# Titanium Dioxide in Chromogenic Devices: Synthesis, Toxicological Issues, and Fabrication Methods
## 3 Toxicity Assessment of TiO\({}_{2}\) NPs and Films  
The strategies adopted to evaluate the toxicity and the conceivable safety of nanomaterials are the same as those used for conventional drugs [117]. However, nanosized materials exhibit unique physico-chemical properties that require specific methods in order to assess the in vivo response and the uptake effectiveness, drug release, and kinetics [118; 119]. So far, no specific protocols have been developed in the preclinical studies, although regulatory institutions from the USA (Food and Drugs Administration, FDA), EU (European Medicines Agency, EMA), and Japan (Pharmaceuticals and Medical Devices Agency, PDMA) are making strong efforts to produce a clear proposition regarding nanomaterial safety [120; 121]. The National Center for Advancing Translational Sciences (NCATS/NIH) in the United States and the Innovative Medicines Initiative (IMI) in Europe are the relevant institutions who are aiming to develop regulatory protocols [122]. NPs can enter into living organisms through different routes, which are inhalation, ingestion, and skin penetration. Many in vitro studies have shown the potential toxicity of TiO\({}_{2}\) NPs on different cell lines; these effects are strongly dependent on NP shape, size, aggregation, and crystalline forms. In general, the anatase phase was found to be more toxic than the rutile phase; in addition, the small size and tubular shape were associated to higher TiO\({}_{2}\) toxicity. However, many variables in the safety assessment are introduced by cell line models, which are different in terms of responsiveness, surface receptors, uptake mechanisms, and sensibility due to the activation of different biochemical pathways during the NP exposure time. For this reason, more complete studies devoted to the understanding of toxicity mechanisms are derived from in vivo studies in which it is also possible to study NP distribution in the tissues and organs once they have entered the body. Several studies were performed in order to understand the toxicity of TiO\({}_{2}\) NPs using different animal models and different administration routes. In order to mimic the inhalation process, a micro-syringe was used to administer rutile 500 ug of TiO\({}_{2}\) NPs (80 nm) and anatase (155 nm) every day through the nose of female mice, showing the accumulation in the brain [123]. The authors analyzed the inflammation response and oxidative stress (up to30 days of exposure) with daily TiO\({}_{2}\) NPs administration. The boost of lipid peroxidation induced a strong increase of tumor necrosis factor alpha (TNF-alpha) and interleukin (IL-1 beta), particularly after anatase phase exposure. Additionally, hamsters, rats, and mice treated by TiO\({}_{2}\) NPs (P25) for up to 13 weeks showed macrophages and neutrophils activation, followed by strong pulmonary inflammation [124]. The same NPs, at a concentration of 4.1 mg/m\({}^{3}\), were used to treat Wistar rats by aerosol administration for four weeks, and an increase of neutrophils in the bronchoalveolar lavage fluid (BALF) was recorded [125]. TiO\({}_{2}\) NPs are also commonly used as white food additives (E171) in a lot of commercial products such as sweets, chewing gums, and puddings [21]; gastro-intestinal accumulation has been studied as well as possible accumulation in several organs. In mice [126], the oral exposure to TiO\({}_{2}\) NPs at different concentrations (0, 324, 648, 972, 1296, 1944, 2592 mg/kg) up to 14 days provoked lethurgy, loss of appetite, and passive behavior; in addition, accumulation of TiO\({}_{2}\) NPs was shown in the liver, kidneys, lungs, and spleen. The latter organ was also investigated in another work, after the administration of TiO\({}_{2}\) NPs into the stomach for one month (5, 50, and 150 mg/kg body weight). The spleen underwent a damage and inflammation process due to the activation of a strong lipid peroxidation triggered by ROS production [127].