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The genotoxicity of anatase/rutile TiO₂ nanoparticles (TiO₂ NPs, NM105 at 3, 15 and 75 &#181;g/cm²) was assessed with the mammalian in-vitro Hypoxanthine guanine phosphoribosyl transferase (<i>Hprt</i>) gene mutation test in Chinese hamster lung (V79) fibroblasts after 24 h exposure. Two dispersion procedures giving different size distribution and dispersion stability were used to investigate whether the effects of TiO₂ NPs depend on the state of agglomeration. TiO₂ NPs were fully characterised in the previous European FP7 projects NanoTEST and NanoREG2. Uptake of TiO₂ NPs was measured by transmission electron microscopy (TEM). TiO₂ NPs were found in cytoplasmic vesicles, as well as close to the nucleus. The internalisation of TiO₂ NPs did not depend on the state of agglomeration and dispersion used. The cytotoxicity of TiO₂ NPs was measured by determining both the relative growth activity (RGA) and the plating efficiency (PE). There were no substantial effects of exposure time (24, 48 and 72 h), although a tendency to lower RGA at longer exposure was observed. No significant difference in PE values and no increases in the <i>Hprt</i> gene mutant frequency were found in exposed relative to unexposed cultures in spite of evidence of uptake of NPs by cells.