Is aggregated synthetic amorphous silica toxicologically relevant?

Abstract

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.

Keywords: Aggregates; Biological activity; In vitro toxicity; Nanomaterials; Synthetic amorphous silica.

Fig. 1

Preparation of synthetic amorphous silica suspensions (SAS). DE-AGGR - 2.56 mg/mL of SAS sonicated with an energy of 7056 J (a); AGGR - 2.56 mg/mL of SAS vortexed for 10 s (b); SuperN (0.64 mg/mL) - AGGR dispersion left undisturbed for 15 min after vortexing and fractionated in supernatant (c) and PREC (1.92 mg/mL) - precipitates of AGGR (d)

Fig. 2

Representative TEM micrographs of freshly prepared SAS stock suspensions. DE-AGGR (a), AGGR (b), SuperN (c) and PREC (d)

Fig. 3

Representative bright field microscopic images of freshly prepared SAS stock suspensions. DE-AGGR (a), AGGR (b), SuperN (c) and PREC (d). Scale bar -20 μm

Fig. 4

Estimated concentration reaching the bottom of the wells after 24 h as a function of nominal concentrations applied in cell based assays. Dosimetry simulation was performed by a distorted grid (DG) model and compared for different SAS suspensions using parameters obtained from exposure media DMEM/F12 (a and b) and RPMI 1640 (c and d). The slope values are indicated near the respective lines. R² > 0.9 for all the suspensions. The percentage of dose delivered to the cells did not differ in 96 or 24 well plates, as the height of the liquid column was equal (6 mm)