Use of the dustiness index in combination with the handling energy factor for exposure modelling of nanomaterials.

The use of modelling tools in the occupational hygiene community has increased in the last years to comply with the different existing regulations. However, limitations still exist mainly due to the difficulty to obtain certain key parameters such as the emission rate, which in the case of powder handling can be estimated using the dustiness index (DI). The goal of this work is to explore the applicability and usability of the DI for emission source characterization and occupational exposure prediction to particles during nanomaterial powder handling. Modelling of occupational exposure concentrations of 13 case scenarios was performed using a two-box model as well as three nano-specific tools (Stoffenmanager nano, NanoSafer and GUIDEnano). The improvement of modelling performance by using a derived handling energy factor (H) was explored. Results show the usability of the DI for emission source characterization and respirable mass exposure modelling of powder handling scenarios of nanomaterials. A clear improvement in modelling outcome was obtained when using derived quartile-3 H factors with, 1) Pearson correlations of 0.88 vs. 0.52 (not using H), and 2) ratio of modelled/measured concentrations ranging from 0.9 to 10 in 75% cases vs. 16.7% of the cases when not using H. Particle number concentrations were generally underpredicted. Using the most conservative H values, predictions with ratios modelled/measured concentrations of 0.4-3.6 were obtained.

The State of the Art and Challenges of In Vitro Methods for Human Hazard Assessment of Nanomaterials in the Context of Safe-by-Design.

The Safe-by-Design (SbD) concept aims to facilitate the development of safer materials/products, safer production, and safer use and end-of-life by performing timely SbD interventions to reduce hazard, exposure, or both. Early hazard screening is a crucial first step in this process. In this review, for the first time, commonly used in vitro assays are evaluated for their suitability for SbD hazard testing of nanomaterials (NMs). The goal of SbD hazard testing is identifying hazard warnings in the early stages of innovation. For this purpose, assays should be simple, cost-effective, predictive, robust, and compatible. For several toxicological endpoints, there are indications that commonly used in vitro assays are able to predict hazard warnings. In addition to the evaluation of assays, this review provides insights into the effects of the choice of cell type, exposure and dispersion protocol, and the (in)accurate determination of dose delivered to cells on predictivity. Furthermore, compatibility of assays with challenging advanced materials and NMs released from nano-enabled products (NEPs) during the lifecycle is assessed, as these aspects are crucial for SbD hazard testing. To conclude, hazard screening of NMs is complex and joint efforts between innovators, scientists, and regulators are needed to further improve SbD hazard testing.

Food contact of paper and plastic products containing SiO2, Cu-Phthalocyanine, Fe2O3, CaCO3: Ranking factors that control the similarity of form and rate of release.

The paper industry is an important sector annually consuming kilotons of nanoforms and non-nanoforms of fillers and pigments. Fillers accelerate the rate of drying (less energy needed) and product cost (increasing the load of low-cost fillers). The plastic industry is another use sector, where coloristic pigments can be in nanoform, and many food containers are made of plastic. Use of paper to wrap both wet and dry food is consumer practice, but not always intended by producers. Here we compare the release behavior of different nano-enabled products (NEPs) by changing a) nanoform (NF) characteristics, b) NF load, c) the nano-enabled product (NEP) matrix, and d) food simulants. The ranking of these factors enables an assessment of food contact by concepts of analogy, specifically via the similarities of the rate and form of release in food during contact. Three types of matrices were used: Paper, plastic ((Polylactic Acid (PLA), Polyamide (PA6), and Polyurethane (PU)), and a paint formulation. Two nanoforms each of SiO2, Fe2O3, Cu-Phthalocyanine were incorporated, additionally to the conventional form of CaCO3 that is always contained in paper to reduce cellulose consumption. Tests were guided by the European Regulation EC 1935/2004 and EU 10/2011. No evidence of particle release was observed: the qualitative similarity (the form of release) was high regarding the food contact of all NEPs with embedded NFs. Quantitative similarity of releases depended primarily on the NEP matrix, as this controls the penetration of the simulant fluid into the NEP. The solubility of the NF and impurities in the simulant fluid was the second decisive factor, as dissolution of the NF inside the NEP is the main mechanism of release. This led to complete removal of CaCO3 in acidic medium, whereas Fe and Si signals remained in the paper, consistent with the low release rates in an ionic form. In our set of 16 NEPs, only one NEP showed a dependence on the REACH NF descriptors (substance, size, shape, surface treatment, crystallinity, impurities), specifically attributed to differences in soluble impurities, whereas for all others the substance of the nanoform was sufficient to predict a similarity of food contact release, without influences of size, shape, surface treatment and crystallinity.

Reproducibility of methods required to identify and characterize nanoforms of substances.

Nanoforms (NFs) of a substance may be distinguished from one another through differences in their physicochemical properties. When registering nanoforms of a substance for assessment under the EU REACH framework, five basic descriptors are required for their identification: composition, surface chemistry, size, specific surface area and shape. To make the risk assessment of similar NFs efficient, a number of grouping frameworks have been proposed, which often require assessment of similarity on individual physicochemical properties as part of the group justification. Similarity assessment requires an understanding of the achievable accuracy of the available methods. It must be demonstrated that measured differences between NFs are greater than the achievable accuracy of the method, to have confidence that the measured differences are indeed real. To estimate the achievable accuracy of a method, we assess the reproducibility of six analytical techniques routinely used to measure these five basic descriptors of nanoforms: inductively coupled plasma mass spectrometry (ICP-MS), Thermogravimetric analysis (TGA), Electrophoretic light scattering (ELS), Brunauer-Emmett-Teller (BET) specific surface area and transmission and scanning electron microscopy (TEM and SEM). Assessment was performed on representative test materials to evaluate the reproducibility of methods on single NFs of substances. The achievable accuracy was defined as the relative standard deviation of reproducibility (RSDR) for each method. Well established methods such as ICP-MS quantification of metal impurities, BET measurements of specific surface area, TEM and SEM for size and shape and ELS for surface potential and isoelectric point, all performed well, with low RSDR, generally between 5 and 20%, with maximal fold differences usually <1.5 fold between laboratories. Applications of technologies such as TGA for measuring water content and putative organic impurities, additives or surface treatments (through loss on ignition), which have a lower technology readiness level, demonstrated poorer reproducibility, but still within 5-fold differences. The expected achievable accuracy of ICP-MS may be estimated for untested analytes using established relationships between concentration and reproducibility, but this is not yet the case for TGA measurements of loss on ignition or water content. The results here demonstrate an approach to estimate the achievable accuracy of a method that should be employed when interpreting differences between NFs on individual physicochemical properties.

Occupational Exposure and Environmental Release: The Case Study of Pouring TiO2 and Filler Materials for Paint Production.

Pulmonary exposure to micro- and nanoscaled particles has been widely linked to adverse health effects and high concentrations of respirable particles are expected to occur within and around many industrial settings. In this study, a field-measurement campaign was performed at an industrial manufacturer, during the production of paints. Spatial and personal measurements were conducted and results were used to estimate the mass flows in the facility and the airborne particle release to the outdoor environment. Airborne particle number concentration (1 × 103-1.0 × 104 cm-3), respirable mass (0.06-0.6 mg m-3), and PM10 (0.3-6.5 mg m-3) were measured during pouring activities. In overall; emissions from pouring activities were found to be dominated by coarser particles >300 nm. Even though the raw materials were not identified as nanomaterials by the manufacturers, handling of TiO2 and clays resulted in release of nanometric particles to both workplace air and outdoor environment, which was confirmed by TEM analysis of indoor and stack emission samples. During the measurement period, none of the existing exposure limits in force were exceeded. Particle release to the outdoor environment varied from 6 to 20 g ton-1 at concentrations between 0.6 and 9.7 mg m-3 of total suspended dust depending on the powder. The estimated release of TiO2 to outdoors was 0.9 kg per year. Particle release to the environment is not expected to cause any major impact due to atmospheric dilution.

In vitro assessment of CeO2 nanoparticles effects on intestinal microvilli morphology.

Some nanoparticles (NPs) have been shown to disrupt intestinal microvilli morphology in vitro, an alteration that could potentially affect nutrient absorption and barrier properties. This study aimed at evaluating the potential effect of CeO2 NPs (4-8 nm, citrate stabilized) on Caco-2 microvilli morphology. In addition to the standard Caco-2 cell clone, the C2BBe1 clone was used, as it is considered to develop a more homogeneous cellular morphology. Semiautomated microvilli density quantification and a new cell scoring approach were used to evaluate scanning electron microscopy (SEM) images. The quantification method made use of the whole micrograph surface, avoiding the need to choose subareas for analysis, and increasing the representativeness of the results when compared to previous studies. The main advantage of the scoring system is that it informs on the intercellular variability within a cell preparation. Benzalkonium was used as a positive control inducing toxicity and morphological alterations on microvilli. After three-week differentiation, Caco-2 cells were exposed to 100 µg/mL of CeO2 NPs for 24 h. The integrity of the membrane was evaluated by transepithelial electrical resistance (TEER) and thereafter processed for its observation by SEM. Results showed that both the standard Caco-2 clone and the C2BBe1 clone present notable morphological heterogeneity. The two evaluation approaches were able to identify morphological effects caused by the positive control, but did not detect statistically significant morphological alterations after exposure to CeO2 NPs.

Multiple endpoints to evaluate pristine and remediated titanium dioxide nanoparticles genotoxicity in lung epithelial A549 cells.

Titanium dioxide nanoparticles (TiO2 NP) are broadly used in a wide range of applications. Several studies have reported that TiO2 NP possess cytotoxic and genotoxic properties that could induce adverse health effects in humans. The FP7 Sanowork project was aimed to minimize occupational hazard and exposure to engineered nanomaterials (ENM), including TiO2 NP, through the surface modification in order to avoid possible adverse toxic effects for humans. In this study we investigated cytotoxicity, genotoxicity and epigenetic properties of TiO2 NP uncoated and coated with silica or citrate, as well as of the benchmark material P25. We used a panel of in vitro assays in the human lung epithelial cell line A549, in order to better understand if the remediation strategy adopted was able to counteract possible toxic effects of uncoated TiO2 NP. Our results showed that the uncoated TiO2 NP were both cytotoxic and genotoxic, and the remediation strategy adopted did not reduce the adverse effects of uncoated TiO2 NP. In particular, the presence of citrate was able to increase their cytotoxicity and genotoxicity, exerting also epigenotoxic effects, as evaluated by the marked reduction of LINE-1 methylation levels.

Hollow-fiber flow field-flow fractionation and multi-angle light scattering investigation of the size, shape and metal-release of silver nanoparticles in aqueous medium for nano-risk assessment.

Due to the increased use of silver nanoparticles in industrial scale manufacturing, consumer products and nanomedicine reliable measurements of properties such as the size, shape and distribution of these nano particles in aqueous medium is critical. These properties indeed affect both functional properties and biological impacts especially in quantifying associated risks and identifying suitable risk-mediation strategies. The feasibility of on-line coupling of a fractionation technique such as hollow-fiber flow field flow fractionation (HF5) with a light scattering technique such as MALS (multi-angle light scattering) is investigated here for this purpose. Data obtained from such a fractionation technique and its combination thereof with MALS have been compared with those from more conventional but often complementary techniques e.g. transmission electron microscopy, dynamic light scattering, atomic absorption spectroscopy, and X-ray fluorescence. The combination of fractionation and multi angle light scattering techniques have been found to offer an ideal, hyphenated methodology for a simultaneous size-separation and characterization of silver nanoparticles. The hydrodynamic radii determined by fractionation techniques can be conveniently correlated to the mean average diameters determined by multi angle light scattering and reliable information on particle morphology in aqueous dispersion has been obtained. The ability to separate silver (Ag(+)) ions from silver nanoparticles (AgNPs) via membrane filtration during size analysis is an added advantage in obtaining quantitative insights to its risk potential. Most importantly, the methodology developed in this article can potentially be extended to similar characterization of metal-based nanoparticles when studying their functional effectiveness and hazard potential.