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.

Koi herpesvirus (KHV), recently designated Cyprinid herpesvirus 3 , is the causative agent of a lethal disease in koi and common carp. In the present study, we investigated the portal of entry of KHV in carp by using bioluminescence imaging. Taking advantage of the recent cloning of the KHV genome as a bacterial artificial chromosome (BAC), we produced a recombinant plasmid encoding a firefly luciferase (LUC) expression cassette inserted in the intergenic region between open reading frame (ORF) 136 and ORF 137. Two viral strains were then reconstituted from the modified plasmid, the FL BAC 136 LUC excised strain and the FL BAC 136 LUC TK revertant strain, including a disrupted and a wild-type thymidine kinase (TK) locus, respectively. In vitro, the two recombinant strains replicated comparably to the parental FL strain. The FL BAC 136 LUC TK revertant strain was shown in vitro to induce a bioluminescent signal allowing the detection of single positive cells as early as 24 h postinfection, while in vivo, it induced KHV infection in carp that was indistinguishable from that induced by the parental FL strain. To identify the KHV portal of entry, carp were analyzed by bioluminescence imaging at different times postinfection with the FL BAC 136 LUC TK revertant strain. These analyses demonstrated that the skin of the fish covering the fins and also the body is the major portal of entry for KHV in carp. Finally, to further demonstrate the role of the skin as the KHV portal of entry, we constructed an original system, nicknamed “U-tube,” to perform percutaneous infection restricted to the posterior part of the fish. All the data obtained in the present study demonstrate that the skin, and not the gills, is the major portal of entry for KHV in carp.

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-1β 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.

An approach for the size measurement of particulate (nano)materials by transmission electron microscopy was evaluated. The approach combines standard operating procedures for specimen preparation, imaging, and image analysis, and it was evaluated on a series of certified reference materials and representative test materials with varying physical properties, including particle size, shape, and agglomeration state. The measurement of the median value of the minimal external particle diameter distribution was intra-laboratory validated. The validation study included an assessment of the limit of detection, working range, selectivity, precision, trueness, robustness, and ruggedness. An uncertainty that was associated to intermediate precision in the range of 1–7% and an expanded measurement uncertainty in the range of 7–20% were obtained, depending on the material and image analysis mode. No bias was observed when assessing the trueness of the approach on the certified reference materials ERM-FD100 and ERM-FD304. The image analysis method was validated in an inter-laboratory study by 19 laboratories, which resulted in a within-laboratory precision in the range of 2–8% and a between-laboratory precision of between 2% and 14%. The automation and standardization of the proposed approach significantly improves labour and cost efficiency for the accurate and precise size measurement of the particulate materials. The approach is shown to be implementable in many other electron microscopy laboratories.

E171 (titanium dioxide) is a food additive that has been authorized for use as a food colorant in the European Union. The application of E171 in food has become an issue of debate, since there are indications that it may alter the intestinal barrier. This work applied standardized and validated methodologies to characterize representative samples of 15 pristine E171 materials based on transmission electron microscopy (TEM) and single-particle inductively coupled plasma mass spectrometry (spICP-MS). The evaluation of selected sample preparation protocols allowed identifying and optimizing the critical factors that determine the measurement of the particle size distribution by TEM. By combining optimized sample preparation with method validation, a significant variation in the particle size and shape distributions, the crystallographic structure (rutile versus anatase), and the physicochemical form (pearlescent pigments versus anatase and rutile E171) was demonstrated among the representative samples. These results are important for risk assessment of the E171 food additive and can contribute to the implementation of the European Food Safety Authority (EFSA) guidance on risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain.

A recombinant GroES nanopore reveals the dynamics and kinetics of the allosteric intermediates of the GroEL protein-folding reaction.