Inhaled titanium dioxide (TiO2) nanoparticles (NPs) can have negative health effects, and have been shown to cause respiratory tract cancer in rats. Inflammation has been linked to oxidative stress, and both have been described as possible mechanisms for genotoxicity of NPs, but rarely examined side-by-side in animal studies. In the present study, a wide range of complementary endpoints have been performed to study TiO2 P25 NP-induced genotoxicity in lung overload and non-overload conditions. Additionally, lung burden, inflammation, cytotoxicity and oxidative stress have also been evaluated in order to link genotoxicity with these responses. To assess quick and delayed responses after recovery, endpoints were evaluated at two time points: 2 h and 35 days after three repeated instillations. This study confirmed the previously described lung overload threshold at approximately 200-300 cm2 of lung burden for total particle surface area lung deposition or 4.2 µl/kg for volume-based cumulative lung exposure dose, above which lung clearance is impaired and inflammation is induced. Our results went on to show that these overload doses induced delayed genotoxicity in lung, associated with persistent inflammation only at the highest dose. The lowest tested doses had no toxicity or genotoxicity effects in the lung. In blood, no lymphocyte DNA damage, erythrocytes chromosomal damage or gene mutation could be detected. Our data also demonstrated that only overload doses induced liver DNA lesions irrespective of the recovery time. Tested doses of TiO2 P25 NPs did not induce glutathione changes in lung, blood or liver at both recovery times.

The characterization of nanoparticles in dispersions, in particular measuring their size and size distribution, is a prerequisite before they can be used in toxicological testing. Such characterization requires reliable methods with good reproducibility. The aim of this study was to evaluate the reproducibility, and thus the potential of Dynamic Light Scattering (DLS) for nanoparticle size determination. DLS is easy to use and well established in most nanotoxicology laboratories. However, reproducibility and in particular variability between measurements done using different instrumental setups have not been addressed systematically before. Here we performed initial experiments with rather monodisperse dispersions of spherical particles in water. Significant discrepancies in the measured distributions were obtained with different DLS instruments, especially when fitting the data using mathematical inversion methods. Significant errors can be made due to different settings being used for fitting the data. These were even more prominent when working with dilute dispersions of very small particles. Our study has identified several important points to be taken into consideration in order to overcome possible issues in measurement and analysis of nanoparticles using DLS.In practice, however, nanoparticles may have significant polydispersities and/or can be non-spherical. We extend the comparative work on spherical particles, to show how to characterize polydisperse and/or high aspect ratio particles using DLS instruments.