Histopathology of the broad class of carbon nanotubes and nanofibers used or produced in U.S. facilities in a murine model.

BACKGROUND: Multi-walled carbon nanotubes and nanofibers (CNT/F) have been previously investigated for their potential toxicities; however, comparative studies of the broad material class are lacking, especially those with a larger diameter. Additionally, computational modeling correlating physicochemical characteristics and toxicity outcomes have been infrequently employed, and it is unclear if all CNT/F confer similar toxicity, including histopathology changes such as pulmonary fibrosis. Male C57BL/6 mice were exposed to 40 µg of one of nine CNT/F (MW #1-7 and CNF #1-2) commonly found in exposure assessment studies of U.S. facilities with diameters ranging from 6 to 150 nm. Human fibroblasts (0-20 µg/ml) were used to assess the predictive value of in vitro to in vivo modeling systems. RESULTS: All materials induced histopathology changes, although the types and magnitude of the changes varied. In general, the larger diameter MWs (MW #5-7, including Mitsui-7) and CNF #1 induced greater histopathology changes compared to MW #1 and #3 while MW #4 and CNF #2 were intermediate in effect. Differences in individual alveolar or bronchiolar outcomes and severity correlated with physical dimensions and how the materials agglomerated. Human fibroblast monocultures were found to be insufficient to fully replicate in vivo fibrosis outcomes suggesting in vitro predictive potential depends upon more advanced cell culture in vitro models. Pleural penetrations were observed more consistently in CNT/F with larger lengths and diameters. CONCLUSION: Physicochemical characteristics, notably nominal CNT/F dimension and agglomerate size, predicted histopathologic changes and enabled grouping of materials by their toxicity profiles. Particles of greater nominal tube length were generally associated with increased severity of histopathology outcomes. Larger particle lengths and agglomerates were associated with more severe bronchi/bronchiolar outcomes. Spherical agglomerated particles of smaller nominal tube dimension were linked to granulomatous inflammation while a mixture of smaller and larger dimensional CNT/F resulted in more severe alveolar injury.

Physicochemical characterization and genotoxicity of the broad class of carbon nanotubes and nanofibers used or produced in U.S. facilities.

BACKGROUND: Carbon nanotubes and nanofibers (CNT/F) have known toxicity but simultaneous comparative studies of the broad material class, especially those with a larger diameter, with computational analyses linking toxicity to their fundamental material characteristics was lacking. It was unclear if all CNT/F confer similar toxicity, in particular, genotoxicity. Nine CNT/F (MW #1-7 and CNF #1-2), commonly found in exposure assessment studies of U.S. facilities, were evaluated with reported diameters ranging from 6 to 150 nm. All materials were extensively characterized to include distributions of physical dimensions and prevalence of bundled agglomerates. Human bronchial epithelial cells were exposed to the nine CNT/F (0-24 µg/ml) to determine cell viability, inflammation, cellular oxidative stress, micronuclei formation, and DNA double-strand breakage. Computational modeling was used to understand various permutations of physicochemical characteristics and toxicity outcomes. RESULTS: Analyses of the CNT/F physicochemical characteristics illustrate that using detailed distributions of physical dimensions provided a more consistent grouping of CNT/F compared to using particle dimension means alone. In fact, analysis of binning of nominal tube physical dimensions alone produced a similar grouping as all characterization parameters together. All materials induced epithelial cell toxicity and micronuclei formation within the dose range tested. Cellular oxidative stress, DNA double strand breaks, and micronuclei formation consistently clustered together and with larger physical CNT/F dimensions and agglomerate characteristics but were distinct from inflammatory protein changes. Larger nominal tube diameters, greater lengths, and bundled agglomerate characteristics were associated with greater severity of effect. The portion of tubes with greater nominal length and larger diameters within a sample was not the majority in number, meaning a smaller percentage of tubes with these characteristics was sufficient to increase toxicity. Many of the traditional physicochemical characteristics including surface area, density, impurities, and dustiness did not cluster with the toxicity outcomes. CONCLUSION: Distributions of physical dimensions provided more consistent grouping of CNT/F with respect to toxicity outcomes compared to means only. All CNT/F induced some level of genotoxicity in human epithelial cells. The severity of toxicity was dependent on the sample containing a proportion of tubes with greater nominal lengths and diameters.

Lung cell exposure to secondary photochemical aerosols generated from OH oxidation of cyclic siloxanes.

To study the fate of cyclic volatile methyl siloxanes (cVMS) undergoing photooxidation in the environment and to assess the acute toxicity of inhaled secondary aerosols from cVMS, we used an oxidative flow reactor (OFR) to produce aerosols from oxidation of decamethylcyclopentasiloxane (D5). The aerosols produced from this process were characterized for size, shape, and chemical composition. We found that the OFR produced aerosols composed of silicon and oxygen, arranged in chain agglomerates, with primary particles of approximately 31 nm in diameter. Lung cells were exposed to the secondary organosilicon aerosols at estimated doses of 54-116 ng/cm2 using a Vitrocell air-liquid interface system, and organic gases and ozone exposure was minimized through a series of denuders. Siloxane aerosols were not found to be highly toxic.

Characterization of aerosolized particles from nanoclay-enabled composites during manipulation processes.

Manufacturing, processing, use, and disposal of nanoclay-enabled composites potentially lead to the release of nanoclay particles from the polymer matrix in which they are embedded; however, exposures to airborne particles are poorly understood. The present study was conducted to characterize airborne particles released during sanding of nanoclay-enabled thermoplastic composites. Two types of nanoclay, Cloisite® 25A and Cloisite® 93A, were dispersed in polypropylene at 0%, 1%, and 4% loading by weight. Zirconium aluminum oxide (P100/P180 grits) and silicon carbide (P120/P320 grits) sandpapers were used to abrade composites in controlled experiments followed by real-time and offline particle analyses. Overall, sanding the virgin polypropylene with zirconium aluminum oxide sandpaper released more particles compared to silicon carbide sandpaper, with the later exhibiting similar or lower concentrations than that of polypropylene. Thus, a further investigation was performed for the samples collected using the zirconium aluminum oxide sandpaper. The 1% 25A, 1% 93A, and 4% 93A composites generated substantially higher particle number concentrations (1.3-2.6 times) and respirable mass concentrations (1.2-2.3 times) relative to the virgin polypropylene, while the 4% 25A composite produced comparable results, regardless of sandpaper type. It was observed that the majority of the inhalable particles were originated from composite materials with a significant number of protrusions of nanoclay (18-59%). These findings indicate that the percent loading and dispersion of nanoclay in the polypropylene modified the mechanical properties and thus, along with sandpaper type, affected the number of particles released during sanding, implicating the cause of potential adverse health effects.

Mitsui-7, heat-treated, and nitrogen-doped multi-walled carbon nanotubes elicit genotoxicity in human lung epithelial cells.

BACKGROUND: The unique physicochemical properties of multi-walled carbon nanotubes (MWCNT) have led to many industrial applications. Due to their low density and small size, MWCNT are easily aerosolized in the workplace making respiratory exposures likely in workers. The International Agency for Research on Cancer designated the pristine Mitsui-7 MWCNT (MWCNT-7) as a Group 2B carcinogen, but there was insufficient data to classify all other MWCNT. Previously, MWCNT exposed to high temperature (MWCNT-HT) or synthesized with nitrogen (MWCNT-ND) have been found to elicit attenuated toxicity; however, their genotoxic and carcinogenic potential are not known. Our aim was to measure the genotoxicity of MWCNT-7 compared to these two physicochemically-altered MWCNTs in human lung epithelial cells (BEAS-2B & SAEC). RESULTS: Dose-dependent partitioning of individual nanotubes in the cell nuclei was observed for each MWCNT material and was greatest for MWCNT-7. Exposure to each MWCNT led to significantly increased mitotic aberrations with multi- and monopolar spindle morphologies and fragmented centrosomes. Quantitative analysis of the spindle pole demonstrated significantly increased centrosome fragmentation from 0.024-2.4 µg/mL of each MWCNT. Significant aneuploidy was measured in a dose-response from each MWCNT-7, HT, and ND; the highest dose of 24 µg/mL produced 67, 61, and 55%, respectively. Chromosome analysis demonstrated significantly increased centromere fragmentation and translocations from each MWCNT at each dose. Following 24 h of exposure to MWCNT-7, ND and/or HT in BEAS-2B a significant arrest in the G1/S phase in the cell cycle occurred, whereas the MWCNT-ND also induced a G2 arrest. Primary SAEC exposed for 24 h to each MWCNT elicited a significantly greater arrest in the G1 and G2 phases. However, SAEC arrested in the G1/S phase after 72 h of exposure. Lastly, a significant increase in clonal growth was observed one month after exposure to 0.024 µg/mL MWCNT-HT & ND. CONCLUSIONS: Although MWCNT-HT & ND cause a lower incidence of genotoxicity, all three MWCNTs cause the same type of mitotic and chromosomal disruptions. Chromosomal fragmentation and translocations have not been observed with other nanomaterials. Because in vitro genotoxicity is correlated with in vivo genotoxic response, these studies in primary human lung cells may predict the genotoxic potency in exposed human populations.

In Vivo Toxicity Assessment of Occupational Components of the Carbon Nanotube Life Cycle To Provide Context to Potential Health Effects.

Pulmonary toxicity studies on carbon nanotubes focus primarily on as-produced materials and rarely are guided by a life cycle perspective or integration with exposure assessment. Understanding toxicity beyond the as-produced, or pure native material, is critical, due to modifications needed to overcome barriers to commercialization of applications. In the first series of studies, the toxicity of as-produced carbon nanotubes and their polymer-coated counterparts was evaluated in reference to exposure assessment, material characterization, and stability of the polymer coating in biological fluids. The second series of studies examined the toxicity of aerosols generated from sanding polymer-coated carbon-nanotube-embedded or neat composites. Postproduction modification by polymer coating did not enhance pulmonary injury, inflammation, and pathology or in vitro genotoxicity of as-produced carbon nanotubes, and for a particular coating, toxicity was significantly attenuated. The aerosols generated from sanding composites embedded with polymer-coated carbon nanotubes contained no evidence of free nanotubes. The percent weight incorporation of polymer-coated carbon nanotubes, 0.15% or 3% by mass, and composite matrix utilized altered the particle size distribution and, in certain circumstances, influenced acute in vivo toxicity. Our study provides perspective that, while the number of workers and consumers increases along the life cycle, toxicity and/or potential for exposure to the as-produced material may greatly diminish.

Occupational exposure to nanoparticles at commercial photocopy centers.

Photocopiers emit high levels of nanoparticles (PM0.1). To-date little is known of physicochemical composition of PM0.1 in real workplace settings. Here we perform a comprehensive physicochemical and morphological characterization of PM0.1 and raw materials (toners and paper) at eight commercial photocopy centers that use color and monochrome photocopiers over the course of a full week. We document high PM0.1 exposures with complex composition and several ENM in toners and PM0.1. Daily geometric mean PM0.1 concentrations ranged from 3700 to 34000 particles/cubic-centimeter (particles/cm(3)) (GSD 1.4-3.3), up to 12 times greater than background, with transient peaks >1.4 million particles/cm(3). PM0.1 contained 6-63% organic carbon, <1% elemental carbon, and 2-8% metals, including iron, zinc, titania, chromium, nickel and manganese, typically in the <0.01-1% range, and in agreement with toner composition. These findings document widespread ENM in toner formulations and high nanoparticle exposures are an industry-wide phenomenon. It further calls attention to the need to substantially redesign the interface of this technology with workers and consumers.

Consumer exposures to laser printer-emitted engineered nanoparticles: A case study of life-cycle implications from nano-enabled products.

It is well established that printers emit nanoparticles during their operation. To-date, however, the physicochemical and toxicological characterization of "real world" printer-emitted nanoparticles (PEPs) remains incomplete, hampering proper risk assessment efforts. Here, we investigate our earlier hypothesis that engineered nanomaterials (ENMs) are used in toners and ENMs are released during printing (consumer use). Furthermore, we conduct a detailed physicochemical and morphological characterization of PEPs in support of ongoing toxicological assessment. A comprehensive suite of state of the art analytical methods and tools was employed for the physicochemical and morphological characterization of 11 toners widely utilized in printers from major printer manufacturers and their PEPs. We confirmed that a number of ENMs incorporated into toner formulations (e.g. silica, alumina, titania, iron oxide, zinc oxide, copper oxide, cerium oxide, carbon black among others) and released into the air during printing. All evaluated toners contained large amounts of organic carbon (OC, 42-89%), metals/metal oxides (1-33%), and some elemental carbon (EC, 0.33-12%). The PEPs possess a composition similar to that of toner and contained 50-90% OC, 0.001-0.5% EC and 1-3% metals. While the chemistry of the PEPs generally reflected that of their toners, considerable differences are documented indicative of potential transformations taking place during consumer use (printing). We conclude that: (i) Routine incorporation of ENMs in toners classifies them as nano-enabled products (NEPs); (ii) These ENMs become airborne during printing; (iii) The chemistry of PEPs is complex and it reflects that of the toner and paper. This work highlights the importance of understanding life-cycle (LC) nano-EHS implications of NEPs and assessing real world exposures and associated toxicological properties rather than focusing on "raw" materials used in the synthesis of an NEP.

Genotoxicity of multi-walled carbon nanotubes at occupationally relevant doses.

Carbon nanotubes are commercially-important products of nanotechnology; however, their low density and small size makes carbon nanotube respiratory exposures likely during their production or processing. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to single-walled carbon nanotubes (SWCNT). In this study, we examined whether multi-walled carbon nanotubes (MWCNT) cause mitotic spindle damage in cultured cells at doses equivalent to 34 years of exposure at the NIOSH Recommended Exposure Limit (REL). MWCNT induced a dose responsive increase in disrupted centrosomes, abnormal mitotic spindles and aneuploid chromosome number 24 hours after exposure to 0.024, 0.24, 2.4 and 24 µg/cm² MWCNT. Monopolar mitotic spindles comprised 95% of disrupted mitoses. Three-dimensional reconstructions of 0.1 µm optical sections showed carbon nanotubes integrated with microtubules, DNA and within the centrosome structure. Cell cycle analysis demonstrated a greater number of cells in S-phase and fewer cells in the G2 phase in MWCNT-treated compared to diluent control, indicating a G1/S block in the cell cycle. The monopolar phenotype of the disrupted mitotic spindles and the G1/S block in the cell cycle is in sharp contrast to the multi-polar spindle and G2 block in the cell cycle previously observed following exposure to SWCNT. One month following exposure to MWCNT there was a dramatic increase in both size and number of colonies compared to diluent control cultures, indicating a potential to pass the genetic damage to daughter cells. Our results demonstrate significant disruption of the mitotic spindle by MWCNT at occupationally relevant exposure levels.

Physicochemical and morphological characterisation of nanoparticles from photocopiers: implications for environmental health.

Several reports link printing and photocopying with genotoxicity, immunologic and respiratory diseases. Photocopiers and printers emit nanoparticles, which may be involved in these diseases. The physicochemical and morphological composition of these emitted nanoparticles, which is poorly understood and is critical for toxicological evaluations, was assessed in this study using both real-time instrumentation and analytical methods. Tests included elemental composition (40 metals), semi-volatile organics (100 compounds) and single particle analysis, using multiple high-sensitivity/resolution techniques. Identical analyses were performed on the toners and dust collected from copier's exhaust filter. Engineered nanoparticles, including titanium dioxide, iron oxide and fumed silica, and several metals were found in toners and airborne nanoscale fraction. Chemical composition of airborne nanoscale fraction was complex and reflected toner chemistry. These findings are important in understanding the origin and toxicology of such nanoparticles. Further investigation of their chemistry, larger scale exposure studies and thorough toxicological characterisation of emitted nanoparticles is needed.

Characterization of exposures to airborne nanoscale particles during friction stir welding of aluminum.

Friction stir welding (FSW) is considered one of the most significant developments in joining technology over the last half century. Its industrial applications are growing steadily and so are the number of workers using this technology. To date, there are no reports on airborne exposures during FSW. The objective of this study was to investigate possible emissions of nanoscale (<100 nm) and fine (<1 microm) aerosols during FSW of two aluminum alloys in a laboratory setting and characterize their physicochemical composition. Several instruments measured size distributions (5 nm to 20 microm) with 1-s resolution, lung deposited surface areas, and PM(2.5) concentrations at the source and at the breathing zone (BZ). A wide range aerosol sampling system positioned at the BZ collected integrated samples in 12 stages (2 nm to 20 microm) that were analyzed for several metals using inductively coupled plasma mass spectrometry. Airborne aerosol was directly collected onto several transmission electron microscope grids and the morphology and chemical composition of collected particles were characterized extensively. FSW generates high concentrations of ultrafine and submicrometer particles. The size distribution was bimodal, with maxima at approximately 30 and approximately 550 nm. The mean total particle number concentration at the 30 nm peak was relatively stable at approximately 4.0 x 10(5) particles cm(-3), whereas the arithmetic mean counts at the 550 nm peak varied between 1500 and 7200 particles cm(-3), depending on the test conditions. The BZ concentrations were lower than the source concentrations by 10-100 times at their respective peak maxima and showed higher variability. The daylong average metal-specific concentrations were 2.0 (Zn), 1.4 (Al), and 0.24 (Fe) microg m(-3); the estimated average peak concentrations were an order of magnitude higher. Potential for significant exposures to fine and ultrafine aerosols, particularly of Al, Fe, and Zn, during FSW may exist, especially in larger scale industrial operations.