number) with doses up to 5000 mg/kg and evaluated over a 14-day post-exposure period. Under the conditions of this study, the oral LD50 for the test substance was >5000 mg/kg bw. In summary, the results from these three toxicity studies - each with different TiO2 particulate-types, demonstrated an absence of adverse toxicological effects. Apart from reporting the findings of these three studies, this publication also focuses on additional critical issues associated with particle and nanotoxicology studies. First, describing the detailed methodology requirements and rigor upon which the standardized OECD 408 guideline subchronic oral toxicity studies are conducted. Moreover, an attempt is made to reconcile the complex issue of particle size distribution as it relates to measurements of nanoscale and pigment-grade TiO2 particles. Clearly this has been a confusing issue and often misrepresented in the media and the scientific literature. It is clear that the particle-size distribution for pigment-grade TiO2, contains a small ("tail") component of nanoscale particles (i.e., 21% by particle number and <1% by weight in the test material used in the 90-day study). However, this robust particle characterization finding should not be confused with mislabeling the test materials as exclusively in the nanoscale range. Moreover, based upon the findings presented herein, there appears to be no significant oral toxicity impact contributed by the nanoscale component of the TiO2 Test Material sample in the 90-day study. Finally, it seems reasonable to conclude that the study findings should be considered for read-across purposes to food-grade TiO2 particles (e.g., E171), as the physicochemical characteristics are quite similar., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.

Man-made organic fibers (MMOFs) have been manufactured for over 50 years. Until recently, there have been few concerns raised regarding the safety of organic fiber dusts. This is due, in large part, to the perception that the dimensions of most, if not all, of these products were too large to be inhaled into the distal lungs of workers, i.e., were considered to be nonrespirable. A brief review of some of the issues related to organic fiber toxicology is presented herein. Some of the organic fiber-types used in commerce are identified and some fundamental tenets of fiber toxicology are discussed. In addition, the European Union, in their recent consideration for banning chrysotile asbestos fibers, evaluated some organic fiber substitutes and compared them to the hazards of asbestos. A brief review of their conclusions is described below. Finally, the results of some recent studies assessing the mechanisms of biodegradability of para-aramid respirable-sized, fiber-shaped particulates (RFP) are presented. Para-aramid (p-aramid) RFP are the most extensively-studied respirable organic fiber-type and RFP is the new term which describes respirable-sized organic fibers (ECETOC, 1996) (1). The results of these studies provide clues regarding the mechanism(s) of p-aramid RFP shortening in the lungs of exposed animals, and may be relevant for humans.

This study describes a short term inhalation bioassay in rats to predict the potential for inhaled particles to produce chronic lung disease in humans (e.g., pulmonary fibrosis). To validate the method, rats were exposed for 6 h or 3 days to various concentrations of two reference materials: (1) a known fibrogenic material (i.e., aerosolized alpha-quartz silica particles in the form of Berkeley Min-U-Sil (Pennsylvania Glass and Sand Company, Pittsburgh, PA), or (2) carbonyl iron (CI) particles, as a negative control. Cells and fluids from groups of sham and dust exposed animals were recovered by bronchoalveolar lavage (BAL). Alkaline phosphatase, lactate dehydrogenase and protein values were measured in BAL fluids at several times postexposure. Cells were identified, counted, and evaluated for viability. The lungs of additional exposed animals were processed for histopathology. Although particle deposition patterns for the two dusts were similar, brief exposures to silica particles produced a persistent pulmonary inflammatory response characterized by neutrophil recruitment at sites of particle deposition and consistently elevated biomarkers of cytotoxicity in BAL fluids. In addition, alveolar macrophage clearance functions were impaired. Progressive histopathologic lesions were observed within 1 mo after a 3-day exposure. Light and electron microscopy of silica exposed lung tissue revealed a chronically active pulmonary inflammatory response characterized by hyperplasia of Type II alveolar epithelial cells and the infiltration of macrophages and neutrophils into alveoli and interstitial compartments. The lesions were progressive, leading to the development of a multifocal, granulomatous-type pneumonitis within 2 mo postexposure. In contrast to the observed effects of silica, 3-day exposures to CI particles produced no significant adverse biochemical or histopathological effects on pulmonary tissues. These results 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 exposure to new and untested respirable materials.

Pulmonary responses in rats were compared after short-term inhalation exposure to polymorphs of silica dust. Groups of CD rats were exposed 6 h a day for 3 d to crystalline silica or amorphous silica. Another group was exposed to Ludox colloidal silica for 6 h a day, 5 d a week for two or four weeks. Thereafter the groups were killed, and the lungs washed at several postexposure times. The crystalline silica produced persistent pulmonary inflammatory responses characterized by neutrophil recruitment and consistently elevated biomarkers of cytotoxicity in bronchoalveolar lavage fluids, and progressive histopathological lesions were observed within one month of the exposure. Amorphous silica produced a transient pulmonary inflammatory response, and Ludox elicited transient pulmonary inflammatory responses at 50 or 150 mg center dot m-3 but not at 10 mg center dot m-3. After three months most of the biochemical values of the Ludox-exposed animals had returned to the control level. These results demonstrate that crystalline silica dust is more potent in producing pulmonary toxicity when compared with amorphous or colloidal 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 x 10(6) PMNs immediately after exposure for 3 days, compared to 0.01 x 10(6) PMNs for both normal or PMN-depleted, sham-exposed rats. Bronchoalveolar lavage fluids from successfully PMN-depleted, exposed rats contained significantly fewer (0.7 x 10(6)) 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.

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.

Evaluation of the pulmonary hazards associated with exposure to fibrous materials tends to be more complicated than assessments required for particulate materials. Fibers are defined by aspect ratios and it is generally considered that physical dimensions play an important role in the pathogenesis of fiber-related lung diseases. Several digestion techniques have been used to recover fibers from exposed lung tissue for clearance studies. Because many of the digestion fluids are corrosive (e.g., bleach, KOH), it is conceivable that the dimensions of recovered fibers are modified during the tissue digestion methods to assess whether the physical dimensions of bulk samples of fibers were altered following simulated digestion processing. Aliquots of crocidolite and chrysotile asbestos, Kevlar aramid, wollastonite, polyacrylonitrile (pan)-based carbon, and glass fibers were incubated with either saline, bleach, or KOH and then filtered. Scanning electron microscopy techniques were utilized to measure the physical dimensions (i.e., lengths and diameters) of at least 160 fibers per treatment group of each fiber type. Our results showed that the lengths and diameters of glass fibers and wollastonite were altered after treatment with KOH. In addition, treatment with bleach produced a small reduction in both asbestos fiber-type diameters, and greater changes in Kevlar and wollastonite diameters and carbon fiber lengths (P less than 0.05). These results indicate that lung digestion methods should be carefully assessed for each fiber type before initiating fiber clearance studies.