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The lack of clear regulations and controls explains that P25TiO₂NPs are still found in many of the commercialized sunscreens in the market. Some of them are coated to reduce the photoactivity of the anatase form, which is known to be responsible for tissue damage, but not enough studies were made on these coated forms. The anatase photoactivity could trigger the production of reactive oxygen species (ROS) generation, as it was stated before. The ROS are chemically reactive species containing oxygen, such as peroxides, superoxide, hydroxyl radical, and singlet oxygen. They are regularly produced in the biological milieu and counterbalanced by physiological antioxidant defense mechanisms. However, an abrupt increase of ROS may result in non-reversible damage to the skin cells. The effects of coated and uncoated P25TiO₂NPs need therefore to be studied, and articles on this topic present different conclusions. [11], [12], [13] Recent literature on this topic found that TiO₂NPs inhalation provokes serious genotoxicity and DNA damage [14], [15], [16], [17]. On the other hand, some studies in rats have reported no significant harm to genetic material [18], [19], [20], [21], [22].

The production of ROS was studied on white blood cells as a model to screen the effect on eukaryotic cells after being exposed to samples and solar simulated irradiation (according to the level of penetration under the skin). For that purpose, the leukocytes were separated from anticoagulated fresh blood using the Ficoll-Hypaque reactive in a well-known technique [33]. Then, 50 μL of suspensions of P25TiO₂NPs (0.2 mg/mL and 0.02 mg/mL), vitaminB2@P25TiO₂NPs (0.2 mg/mL and 0.02 mg/mL) and vitamin B2 (0.2 mg/mL and 0.02 mg/mL) were prepared and mixed with 50 μL of white blood cells suspension. A solution of 3% H₂O₂ was used as positive control and PBS as negative control. Then, the samples were irradiated using the LED panel for 3 and 6 h to simulate the light penetration into the skin. Also, a set of samples was kept in the dark as control. Finally, the ROS were detected through the colorimetric assay employing the nitroblue tetrazolium salt (NBT salt) and the absorbance at 650 nm was measured. The experiment was reproduced twice; the standard deviation was calculated and p-value < 0.05 were considered significant.