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Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate  in vitro  test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The  in vitro  toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO 2  NMs with different surface chemistry – hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO – uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO 2  NMs produced by two different manufacturing techniques – precipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO 2 >TiO 2 ). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO 2  and SiO 2 , while the embryonic stem cell test (EST) classified the TiO 2  NMs as potentially ‘weak-embryotoxic’ and ZnO and SiO 2  NMs as ‘non-embryotoxic’. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO 2  NM-203 > SiO 2  NM-200 > TiO 2  NM-104 > TiO 2  NM-103). This ranking was different in the case of embryonic tissues, for which TiO 2  displayed higher toxicity compared with ZnO and SiO 2 . Importantly, the  in vitro  methodology applied could identify cell- and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects.

Comprehensive In Vitro Toxicity Testing of a Panel of Representative Oxide Nanomaterials: First Steps towards an Intelligent Testing Strategy