UV–vis spectra were carried out in the supernatant of both vitamins@P25TiO₂NPs samples after centrifugation to measure the amount of unbound vitamin. Standard curves at 375 nm and 255 nm were done for vitamin B2 and C, respectively, using a Synergy BioTeK multi-mode microplate reader.

The most significant uncertainty identified by the EU experts was the concern that TiO₂ particles may have genotoxic effects. Genotoxicity refers to the ability of a chemical to directly damage genetic material within a cell (DNA), which may lead to cancer in certain situations. Although the experts did not conclude that TiO₂ particles in E171 are genotoxic, they could not rule out the concern that they might be.

Lithopone: an alternative to titanium dioxide