A few non-dietary studies have reported adverse effects in the gastrointestinal tract of laboratory animals given food-grade TiO2. However, these same effects were not seen when the same or higher doses of food-grade TiO2 were administered in the animals' diet. Dietary studies best reflect how humans are exposed to TiO2 from food. Thus, the Food Directorate placed the most emphasis on the results of these studies in the state of the science report.
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.