Differences in the toxicity of cerium dioxide nanomaterials after inhalation can be explained by lung deposition,animal species and nanoforms

Abstract

Considerable differences in pulmonary responses have been observed in animals exposed to cerium dioxide nanoparticles via inhalation. These differences in pulmonary toxicity might be explained by differences in lung deposition, species susceptibility or physicochemical characteristics of the tested cerium dioxide nanoforms (i.e. same chemical substance, different size, shape, surface area or surface chemistry). In order to distinguish the relative importance of these different influencing factors, we performed a detailed analysis of the data from several inhalation studies with different exposure durations, species and nanoforms, namely published data on NM211 and NM212 (JRC repository), NanoAmor (commercially available) and our published and unpublished data on PROM (industry provided). Data were analyzed by comparing the observed pulmonary responses at similar external and internal dose levels. Our analyses confirm that rats are more sensitive to developing pulmonary inflammation compared to mice. The observed differences in responses do not result purely from differences in the delivered and retained doses (expressed in particle mass as well as surface area). In addition, the different nanoforms assessed showed differences in toxic potency likely due to differences in their physicochemical parameters. Primary particle and aggregate/agglomerate size distributions have a substantial impact on the deposited dose and consequently on the pulmonary response. However, in our evaluation size could not fully explain the difference observed in the analyzed studies indicating that the pulmonary response also depends on other physicochemical characteristics of the particles. It remains to be determined to what extent these findings can be generalized to other poorly soluble nanomaterials.     

Comparative pulmonary toxicity of blasting sand and five substitute abrasive blasting agents

Blasting sand is used for abrasive blasting, but its inhalation is associated with pulmonary inflammation and fibrosis. Consequently, safer substitute materials for blasting sand are needed. In a previous study from this laboratory, the comparative pulmonary toxicity of five abrasive blasting substitutes and blasting sand was reported. In this study, the pulmonary toxicity of blasting sand was compared to five additional abrasive blasting substitutes: steel grit, copper slag, nickel slag, crushed glass, and olivine. Exposed rats received by intratracheal instillation 10 mg of respirable-size particles of blasting sand or an abrasive blasting substitute, while controls were instilled with vehicle. Pulmonary inflammation, damage, and fibrosis were examined 28 d postexposure. Pulmonary inflammation was monitored by determining bronchoalveolar lavage polymorphonuclear cell counts and alveolar macrophage activation by chemiluminescence. Pulmonary damage was assessed by acellular bronchoalveolar (BAL) fluid serum albumin concentrations and lactate dehydrogenase activities. Histological examination of lung tissue samples was made to assess the severity and distribution of pulmonary fibrosis, alveolitis, and alveolar epithelial cell hypertrophy and hyperplasia. In comparison to blasting sand, olivine exposed rats had higher levels of pulmonary inflammation and damage with a similar level of fibrosis. Steel grit-exposed rats had lower levels of pulmonary inflammation and damage, and did not develop fibrosis. However, steel grit-exposed rats had a level of epithelial cell hypertrophy and hyperplasia similar to blasting sand. The other abrasive blasting substitutes gave a mixed profile of toxicity. The data demonstrate that steel grit produced less acute pulmonary toxicity than blasting sand or any of the other abrasive blasting substitutes. Notwithstanding, the data also suggest that chronic exposure to steel grit may pose a health risk due to its effects on epithelial cell proliferation in the lung.     

Pulmonary toxicity of aerosolized oil-formulated fenitrothion in rats

To evaluate the potential risk of pulmonary damage due to aerial spraying of the insecticide fenitrothion, rat lungs were examined under light and electron microscopy at 3, 7, 21, and 60 days after exposure. Rats were exposed by a "nose-only" apparatus for 1 hr to 2 or 500 mg/m3 of aerosolized fenitrothion (15%) mixed with solvent Cyclosol 63 (35%) and diluent oil 585 (50%). Aerosol size particles were monitored by a light scattering apparatus. Only minor modifications of lung alveolar tissues were observed after exposure to the high concentration. At 3 days, discrete foci of mild inflammation were detected, including interstitial edema, cellular infiltration, and increased number of alveolar macrophages. At 7 days, signs of irritation were diminished while at 21 and 60 days alveolar tissues were essentially normal. Exposure to lower concentration induced more limited changes at 3 days; no modifications were seen at later periods. It is concluded that a single exposure to this fenitrothion mixture at 500 mg/m3 presents no serious hazard of pulmonary toxicity.     

Short-term rat inhalation study with aerosols of acrylic ester-based polymer dispersions containing a fraction of nanoparticles

Aqueous polymer dispersions are important raw materials used in a variety of industrial processes. They may contain particles with diameters ranging from 10 to 1500 nm. Polymer exposure alone may cause pulmonary lesions after inhalation exposure. Polymer dispersions with increased proportions of nano-sized particles are being developed for improved material characteristics, and this may pose even increased pulmonary hazards upon potential inhalation exposure. In a 5-day screening study, male rats were nose-only exposed to aerosols generated from 2 dispersions of acrylic ester polymers with identical chemical composition but different nano-sized particle proportions at particle concentrations of 3 and 10 mg/m3. Immediately and 19 days after the end of inhalation, necropsies were conducted with major emphasis on respiratory tract histopathology. Three and 23 days after the end of inhalation, bronchoalveolar lavage was performed to screen for early pulmonary injury and inflammation. In contrast to the adverse effects known for other materials in short-term inhalation studies, none of the tested preparations of acrylic ester polymers elicited any adverse effect at the end of the inhalation or postinhalation periods. No shift in toxicity could be observed by the increased proportion of nano-sized polymer particles. Under the conditions of this study, the no observable adverse effect levels for both preparations were >10 mg/m3, that is 2- to 3-fold beyond current nuisance dust threshold limit values.     

Comparison of acute phase response in mice after inhalation and intratracheal instillation of molybdenum disulphide and tungsten particles

Inhalation studies are the gold standard for assessing the toxicity of airborne materials. They require considerable time, special equipment, and large amounts of test material. Intratracheal instillation is considered a screening and hazard assessment tool as it is simple, quick, allows control of the applied dose, and requires less test material. The particle-induced pulmonary inflammation and acute phase response in mice caused by intratracheal instillation or inhalation of molybdenum disulphide or tungsten particles were compared. End points included neutrophil numbers in bronchoalveolar lavage fluid, Saa3 mRNA levels in lung tissue and Saa1 mRNA levels in liver tissue, and SAA3 plasma protein. Acute phase response was used as a biomarker for the risk of cardiovascular disease. Intratracheal instillation of molybdenum disulphide or tungsten particles did not produce pulmonary inflammation, while molybdenum disulphide particles induced pulmonary acute phase response with both exposure methods and systemic acute phase response after intratracheal instillation. Inhalation and intratracheal instillation showed similar dose–response relationships for pulmonary and systemic acute phase response when molybdenum disulphide was expressed as dosed surface area. Both exposure methods showed similar responses for molybdenum disulphide and tungsten, suggesting that intratracheal instillation can be used for screening particle-induced acute phase response and thereby particle-induced cardiovascular disease.     

Pulmonary toxicity of indium‐tin oxide production facility particles in rats

Indium‐tin oxide (ITO) is used to make transparent conductive coatings for touch‐screen and liquid crystal display electronics. Occupational exposures to potentially toxic particles generated during ITO production have increased in recent years as the demand for consumer electronics continues to rise. Previous studies have demonstrated cytotoxicity in vitro and animal models have shown pulmonary inflammation and injury in response to various indium‐containing particles. In humans, pulmonary alveolar proteinosis (PAP) and fibrotic interstitial lung disease have been observed in ITO facility workers. However, which indium materials or specific processes in the workplace may be the most toxic to workers is unknown. Here we examined the pulmonary toxicity of three different particle samples that represent real‐life worker exposures, as they were collected at various production stages throughout an ITO facility. Indium oxide (In₂O₃), sintered ITO (SITO) and ventilation dust (VD) particles each caused pulmonary inflammation and damage in rats over a time course (1, 7 and 90 days post‐intratracheal instillation), but SITO and VD appeared to induce greater toxicity in rat lungs than In₂O₃ at a dose of 1 mg per rat. Downstream pathological changes such as PAP and fibrosis were observed in response to all three particles 90 days after treatment, with a trend towards greatest severity in animals exposed to VD when comparing animals that received the same dose. These findings may inform workplace exposure reduction efforts and provide a better understanding of the pathogenesis of an emerging occupational health issue. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.     

Comparative pulmonary toxicity of 6 abrasive blasting agents.

Inhalation of silica dust is associated with pulmonary fibrosis. Therefore, substitute abrasive materials have been suggested for use in abrasive blasting operations. To date, toxicological evaluation of most substitute abrasives has been incomplete. Therefore, the objective of this study was to compare the pulmonary toxicity of a set of substitute abrasives (garnet, staurolite, coal slag, specular hematite, and treated sand) to that of blasting sand. Rats were exposed to blasting sand or an abrasive substitute by intratracheal instillation and pulmonary responses to exposure were monitored 4 weeks postexposure. Pulmonary damage was monitored as lactate dehydrogenase (LDH) in the acellular lavage fluid. Pulmonary inflammation was evaluated from the yield of polymorphonuclear leukocytes (PMN) obtained by bronchoalveolar lavage. The activity of alveolar macrophages was determined by measuring zymosan-stimulated chemiluminescence. Blasting sand caused lung damage and showed histologic evidence for inflammation and fibrosis. Garnet, staurolite, and treated sand exhibited toxicity and inflammation that were similar to blasting sand, while coal slag caused greater pulmonary damage and inflammation than blasting sand. In contrast, specular hematite did not significantly elevate LDH or PMN levels and did not stimulate macrophage activity 4 weeks postexposure.     

A quantitative framework to group nanoscale and microscale particles by hazard potency to derive occupational exposure limits: Proof of concept evaluation

The large and rapidly growing number of engineered nanomaterials (ENMs) presents a challenge to assessing the potential occupational health risks. An initial database of 25 rodent studies including 1929 animals across various experimental designs and material types was constructed to identify materials that are similar with respect to their potency in eliciting neutrophilic pulmonary inflammation, a response relevant to workers. Doses were normalized across rodent species, strain, and sex as the estimated deposited particle mass dose per gram of lung. Doses associated with specific measures of pulmonary inflammation were estimated by modeling the continuous dose-response relationships using benchmark dose modeling. Hierarchical clustering was used to identify similar materials. The 18 nanoscale and microscale particles were classified into four potency groups, which varied by factors of approximately two to 100. Benchmark particles microscale TiO₂ and crystalline silica were in the lowest and highest potency groups, respectively. Random forest methods were used to identify the important physicochemical predictors of pulmonary toxicity, and group assignments were correctly predicted for five of six new ENMs. Proof-of-concept was demonstrated for this framework. More comprehensive data are needed for further development and validation for use in deriving categorical occupational exposure limits.     

Assessing toxicity of fine and nanoparticles: comparing in vitro measurements to in vivo pulmonary toxicity profiles.

Previous studies have reported little correlation between the relative toxicity of particle types when comparing lung toxicity rankings following in vivo instillation versus in vitro cell culture exposures. This study was designed to assess the capacity of in vitro screening studies to predict in vivo pulmonary toxicity of several fine or nanoscale particle types in rats. In the in vivo component of the study, rats were exposed by intratracheal instillation to 1 or 5 mg/kg of the following particle types: (1) carbonyl iron (CI), (2) crystalline silica (CS) (Min-U-Sil 5, alpha-quartz), (3) precipitated amorphous silica (AS), (4) nano-sized zinc oxide (NZO), or (5) fine-sized zinc oxide (FZO). Depending on particle type and solution state, these particles range in size from 90 to 500 nm in size. Following exposures, the lungs of exposed rats were lavaged and inflammation (neutrophil recruitment) and cytotoxicity end points (bronchoalveolar lavage [BAL] fluid lactate dehydrogenase [LDH] values) were measured at 24 h, 1 week, 1 and 3 months postexposure. For the in vitro component of the study, three different culture conditions were utilized. Cultures of (1) rat L2 lung epithelial cells, (2) primary alveolar macrophages (AMs) (collected via BAL from unexposed rats), as well as (3) AM-L2 lung epithelial cell cocultures were incubated with the particle types listed above, and the culture fluids were evaluated for cytotoxicity end points (LDH, 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan [MTT]) as well as inflammatory cytokines (macrophage inflammatory 2 protein [MIP-2], tumor necrosis factor alpha [TNF-alpha], and interleukin-6 [IL-6]) at one (i.e., cytokines) or several (cytotoxicity) time periods. Results of in vivo pulmonary toxicity studies demonstrated that instilled CI particles produced little toxicity. CS particles produced sustained inflammation and cytotoxicity. AS particles produced reversible and transient inflammatory responses. NZO or FZO particles produced potent but reversible inflammation which was resolved by 1 month postinstillation exposure. Results of in vitro pulmonary cytotoxicity studies demonstrated a variety of responses to the different particle types, primarily at high doses. With respect to the LDH results, L2 cells were the most sensitive and exposures to nano- or fine-sized ZnO for 4 or 24 h were more cytotoxic than exposures to CS or AS particles. Macrophages essentially were resistant and epithelial macrophage cocultures generally reflected the epithelial results at 4 and 24 h incubation, but not at 48 h incubation. MTT results were also interesting but, except for nano- and fine-sized ZnO, did not correlate well with LDH results. Results of in vitro pulmonary inflammation studies demonstrated that L2 cells did not produce MIP-2 cytokines, but CS- or AS-exposed AMs and, to a lesser degree, cocultures secreted these chemotactic factors into the culture media. Measurements of TNF-alpha in the culture media by particle-exposed cells demonstrated little activity. In addition, IL-6 secretion was measured in CS, AS, and nano-sized ZnO-exposed cocultures. When considering the range of toxicity end points to five different particle types, the comparisons of in vivo and in vitro measurements demonstrated little correlation, particularly when considering many of the variables assessed in this study-such as cell types to be utilized, culture conditions and time course of exposure, as well as measured end points. It seems clear that in vitro cellular systems will need to be further developed, standardized, and validated (relative to in vivo effects) in order to provide useful screening data on the relative toxicity of inhaled particle types.     

Pulmonary diseases caused by airborne contaminants in swine confinement buildings

Exposure to toxic gases and particles or dusts while working or living in confinement animal systems pose a pulmonary health hazard. The severity of lung impairment from exposure to such environment is investigated using intratracheal instillation, intratracheal nebulization, and inhalation procedures. Ability to deliver particles with intratracheal instillation that are evenly distributed throughout the lung depends on the material used for injection. Pulmonary histopathology reflects anatomic changes following inhalation or instillation of chemicals or particles. Endobronchial saline washings of bronchioles and alveoli allow measurement of markers of pulmonary inflammation such as total nucleated cell (leukocyte) counts and those of macrophages, neutrophils and lymphocytes; TNF-alpha, and collagen concentration are used to further evaluate pulmonary response to endotoxin or dust exposure. Alveolar epithelial cells have an important role in clearing pulmonary fluid and maintaining the structure of lung tissue. After repeated exposure, damage to epithelial cells may result in their death, causing edema and collagen deposition that may lead to fibrosis.     

Urinary levels,tissue concentrations and lung inflammation after nose‐only exposure to three different chemical forms of beryllium

This study aimed to determine the toxicity and toxicokinetic of three Be chemical species A total of 120 mice (four groups of 30) were nose‐only exposed. The first group was used as a control while the three others were exposed to 250 μg m^?3 of fine particles of three different Be species (Be metal, Be–F; Be oxide, BeO–F; Be aluminium, BeAl–F). Exposure lasted over three consecutive weeks, five days per week and 6 h per day. Blood and several tissues were collected one week after exposure. Urines were collected before the beginning of exposure, at the end of every week of exposure and one week after exposure. Results showed that urine concentrations were different from one Be species to another and that excretion continued after the end of exposure. Except for BeO–F, where Be urine concentrations were stable during the three weeks of exposure, concentrations of Be–F and BeAl–F reached a peak after the first week. According to particle size, BeO–F obtained the highest theoretical pulmonary deposition rate, which partially led to the highest Be lung concentration. This group also presented the lowest urine concentration but that did not lead to more severe lung inflammation. Moreover, even if BeAl–F obtained the lowest percentage theoretical pulmonary deposition, it showed the highest Be urinary concentration, the lowest Be lung concentration and the lowest lung toxicity. In this specific case, a high Be concentration in urine did not reflect a high exposure or a severe toxic effect. Copyright © 2010 John Wiley & Sons, Ltd.     

A role for surface reactivity in TiO2 and quartz-related nanoparticle pulmonary toxicity

A variety of pulmonary hazard studies in rats have demonstrated that exposures to ultrafine or nanoparticles (generally defined as particles in the size range < 100 nm) produce more intensive inflammatory responses when compared with bulk-sized particle-types of similar chemical composition. However, this common perception of greater nanoparticle toxicity is based on a limited number of studies, conducted primarily with titanium dioxide and carbon black particle-types. Apart from variables such as particle size and surface area, it is conceivable that several additional physicochemical particle characteristics could play more significant roles in facilitating the development of nanoparticle-related toxicity; particularly when considering particle surface-cell interactions. These include but are not limited to: (i) Surface reactivity of particle-types; (ii) surface coatings; (iii) aggregation/disaggregation potential; and (iv) the method of nanoparticle synthesis. We present results of pulmonary bioassay hazard/safety studies with quartz particles of varying sizes/surface areas. These demonstrated that intratracheal instillation exposures to fine-sized, Min-U-Sil quartz particles (0.5 µm [particle size] – 5 m²/g [surface area]) produced (persistent) enhanced pulmonary toxicity (inflammation, cytotoxicity, cell proliferation and/or histopathology) in rats when compared to nanoscale quartz particles (50 nm–31 m²/g), but not when compared to smaller nanoscale quartz sizes (e.g., 12 nm–91 m²/g). The toxicity results correlated with red blood cell hemolytic potency as a measure of particle surface reactivity. In a second pulmonary bioassay study in rats, pulmonary hazard effects were measured following exposures to three different ultrafine (nano) TiO₂ particle-types, each with similar particle size distributions. The various TiO₂ particles differed in their crystal structures and surface reactivity endpoints as measured by the Vitamin C yellowing assay. Moreover, the surface activity characteristics correlated with potency of hazard biomarkers as described above, in these dose/response, time-course studies. It is concluded that particle surface reactivity, rather than particle size/surface area endpoints correlated best with lung inflammatory potency following exposures to particles.     

Apoptosis of lung cells regulated by mitochondrial signal pathway in crotonaldehyde‐induced lung injury

Crotonaldehyde, a highly toxic α, β‐unsaturated aldehyde, is a ubiquitous hazardous pollutant. Because of its extreme toxicity and ubiquity in all types of smoke, most current research focuses on the lung toxicity of such air pollutants. However, the specific mechanism of pulmonary toxicity caused by crotonaldehyde remains unclear, especially after long‐term exposure to crotonaldehyde at low dose. Therefore, the aim of the present study is to determine whether crotonaldehyde‐induced oxidative damage and inflammation promote apoptosis in rats via the mitochondrial pathway using histopathology, immunohistochemistry, biochemistry analysis and Western blot analysis. The results show that crotonaldehyde elicited oxidative damage and inflammation in rats in a concentration‐dependent manner. Crotonaldehyde‐induced lung injury which was confirmed by H&E, Masson's trichrome staining and TUNEL. And crotonaldehyde‐induced lung cell apoptosis showed a concentration‐response relationship. Immunohistochemistry and Western blot results showed that apoptotic mitochondrial signaling pathway is abnormally activated in crotonaldehyde‐induced lung injury. Collectively, this study demonstrates that exposure of rats to crotonaldehyde induces lung injury by inducing apoptosis, which is related to oxidative damage and inflammation through mitochondrial pathway.     

REGULATION OF LEUKOCYTE FUNCTION BY NITRIC OXIDE DONORS: THE EFFECT OF S-NITROSO-THIOL COMPLEXES

The pulmonary toxicity of particles is often studied using a single intratracheal instillation of the material. It was hypothesized that smaller multiple intratracheal administrations of silica would result in differences in pulmonary responses as compared to a single large intratracheal administration. In the first of a series of experiments, the pulmonary responses in male F344 rats to a single intratracheal instillation of crystalline silica (5 mg/100 g body weight) given on d 0 were compared with those resulting from 5 consecutive daily intratracheal administrations of the dust (1 mg/100 g body weight/d) with the initial dose given on d 0. Controls received saline intratracheally. In the second experiment, the dose was reduced to 1 mg/100 g body weight for the single-dose protocol and 0.2 mg/100 g body weight/d for 5 consecutive days for the multiple-dose protocol. In both experiments, responses were assessed on d 14. In the third experiment, the doses were the same as the first experiment, but the responses were assessed on d 28. The indices of toxicity were cellular differentials recovered by bronchoalveolar lavage, which is an index of inflammation, and the level of albumin in the bronchoalveolar lavage fluid, a measure of damage to the capillary-epithelial barrier. At the higher dose of silica, similar levels of inflammation and lung damage were evident in both dosing protocols. Less severe responses occurred at the lower dose. The comparative pattern between the single and multiple dosing protocols was similar in all three experiments. Since only minor differences were noted in the pulmonary responses when the responses to the single- and multiple-dose protocols were compared, data indicate that the multiple-dose protocol does not offer any advantages over the single-dose protocol.     

Pulmonary responses to single versus multiple intratracheal instillations of silica in rats

The pulmonary toxicity of particles is often studied using a single intratracheal instillation of the material. It was hypothesized that smaller multiple intratracheal administrations of silica would result in differences in pulmonary responses as compared to a single large intratracheal administration. In the first of a series of experiments, the pulmonary responses in male F344 rats to a single intratracheal instillation of crystalline silica (5 mg/100 g body weight) given on d 0 were compared with those resulting from 5 consecutive daily intratracheal administrations of the dust (1 mg/100 g body weight/d) with the initial dose given on d 0. Controls received saline intratracheally. In the second experiment, the dose was reduced to 1 mg/100 g body weight for the single-dose protocol and 0.2 mg/100 g body weight/d for 5 consecutive days for the multiple-dose protocol. In both experiments, responses were assessed on d 14. In the third experiment, the doses were the same as the first experiment, but the responses were assessed on d 28. The indices of toxicity were cellular differentials recovered by bronchoalveolar lavage, which is an index of inflammation, and the level of albumin in the bronchoalveolar lavage fluid, a measure of damage to the capillary-epithelial barrier. At the higher dose of silica, similar levels of inflammation and lung damage were evident in both dosing protocols. Less severe responses occurred at the lower dose. The comparative pattern between the single and multiple dosing protocols was similar in all three experiments. Since only minor differences were noted in the pulmonary responses when the responses to the single- and multiple-dose protocols were compared, data indicate that the multiple-dose protocol does not offer any advantages over the single-dose protocol.     

Siderite (FeCO₃) and magnetite (Fe₃O₄) overload-dependent pulmonary toxicity is determined by the poorly soluble particle not the iron content

The two poorly soluble iron containing solid aerosols of siderite (FeCO?) and magnetite (Fe?O?) were compared in a 4-week inhalation study on rats at similar particle mass concentrations of approximately 30 or 100?mg/m3. The particle size distributions were essentially identical (MMAD ≈1.4 μm). The iron-based concentrations were 12 or 38 and 22 or 66?mg Fe/m3 for FeCO? and Fe?O?, respectively. Modeled and empirically determined iron lung burdens were compared with endpoints suggestive of pulmonary inflammation by determinations in bronchoalveolar lavage (BAL) and oxidative stress in lung tissue during a postexposure period of 3 months. The objective of study was to identify the most germane exposure metrics, that are the concentration of elemental iron (mg Fe/m3), total particle mass (mg PM/m3) or particle volume (μl PM/m3) and their associations with the effects observed. From this analysis it was apparent that the intensity of pulmonary inflammation was clearly dependent on the concentration of particle-mass or -volume and not of iron. Despite its lower iron content, the exposure to FeCO? caused a more pronounced and sustained inflammation as compared to Fe?O?. Similarly, borderline evidence of increased oxidative stress and inflammation occurred especially following exposure to FeCO? at moderate lung overload levels. The in situ analysis of 8-oxoguanine in epithelial cells of alveolar and bronchiolar regions supports the conclusion that both FeCO? and Fe?O? particles are effectively endocytosed by macrophages as opposed to epithelial cells. Evidence of intracellular or nuclear sources of redox-active iron did not exist. In summary, this mechanistic study supports previous conclusions, namely that the repeated inhalation exposure of rats to highly respirable pigment-type iron oxides cause nonspecific pulmonary inflammation which shows a clear dependence on the particle volume-dependent lung overload rather than any increased dissolution and/or bioavailability of redox-active iron.     

Multi-walled carbon nanotubes (Baytubes®): Approach for derivation of occupational exposure limit

Carbon nanotubes come in a variety of types, but one of the most common forms is multi-walled carbon nanotubes (MWCNT). This paper focuses on the dose–response and time course of pulmonary toxicity of Baytubes®, a more flexible MWCNT type with the tendency to form assemblages of nanotubes. This MWCNT has been examined in previous single and repeated exposure 13-week rat inhalation studies. Kinetic endpoints and the potential to translocate to extrapulmonary organs have been examined during postexposure periods of 3 and 6 months, respectively. The focus of both studies was to compare dosimetric endpoints and the time course of pulmonary inflammation characterized by repeated bronchoalveolar lavage and histopathology during the respective follow-up periods. To better understand the etiopathology of pulmonary inflammation and time-related lung remodeling, two metrics of retained lung dose were compared. The first used the mass metric based on the exposure concentration obtained by filter analyses and aerodynamic particle size of airborne MWCNT. The second was based on calculated volumetric lung burdens of retained MWCNT. Kinetic analyses of lung burdens support the conclusion that Baytubes®, in principal, act like poorly soluble agglomerated carbonaceous particulates. However, the difference in pulmonary toxic potency (mass-based) appears to be associated with the low-density (≈0.1–0.3 g/m³) of the MWCNT assemblages. Of note is that assemblages of MWCNT were found predominantly both in the exposure atmosphere and in digested alveolar macrophages. Isolated fibers were not observed in exposure atmospheres or biological specimens. All findings support the conclusion that the low specific density of microstructures was conducive to attaining the volumetric lung overload-related inflammatory response conditions earlier than conventional particles. Evidence of extrapulmonary translocation or toxicity was not found in any study. Thus, pulmonary overload is believed to trigger the cascade of events leading to a stasis of clearance and consequently increased MWCNT doses high enough to trigger sustained pulmonary inflammation. This mechanism served as conceptual basis for the calculation of the human equivalent concentration. Accordingly, multiple interspecies adjustments were necessary which included species-specific differences in alveolar deposition, differences in ventilation, and the time-dependent particle accumulation accounting for the known species-specific differences in particle clearance half-times in rats and humans. Based on this rationale and the NOAEL (no-observed adverse effect level) from the 13-week subchronic inhalation study on rats, an occupational exposure limit (OEL) of 0.05 mg Baytubes/m³ (time weighted average) is considered to be reasonably protective to prevent lung injury to occur in the workplace environment.     

Comparison of low doses of aged and freshly fractured silica on pulmonary inflammation and damage in the rat

Most previous studies of silica toxicity have used relatively high exposure doses of silica. In this study, male rats received by intratracheal instillation either vehicle, aged or freshly fractured silica at a dose of either 5 microg/rat once a week for 12 weeks (total dose=60 microg) or 20 microg/rat once a week for 12 weeks (total dose=240 microg). One week after the last exposure, bronchoalveolar lavage (BAL) was conducted and markers of pulmonary inflammation, alveolar macrophage (AM) activation and pulmonary damage were examined. For rats exposed to a total of 60 microg silica, both aged and freshly fractured silica increased polymorphonuclear leukocytes (PMN) yield and AM activation above control to a similar degree, but no evidence of pulmonary damage, as measured by BAL fluid lactate dehydrogenase activity or albumin concentration, was detected. For rats exposed to 240 microg silica, aged or freshly fractured silica increased PMN yield and AM activation above control. However, zymosan-stimulated and L-NAME sensitive AM chemiluminescence was greater for rats exposed to freshly fractured silica compared to aged silica. Exposure to 240 microg aged or freshly fractured silica also resulted in pulmonary damage, but the extent of this damage did not differ between the two types of silica. The results suggest that exposure of rats to silica levels far lower than those previously examined can cause pulmonary inflammation. In addition, exposure to freshly fractured silica causes greater generation of reactive oxygen species from AM, measured as AM chemiluminescence, in comparison to aged silica, but there is an apparent threshold below which this difference does not occur.     

Assessing nanomaterial exposures in aquatic ecotoxicological testing: Framework and case studies based on dispersion and dissolution

The unique behavior of engineered nanomaterials (ENM) in aqueous media and dynamic changes in particle settling, agglomeration and dissolution rates is a challenge to the consistency, reliability and interpretation of standard aquatic hazard bioassay results. While the toxicological endpoints (e.g., survival, growth, reproduction, etc.) in ecotoxicity bioassays are largely applicable to ENMs, the standard methods as written for dissolved substances are confounded by the dynamic settling, agglomeration and dissolution of particulate ENMs during the bioassay. A testing framework was designed to serve as a starting point to identify approaches for the consistent conduct of aquatic hazard tests that account for the behavior of ENMs in test media and suitable data collection to support representative exposure metrology. The framework was demonstrated by conducting three case studies testing ENMs with functionally distinct characteristics and behaviors. Pretests with a temporal sampling of particle concentration, agglomeration and dissolution were conducted on each ENM in test media. Results indicated that a silver nanoparticle (AgNP) powder was not dispersible, a nano-TiO₂ powder was dispersible but unstable, and a polyvinylpyrrolidinone-coated AgNP was relatively stable in test media. Based on these functional results, Ceriodaphnia dubia bioassays were conducted to compare different exposure summary methods (nominal, arithmetic average, geometric average, time-weighted average) for calculating and expressing toxicity endpoints. Results indicated that while arithmetic means were effective for expressing the toxicity of more stable materials, time-weighted averaged concentrations were appropriate for the unstable nano-TiO₂.