Critical aspects in occupational exposure assessment with different aerosol metrics in an industrial spray coating process.

Engineered Nanomaterials (ENMs) have several uses in various industrial fields and are embedded in a myriad of consumer products. However, there is continued concern over the potential adverse health effects and environmental impacts of ENMs due to their unique physico-chemical characteristics. Currently, there are no specific international regulations for various ENMs. There are also no Occupational Exposure Limits (OEL) regulated by the European Union (EU) for nanomaterials in the form of nano-objects, their aggregates or agglomerates (NOAA). For ENMs the question of which metric to be used (i.e., mass, surface area, number concentrations) to determine the exposure is still not resolved. The aim of this work is to assess the worker exposure by inhalation in an industrial spray coating process by using all three metrics mentioned above. Two target ENMs (N-doped TiO2, TiO2N and AgNPs capped with a quaternized hydroxyethyl-cellulose, AgHEC) generated for industrial-scale spraying processes were considered. Results showed that the averaged particle number concentration (10-100 nm) was below 2.7 104 cm-3 for both materials. The Lung Deposited Surface Area (LDSA) was in the range between 73 and 98 µm2cm-3 and the particle mass concentration (obtained by means of ICP-EOS off-line analysis) resulted below 70 µg m-3 and 0.4 µg m-3 for TiO2 and Ag, respectively. Although, the airborne particles concentration compared well with the NIOSH Recommended Exposure Level (REL) limits the contribution to the background, according to EN 17058 (Annex E) was significant (particularly in the particle number and PM1 mass concentrations). We successfully evaluated the worker exposure by means of the different airborne particles' metrics (number, surface and mass concentrations). We concluded that worker exposure assessment involving ENMs is a complex procedure with requires both real time and off-line measurements and a deep investigation of the background.

Length-dependent toxicity of TiO2 nanofibers: mitigation via shortening.

Length and aspect ratio represent important toxicity determinants of fibrous nanomaterials. We have previously shown that anatase TiO2 nanofibers (TiO2 NF) cause a dose-dependent decrease of cell viability as well as the loss of epithelial barrier integrity in polarized airway cell monolayers. Herein we have investigated the impact of fiber shortening, obtained by ball-milling, on the biological effects of TiO2 NF of industrial origin. Long TiO2 NF (L-TiO2 NF) were more cytotoxic than their shortened counterparts (S-TiO2 NF) toward alveolar A549 cells and bronchial 16HBE cells. Moreover, L-TiO2 NF increased the permeability of 16HBE monolayers and perturbed the distribution of tight-junction proteins, an effect also mitigated by fiber shortening. Raw264.7 macrophages efficiently internalized shortened but not long NF, which caused cell stretching and deformation. Compared with L-TiO2 NF, S-TiO2 NF triggered a more evident macrophage activation, an effect suppressed by the phagocytosis inhibitor cytochalasin B. Conversely, a significant increase of inflammatory markers was detected in either the lungs or the peritoneal cavity of mice exposed to L-TiO2 NF but not to S-TiO2 NF, suggesting that short-term macrophage activation in vitro may not be always a reliable indicator of persistent inflammation in vivo. It is concluded that fiber shortening mitigates NF detrimental effects on cell viability and epithelial barrier competence in vitro as well as inflammation development in vivo. These data suggest that fiber shortening may represent an effective safe-by-design strategy for mitigating TiO2 NF toxic effects.

Dip coating of air purifier ceramic honeycombs with photocatalytic TiO2 nanoparticles: A case study for occupational exposure.

Nanoscale TiO2 (nTiO2) is manufactured in high volumes and is of potential concern in occupational health. Here, we measured workers exposure levels while ceramic honeycombs were dip coated with liquid photoactive nanoparticle suspension and dried with an air blade. The measured nTiO2 concentration levels were used to assess process specific emission rates using a convolution theorem and to calculate inhalation dose rates of deposited nTiO2 particles. Dip coating did not result in detectable release of particles but air blade drying released fine-sized TiO2 and nTiO2 particles. nTiO2 was found in pure nTiO2 agglomerates and as individual particles deposited onto background particles. Total particle emission rates were 420×109min-1, 1.33×109µm2min-1, and 3.5mgmin-1 respirable mass. During a continued repeated process, the average exposure level was 2.5×104cm-3, 30.3µm2cm-3, <116µgm-3 for particulate matter. The TiO2 average exposure level was 4.2µgm-3, which is well below the maximum recommended exposure limit of 300µgm-3 for nTiO2 proposed by the US National Institute for Occupational Safety and Health. During an 8-hour exposure, the observed concentrations would result in a lung deposited surface area of 4.3×10-3cm2g-1 of lung tissue and 13µg of TiO2 to the trachea-bronchi, and alveolar regions. The dose levels were well below the one hundredth of the no observed effect level (NOEL1/100) of 0.11cm2g-1 for granular biodurable particles and a daily no significant risk dose level of 44µgday-1. These emission rates can be used in a mass flow model to predict the impact of process emissions on personal and environmental exposure levels.

Toxicity of surface-modified copper oxide nanoparticles in a mouse macrophage cell line: Interplay of particles, surface coating and particle dissolution.

The rapid dissolution of copper oxide (CuO) nanoparticles (NPs) with release of ions is thought to be one of the main factors modulating their toxicity. Here we assessed the cytotoxicity of a panel of CuO NPs (12 nm ±â€¯4 nm) with different surface modifications, i.e., anionic sodium citrate (CIT) and sodium ascorbate (ASC), neutral polyvinylpyrrolidone (PVP), and cationic polyethylenimine (PEI), versus the pristine (uncoated) NPs, using a murine macrophage cell line (RAW264.7). Cytotoxicity, reactive oxygen species (ROS) production, and cellular uptake were assessed. The cytotoxicity results were analyzed by the benchmark dose (BMD) method and the NPs were ranked based on BMD20 values. The PEI-coated NPs were found to be the most cytotoxic. Despite the different properties of the coating agents, NP dissolution in cell medium was only marginally affected by surface modification. Furthermore, CuCl2 (used as an ion control) elicited significantly less cytotoxicity when compared to the CuO NPs. We also observed that the antioxidant, N-acetylcysteine, failed to protect against the cytotoxicity of the uncoated CuO NPs. Indeed, the toxicity of the surface-modified CuO NPs was not directly linked to particle dissolution and subsequent Cu burden in cells, nor to cellular ROS production, although CuO-ASC NPs, which were found to be the least cytotoxic, yielded lower levels of ROS in comparison to pristine NPs. Hierarchical cluster analysis suggested, instead, that the toxicity in the current in vitro model could be explained by synergistic interactions between the NPs, their dissolution, and the toxicity of the coating agents.

A panel of in vitro tests to evaluate genotoxic and morphological neoplastic transformation potential on Balb/3T3 cells by pristine and remediated titania and zirconia nanoparticles.

The FP7 Sanowork project was aimed to minimise occupational hazard and exposure to engineered nanomaterials (ENM) through the surface modification in order to prevent possible health effects. In this frame, a number of nanoparticles (NP) have been selected, among which zirconium (ZrO2) and titanium (TiO2) dioxide. In this study, we tested ZrO2 NP and TiO2 NP either in their pristine (uncoated) form, or modified with citrate and/or silica on their surface. As benchmark material, Aeroxide® P25 was used. We assessed cytotoxicity, genotoxicity and induction of morphological neoplastic transformation of NP by using a panel of in vitro assays in an established mammalian cell line of murine origin (Balb/3T3). Cell viability was evaluated by means of colony-forming efficiency assay (CFE). Genotoxicity was investigated by cytokinesis-block micronucleus cytome assay (CBMN cyt) and comet assay, and by the use of the restriction enzymes EndoIII and Fpg, oxidatively damaged DNA was detected; finally, the morphological neoplastic transformation of NP was assayed in vitro by cell transformation assay (CTA). Our results show that the surface remediation has not been effective in modifying cyto- and genotoxic properties of the nanomaterials tested; indeed, in the case of remediation of zirconia and titania with citrate, there is a tendency to emphasise the toxic effects. The use of a panel of assays, such as those we have employed, allowing the evaluation of multiple endpoints, including cell transformation, seems particularly advisable especially in the case of long-term exposure effects in the same cell type.

Shape-Related Toxicity of Titanium Dioxide Nanofibres.

Titanium dioxide (TiO2) nanofibres are a novel fibrous nanomaterial with increasing applications in a variety of fields. While the biological effects of TiO2 nanoparticles have been extensively studied, the toxicological characterization of TiO2 nanofibres is far from being complete. In this study, we evaluated the toxicity of commercially available anatase TiO2 nanofibres using TiO2 nanoparticles (NP) and crocidolite asbestos as non-fibrous or fibrous benchmark materials. The evaluated endpoints were cell viability, haemolysis, macrophage activation, trans-epithelial electrical resistance (an indicator of the epithelial barrier competence), ROS production and oxidative stress as well as the morphology of exposed cells. The results showed that TiO2 nanofibres caused a cell-specific, dose-dependent decrease of cell viability, with larger effects on alveolar epithelial cells than on macrophages. The observed effects were comparable to those of crocidolite, while TiO2 NP did not decrease cell viability. TiO2 nanofibres were also found endowed with a marked haemolytic activity, at levels significantly higher than those observed with TiO2 nanoparticles or crocidolite. Moreover, TiO2 nanofibres and crocidolite, but not TiO2 nanoparticles, caused a significant decrease of the trans-epithelial electrical resistance of airway cell monolayers. SEM images demonstrated that the interaction with nanofibres and crocidolite caused cell shape perturbation with the longest fibres incompletely or not phagocytosed. The expression of several pro-inflammatory markers, such as NO production and the induction of Nos2 and Ptgs2, was significantly increased by TiO2 nanofibres, as well as by TiO2 nanoparticles and crocidolite. This study indicates that TiO2 nanofibres had significant toxic effects and, for most endpoints with the exception of pro-inflammatory changes, are more bio-active than TiO2 nanoparticles, showing the relevance of shape in determining the toxicity of nanomaterials. Given that several toxic effects of TiO2 nanofibres appear comparable to those observed with crocidolite, the possibility that they exert length dependent toxicity in vivo seems worthy of further investigation.