’.

Following the filtration, the precipitate is washed thoroughly to remove any impurities and excess reagents. This is a crucial step, as thorough washing ensures that no contaminants interfere with the final mass measurement. Once washed, the precipitate is dried in an oven at a controlled temperature to remove moisture, ensuring that the mass recorded reflects only the titanium content.

At the present JECFA meeting, the committee considered additional toxicological studies relevant to the safety assessment of the chemical that investigated its toxicokinetics, acute toxicity, short-term toxicity, long-term toxicity and carcinogenicity, genotoxicity, and reproductive and developmental toxicity, as well as special studies addressing its short-term initiation/promotion potential for colon cancer. The experts acknowledged that a large number of toxicological studies have been conducted using test materials, including nanoparticles, having size distributions and physico-chemical properties not comparable to real-world uses of titanium dioxide as a food additive. The studies on non-representative materials were evaluated by JECFA, but the committee concluded that such studies are not relevant to the safety assessment of the additive.

Overall, the Food Directorate's comprehensive review of the available science of TiO₂ as a food additive showed:

As mentioned above, these oxide NPs are harmful in part because both anatase and rutile forms are semiconductors and produce ROS. Particularly, P25 kind has band-gap energies estimated of 3.2 and 3.0 eV, equivalent to radiation wavelengths of approximately 388 and 414 nm, respectively. Irradiation at these wavelengths or below produces a separation of charge, resulting in a hole in the valence band and a free electron in the conduction band, due to the electron movement from the valence to conduction bands. These hole–electron pairs generate ROS when they interact with H₂O or O₂ [43,44]. It was described that they can cause an increase in ROS levels after exposure to UV-visible light [45]. The NBT assay in the studied samples showed that bare P25TiO₂NPs produce a large amount of ROS, which is drastically reduced by functionalization with vitamin B2 (Fig. 5). This vitamin, also known as riboflavin, was discovered in 1872 as a yellow fluorescent pigment, [46] but its function as an essential vitamin for humans was established more than sixty years later, and its antioxidant capacity was not studied until the end of the XX century [47,48]. This antioxidant role in cells is partially explained because the glutathione reductase enzyme (GR) requires it for good functionality. This enzyme is the one in charge of the conversion of oxidized glutathione to its reduced form which acts as a powerful inner antioxidant and can quench the ROS [49,50]. The cost of this action is that the glutathione is converted to the oxidized form and needs to be recovered by the GR. Consequently, the cells need more vitamin B2. Another glutathione action is the protection against hydroperoxide. This activity is also mediated by riboflavin. Therefore, local delivery of this vitamin seems to significantly help the cells in their fight to keep the oxidative balance, once they are exposed to high levels of ROS.