Agglomeration of titanium dioxide nanoparticles increases toxicological responses in vitro and in vivo.

BACKGROUND: The terms agglomerates and aggregates are frequently used in the regulatory definition(s) of nanomaterials (NMs) and hence attract attention in view of their potential influence on health effects. However, the influence of nanoparticle (NP) agglomeration and aggregation on toxicity is poorly understood although it is strongly believed that smaller the size of the NPs greater the toxicity. A toxicologically relevant definition of NMs is therefore not yet available, which affects not only the risk assessment process but also hinders the regulation of nano-products. In this study, we assessed the influence of NP agglomeration on their toxicity/biological responses in vitro and in vivo. RESULTS: We tested two TiO2 NPs with different primary sizes (17 and 117 nm) and prepared ad-hoc suspensions composed of small or large agglomerates with similar dispersion medium composition. For in vitro testing, human bronchial epithelial (HBE), colon epithelial (Caco2) and monocytic (THP-1) cell lines were exposed to these suspensions for 24 h and endpoints such as cytotoxicity, total glutathione, epithelial barrier integrity, inflammatory mediators and DNA damage were measured. Large agglomerates of 17 nm TiO2 induced stronger responses than small agglomerates for glutathione depletion, IL-8 and IL-1ß increase, and DNA damage in THP-1, while no effect of agglomeration was observed with 117 nm TiO2. In vivo, C57BL/6JRj mice were exposed via oropharyngeal aspiration or oral gavage to TiO2 suspensions and, after 3 days, biological parameters including cytotoxicity, inflammatory cell recruitment, DNA damage and biopersistence were measured. Mainly, we observed that large agglomerates of 117 nm TiO2 induced higher pulmonary responses in aspirated mice and blood DNA damage in gavaged mice compared to small agglomerates. CONCLUSION: Agglomeration of TiO2 NPs influences their toxicity/biological responses and, large agglomerates do not appear less active than small agglomerates. This study provides a deeper insight on the toxicological relevance of NP agglomerates and contributes to the establishment of a toxicologically relevant definition for NMs.

Is aggregated synthetic amorphous silica toxicologically relevant?

BACKGROUND: The regulatory definition(s) of nanomaterials (NMs) frequently uses the term 'agglomerates and aggregates' (AA) despite the paucity of evidence that AA are significantly relevant from a nanotoxicological perspective. This knowledge gap greatly affects the safety assessment and regulation of NMs, such as synthetic amorphous silica (SAS). SAS is used in a large panel of industrial applications. They are primarily produced as nano-sized particles (1-100 nm in diameter) and considered safe as they form large aggregates (> 100 nm) during the production process. So far, it is indeed believed that large aggregates represent a weaker hazard compared to their nano counterpart. Thus, we assessed the impact of SAS aggregation on in vitro cytotoxicity/biological activity to address the toxicological relevance of aggregates of different sizes. RESULTS: We used a precipitated SAS dispersed by different methods, generating 4 ad-hoc suspensions with different aggregate size distributions. Their effect on cell metabolic activity, cell viability, epithelial barrier integrity, total glutathione content and, IL-8 and IL-6 secretion were investigated after 24 h exposure in human bronchial epithelial (HBE), colon epithelial (Caco2) and monocytic cells (THP-1). We observed that the de-aggregated suspension (DE-AGGR), predominantly composed of nano-sized aggregates, induced stronger effects in all the cell lines than the aggregated suspension (AGGR). We then compared DE-AGGR with 2 suspensions fractionated from AGGR: the precipitated fraction (PREC) and the supernatant fraction (SuperN). Very large aggregates in PREC were found to be the least cytotoxic/biologically active compared to other suspensions. SuperN, which contains aggregates larger in size (> 100 nm) than in DE-AGGR but smaller than PREC, exhibited similar activity as DE-AGGR. CONCLUSION: Overall, aggregation resulted in reduced toxicological activity of SAS. However, when comparing aggregates of different sizes, it appeared that aggregates > 100 nm were not necessarily less cytotoxic than their nano-sized counterparts. This study suggests that aggregates of SAS are toxicologically relevant for the definition of NMs.

Size measurement of silica nanoparticles by Asymmetric Flow Field-Flow Fractionation coupled to Multi-Angle Light Scattering: A comparison exercise between two metrological institutes.

In this work we present a comparison exercise between two metrological institutes for size measurement of silica nanoparticles by Asymmetrical Flow Field-Flow Fractionation (AF4) coupled to static light scattering. The work has been performed in the frame of a French inter-laboratory comparison (ILC) exercise organized by the nanoMetrology Club (CnM). The general aim of this multi-technique comparison was to improve the measurement process for each technique, after establishing a well-defined measurement procedure. The results obtained by two national metrological institutes (NMIs), the LNE (France) and the SMD (Belgium) by AF4-UV-DRI-MALS will be presented and discussed. Three different samples were characterized: the reference material ERM®-FD304, which is a suspension of colloidal silica in aqueous solution and two silica bimodal samples consisting of two populations of SiO2 nanoparticles of unknown size in aqueous solution, with different populations' ratios. The procedure for the preparation of the sample before the analysis, and main separation parameters have been previously defined between the two institutes and will be described. The principals measured parameters were the weight-average (dge_w), number-average (dge_n) and z-average (dge_z) geometric diameter; the average hydrodynamic diameter (dh); and the diameter obtained by external calibration using polystyrene latex standards (dcal). Results between the two NMIs were comparable and coherent with the expected size values of those obtained by other techniques like Scanning Mobility Particle Sizer (SMPS) and Scanning Electron Microscopy (SEM) also involved in this ILC exercise. Where discrepancies are observed, they leave the results compatible within their uncertainties and underpin the challenges in analysing data and reporting results, making AF4 a powerful tool to compare to other measurement techniques.