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The hazard posed to human health by inhaled amorphous silica nanomaterials (aSiO 2  NM) remains uncertain. Herein, we assessed the cyto- and genotoxicity of aSiO 2  NM variants covering different sizes (7, 15, and 40 nm) and surface modifications (unmodified, phosphonate-, amino- and trimethylsilyl-modified) on rat alveolar epithelial (RLE-6TN) cells. Cytotoxicity was evaluated at 24 h after exposure to the aSiO 2  NM variants by the lactate dehydrogenase (LDH) release and WST-1 reduction assays, while genotoxicity was assessed using different endpoints: DNA damage (single- and double-strand breaks [SSB and DSB]) by the comet assay for all aSiO 2  NM variants; cell cycle progression and γ-H2AX levels (DSB) by flow cytometry for those variants that presented higher cytotoxic and DNA damaging potential. The variants with higher surface area demonstrated a higher cytotoxic potential (SiO 2 _7, SiO 2 _15_Unmod, SiO 2 _15_Amino, and SiO 2 _15_Phospho). SiO 2 _40 was the only variant that induced significant DNA damage on RLE-6TN cells. On the other hand, all tested variants (SiO 2 _7, SiO 2 _15_Unmod, SiO 2 _15_Amino, and SiO 2 _40) significantly increased total γ-H2AX levels. At high concentrations (28 µg/cm 2 ), a decrease in G 0 /G 1  subpopulation was accompanied by a significant increase in S and G 2 /M sub-populations after exposure to all tested materials except for SiO 2 _40 which did not affect cell cycle progression. Based on the obtained data, the tested variants can be ranked for its genotoxic DNA damage potential as follows: SiO 2 _7 = SiO 2 _40 = SiO 2 _15_Unmod &gt; SiO 2 _15_Amino. Our study supports the usefulness of multiparametric approaches to improve the understanding on NM mechanisms of action and hazard prediction.

Multiparametric in vitro genotoxicity assessment of different variants of amorphous silica nanomaterials in rat alveolar epithelial cells