Background: Oral exposure to titanium dioxide (TiO₂) is common since it is widely used in food and pharmaceutical products. Concern on the safety of this substance has been recently raised, due to the presence of an ultrafine fraction in food-grade TiO₂. Discrepancy exists among data reported in in vitro and in vivo studies on intestinal acute/chronic toxicity of TiO₂. This might be due to the different biological identity of TiO₂ in traditional in vitro test by respect in vivo conditions. Methods: One food-grade TiO₂ and two nanometric TiO₂ samples were treated with a simulated human digestive dystem (SHDS) in order to investigate the bio-transformation occurring to the particles once ingested in term of size distribution (Dynamic Light Scattering-DLS-, Flow Particle Imaging, Asymmetric Flow Field Flow Fractionation-AF4-) and surface modification (Electrophoretic Light Scattering-ELS-, Electron Paramagnetic Resonance Spectroscopy-EPR-). The effect of SHDS on the cyto-, genotoxicity and potential to induce oxidative stress towards human colorectal carcinoma HCT116 cells was also assessed. Results: Aggregation as a consequence of the high ionic strength of the gastric and intestinal simulated fluids was observed, together with the formation of a partially irreversible bio-corona containing phosphate ions and proteins. Such bio-corona led to a partial masking of the TiO₂ particles surface and reactivity. Pristine and treated TiO₂ nanoparticles showed comparable acute toxicity and genotoxicity toward HCT116 cells, whereas a small decrease of the induction of oxidative stress after treatment was observed. Conclusions: Overall the results underline the importance of SHDS as a tool to improve the predictive power of in vitro tests towards intestinal nanomaterial toxicity.

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