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The development of accurate in vitro screening assays to assess lung hazard potential of nanomaterials is a highly desirable goal. However, some studies have noted little correlation between in vitro and in vivo results. Moreover, a recent National Academy of Sciences report predicts that future hazard testing will be conducted primarily using cell culture assays. The three major objectives of this study were to compare lung toxicity impacts of nanoscale (NZnO) vs fine zinc oxide (FZnO) particulates, assess predictability of in vitro cell culture systems, and compare effects of instillation vs inhalation exposures in rats. Physicochemical aspects of ZnO particle types were rigorously characterized and did not agree with specifications provided by the supplier; i.e., the ZnO particle types were closer in size than advertised. Rats were exposed in vivo either by intratracheal instillation to 1 or 5 mg/kg of nanoscale or fine size zinc oxide particle types or by inhalation to aerosols of 25 or 50 mg/m³ for 1 or 3 h. Lung inflammation, cytotoxicity, and histopathological endpoints were assessed at several time points postexposure. Three different in vitro culture conditions were utilized. Cultures of (1) rat lung epithelial cells, (2) primary alveolar macrophages, and (3) alveolar macrophages-L2 lung epithelial cell cocultures were incubated with fine or nano ZnO particles and evaluated for cytotoxicity biomarkers (LDH) and proinflammatory cytokines (MIP-2 and TNF-α). In vivo exposures to instilled or inhaled fine or nanoscale ZnO produced "metal fume fever" responses, characterized by transient, short-term lung inflammatory or cytotoxic responses. Alternatively, in vitro exposures to fine or nanoscale ZnO particles produced minor cytotoxic responses at 4 and 24 h, only in cocultures and at the highest (particle overload) dose with little detectable proinflammatory cytokine generation (MIP-2, and TNF-α). To summarize, the comparisons of in vivo and in vitro toxicity measurements following nano or fine ZnO particle exposures demonstrated little convergence and few differences in potency. [ABSTRACT FROM AUTHOR]

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]

Genotoxicity evaluations have been proposed as regulatory requirements for establishing German MAK values for inhaled fibrous dusts. The objective of this in vitro assay was to assess the potential for para-aramid (p-aramid) respirable-sized, fiber-shaped particulates (RFP) to induce chromosomal aberrations in cultured human peripheral blood lymphocytes without metabolic activation. The highest concentration tested in this assay was limited by the physical characteristics of p-aramid RFP. The test substance was suspended in fully supplemented RPMI culture medium with 1% Pluronic F68. All dosing was achieved using a dosing volume of 90% (900 µl/ml), and the vehicle control cultures were treated with 900 µl/ml of fully supplemented RPMI culture medium with 1% Pluronic F68. In the chromosomal aberrations assay, the treatments were either 3 or 19 h without metabolic activation. Cultures were harvested 22 h from the initiation of treatment. Replicated cultures of human whole blood lymphocytes were incubated with p-aramid RFP concentrations of 6.30, 12.6, 25.2, 50.4, 101, 201, and 401 µg/ml. Cultures treated with concentrations to 50.4 µg/ml for 3 h and 6.30, 12.6, 25.2, and 201 µg/ml for 19 h were analyzed for structural and numerical chromosomal aberrations. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed. The results demonstrated that p-aramid RFP was negative for inducing chromosomal aberrations in cultured human peripheral blood lymphocytes without metabolic activation. In addition, we conclude that the utility of these tests for evaluating the genotoxicity of fibrous or particulate materials is questionable. [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.

Carbon fibres (CF) are synthetic fibres which are used commercially for their thermal resistance, excellent electrical conductivity, flexibility and high tensile strength. The dimensions of most of the carbon fibre types currently available fall outside the respirable range, having nominal diameters from 5 to 10 μm. The pitch-based experimental fibres used in this study (1–4 fim diameter) have the potential to cause toxic pulmonary effects. Groups of male Crl:CD® BR rats were exposed to aerosols of pitch or polyacrylonitrile (PAN)-based carbon fibres at target concentrations of 50 or 100 mgm−3 for periods ranging from 1 to 5 days. Following the exposure period, cells and fluids from sham and carbon fibre-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 post-exposure. Alveolar macrophages were evaluated for viability and functional capacity. The lungs of additional exposed animals were processed for histopathological analysis. A 5-day exposure to respirable pitch carbon fibres at design concentrations of 50 and 120 mg m−3 (fibre concentrations at 47 and 62 fibres cm−3 of air) produced dose-dependent, transient inflammatory responses in the lungs of exposed rats, manifested by increased numbers of neutrophils. In addition, significant increases in LDH, protein or alkaline phosphatase levels were measured in BAL fluids at early post-exposure time periods but were reversible within 10 days after exposure. No significant differences were measured in the morphology or in vitro phagocytic capacities of macrophages recovered from sham or pitch-based carbon fibre-exposed rats. Results from cell labelling studies in rats exposed to pitch-based carbon fibres for 5 days demonstrated an increased turnover of lung parenchymal cells at 10 days or 1 month after exposure but no increases in cell turnover were measured in the airways at any time post-exposure. The lungs of additional exposed animals were processed for histopathology at 1 or 3 months after a 5-day exposure. Pigment-laden alveolar macrophages as well as minimal Type II epithelial cell hyperplasia was observed primarily at the junctions of terminal bronchioles and alveolar ducts. An additional group of rats was exposed for 1 day (i.e. 6 h) to a sample of polyacrilonitrile (PAN)-based carbon fibres (fibre diameter = 4.4 μm; MMAD = 9 μm). These materials were used as negative controls since the physical dimensions of these fibres fell outside the respirable range. Exposures to the PAN-based fibres produced no cellular, cytotoxic or alveolar-capillary membrane permeability changes at any time post-exposure. The results of this study demonstrate that the pulmonary toxicity of respirable, pitch-based carbon fibres is substantially different from well-known fibrogenic agents such as crystalline silica and asbestos. [ABSTRACT FROM PUBLISHER]

Results from recent studies indicate that anatomical regional differences in inhaled asbestos fibre burdens are inversely related to the pathlengths of intrapulmonary airways of rats, i.e. increased numbers of fibres were retained in cranial regions containing the shortest pathlengths from the main bronchus while fewer fibres were retained in caudal regions at the base of the same lobe. It was not established, however, whether the increased fibre retention was due to enhanced deposition or reduced clearance of inhaled fibres. Thus, the present study was undertaken to test the hypothesis that enhanced particle deposition occurs at cranial regions with the shortest pathlengths. Male Long- Evans rats were exposed to aerosols of carbonyl iron (CI) particles for 1 or 6 h at a concentration of 107 mg m−3. Immediately following exposures, exposed rats were vascularly-fixed for particle deposition studies. Subsequently, the left lobes of all rats were microdissected along the long axes for a minimum of five airway generations; the tissue blocks were then dried and further dissected to the level of the broncho-alveolar junctions. Particle deposition was quantified by scanning electron microscopy. Our data indicated that increased numbers of particles deposited in cranial regions (P <0.05 compared to caudal or costolateral), and this correlated with enhanced alveolar macrophage chemotactic responses. Our results suggest that airway pathlengths influence regional deposition patterns for inhaled particulate materials. [ABSTRACT FROM PUBLISHER]

Interspecies differences in alveolar macrophage (AM) function in vitro may account for differences in responses to inhaled particles in vivo. The current studies were undertaken to compare pulmonary clearance and inflammatory responses in rodents exposed to either carbonyl iron (CI) or silica particles. In the first set of studies, in vitro and in vivo pulmonary clearance responses were evaluated using two strains of rats and one strain each of mouse, hamster and guinea pig. in vitro studies showed that hamster AM had the highest phagocytic activity and that rat AM migrated best to complement-dependent chemotactic factors. Subsequently, groups of animals from each strain were exposed to aerosols of CI for 1 or 6 h at identical concentrations (100 mg m−3). Particle deposition patterns in the distal lung were nearly identical for all species, although increased numbers of CI particles were deposited on alveolar duct bifurcations of either rats or mice compared to hamsters and guinea pigs. Time-course studies showed that enhanced numbers of rat AM migrated to deposition sites and phagocytized particles, and this correlated with increased percentages of phagocytic macrophages recovered by lavage (P < 0.01). ACl-induced PMN inflammatory response was observed only in the mouse and this correlated with the lowest AM clearance rates in vivo and phagocytic rates in vitro. It was concluded from these studies that the rat model may be the most efficient rodent species for clearing inhaled iron particles. In another set of studies the responses of rats and mice to inhaled carbonyl iron or silica particles were evaluated. Three strains of mice and two strains of rats were exposed acutely to CI particles at identical aerosol concentrations. Two of the mouse strains developed transient lung inflammatory responses while CDR and Long-Evans rats did not. Moreover, AM chemotaxis and phagocytosis in rats were significantly increased compared to the mouse strains (P < 0.05). In another experiment, a single exposure to silica particles produced sustained inflammation in the lungs of rats, characterized by neutrophil emigration, and increased levels of extracellular enzymes and protein. In contrast, silica produced only a transient inflammatory response in the lungs of both strains of mice and this finding could not be accounted for by enhanced AM clearance of silica in mice. Our results demonstrate that acute pulmonary responses to the two inhaled particles were different in rats and mice. Whether either species simulates the human response remains to be determined. [ABSTRACT FROM PUBLISHER]

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]

Methods development studies were conducted to develop standardized techniques for quantifying respirable organic fiber aerosol exposures in the workplace. The aim of this study was to compare the light microscopy results of aerosolized organic respirable-sized, fiber-shaped particulate (RFP) counts from three different laboratories and from four different individual counters (two in one laboratory). p-Aramid was utilized in these studies as a representative organic fiber-type. Atmospheres of aerosolized p-aramid RFPs were generated in an inhalation chamber. Fifteen randomly distributed methylcellulose filters were exposed to a p-aramid aerosol for 5 min at estimated concentrations of 20-30 f/ml. Following completion of exposures, filters were prepared for phase-contrast optical microscopy counting using standard preparation methods. The prepared slides containing a portion of the fiber-exposed filters were first counted at DuPont Haskell Laboratory in the USA, and then the same slides were sent to the Denkendorf Institute in Stuttgart, Germany and finally to the Institute of Occupational Medicine, Edinburgh, Scotland, UK. For quantification of fiber counts, the NIOSH 7400 fiber counting method was used at DuPont Haskell, while a WHO/EURO MMF fiber counting method was utilized in the European laboratories. The results demonstrated that the fiber counts from one laboratory (designated here as Lab 1) had consistently lower counts when compared to another laboratory (i.e. Lab 2) (mean values for the 15 filters = 18.4 ± 4.3 versus 27.7 ± 4.3 f/ml). A third laboratory (Lab 3), with two different counters, was frequently intermediate between the counts of Labs 1 and 2 (24.2 ± 1.1 and 22.1 ± 2.2 f/ml). The differences in fiber counts may be related either to the minor differences in counting rules between the US and European methods and/or to variability among counters. With few exceptions, the intralaboratory variability between counts was lower than the inter-laboratory variability among counts. Studies are ongoing to better assess the expected variability for aerosolized organic RFP counts when comparing the results from one laboratory to another and one method to another (NIOSH versus WHO/EURO MMF). [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]

In the first half of the twentieth century epidemiologic evidence linked elevated incidences of pulmonary fibrosis and cancer with inhalation of chrysotile and crocidolite asbestos, a family of naturally occurring inorganic fibrous materials. As the serpentine and amphibole forms of asbestos were phased out, synthetic vitreous fibers (SVFs; fiber glass, mineral wool, and refractory fiber) became increasingly utilized, and concerns were raised that they too might cause adverse health effects. Extensive toxicological research on SVFs has demonstrated that their pulmonary effects are directly related to fiber dose in the lung over time. This is the result of deposition (thin fibers deposit in the lower lung more efficiently than thick fibers) and lung-persistence ('biopersistence' is directly related to fiber length and inversely related to dissolution and fragmentation rates). In rat inhalation studies, asbestos was determined to be 7- to 10-fold more biopersistent in the lung than SVFs. Other than its effect on biopersistence, fiber composition did not appear to play a direct role in thebiological activity of SVFs. Recently, the utilization of man-made organic fibers (MMOFs) (also referred to by some as synthetic organic fibers) has increased rapidly for a variety of applications. In contrast to SVFs, research on the potential pulmonary effects of MMOFs is relatively limited, because traditionally MMOFs were manufactured in diameters too thick to be respirable (inhalable into the lower lung).However, new developments in the MMOF industry have resulted in the production of increasingly fine-diameter fibers for special applications, and certain post-manufacturing processes (e.g., chopping) generate respirable-sized MMOF dust. Until the mid-1990s, there was no consistent evidence of human health affects attributed to occupational exposure to MMOFs. [ABSTRACT FROM AUTHOR]