Infrared analysis showed that the characteristics bands for the bare nanoparticles are still exhibited in the vitamins@P25TiO₂NPs spectra, such as a wide peak in 450–1028 cm⁻¹ related to the stretching vibration of Ti-O-Ti and other peaks in 1630 cm⁻¹ and 3400 cm⁻¹, which represent the surface OH groups stretching. The IR spectrum of vitaminB2@P25TiO₂NPs showed signs of binding between compounds. The OH bending peak (1634 cm⁻¹) corresponding to bare nanoparticles disappeared, and the NH₂ bending band characteristic of vitamin B2 appeared (1650 cm⁻¹). The IR spectrum of vitaminC@P25TiO₂NPs also showed signs of successful functionalization. Bands at 1075 cm⁻¹; 1120 cm⁻¹; 1141 cm⁻¹ were observed, which are originated by CO-C vibrations present in the vitamin C. The intense band at 1672 cm⁻¹ is attributed to the C = O stretching in the lactone ring while the peak at 1026 cm⁻¹ is ascribed to the stretching vibration Ti-O-C. Wide bands at 3880–3600 cm⁻¹ are related to stretching vibration OH groups, but those disappear in the modified nanoparticles spectrum. These observations confirm the interactions between the P25TiO₂NPs and the vitamins [35].

In 2021, the European Food Safety Authority concluded that titanium dioxide is no longer safe in foods due to the same concerns over nanoparticles. As a result, titanium dioxide is now banned as a food additive in the EU. Although studies have shown that the absorption of ingested titanium dioxide is low, evidence suggests that titanium dioxide nanoparticles can accumulate in the body over time. Health Canada deemed it safe in 2022 but noted concerns. Unlike their European counterparts, Canadian officials did not consider studies performed with titanium dioxide nanoparticles alone. 

The toxicity of P25TiO₂NPs was evaluated in both prokaryotic (Fig. 3) and eukaryotic cells (Fig. 4). The XTT assay was chosen to measure the cell viability in bacterial cultures of MSSA, a normal skin microbiota microorganism. The reduction in the viability of samples with bare NPs is notorious, possibly due to the described ROS production from the interaction of P25TiO₂NPs with light [37]. This effect seems to be avoided when they are functionalized with vitamin B2. Also, the most concentrated vitaminB2@P25TiO₂NPs sample (0.2 mg/mL) shows up to 60% more absorbance after 6 h compared to the bare NPs (due to normal cell replication). This may indicate that the antioxidant effect of the vitamin B2 coating is greater than the oxidation damage produced by the NPs. This protective capacity could be attributed to the glutathione redox cycle and the conversion of reduced riboflavin to its oxidized form [38]. Values of cell viability greater than 100% are not rare and could be understood because the XTT assay actually measure metabolic activity when reducing the tetrazole to formazan. It is usually assumed that conversion is dependent on the number of viable cells, but it could also be related to an expected increased enzymatic activity when cells are exposed to small doses of some new substance. Further analysis showed that this effect was not the only one responsible for better cell viability of vitaminB@P25TiO₂NPs treated samples.

The EU expert panel did not identify an immediate health concern linked to TiO₂ when used as a food additive. However, due mainly to uncertainties concerning the safety of TiO₂ nanoparticles, the panel concluded that TiO₂ as a food additive (E171) could no longer be considered safe.

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Genotoxicity Assessment 

Although the evidence for general toxic effects was not conclusive, on the basis of the new data and strengthened methods we could not rule out a concern for genotoxicity and consequently we could not establish a safe level for daily intake of the food additive, commented Matthew Wright, member of the EFSA's Food Additives and Flavourings Panel in a press statement.

Titanium dioxide is a food additive that can be found in over 3,000 different products in EWG’s Food Scores database.

Genotoxicity refers to the ability of a chemical substance to damage DNA , the genetic material of cells. As genotoxicity may lead to carcinogenic effects, it is essential to assess the potential genotoxic effect of a substance to conclude on its safety.  

A 2023 study published in the journal Particle and Fibre Toxicology set out to examine the impact of titanium dioxide nanoparticles in mice “on the course and prognosis of ulcerative colitis,” by creating an ulcerative colitis disease model. Researchers found that the titanium dioxide nanoparticles significantly increased the severity of colitis. They also “decreased the body weight, increased the disease activity index and colonic mucosa damage index scores, shortened the colonic length, increased the inflammatory infiltration in the colon.” Researchers concluded: “Oral intake of TiO2 nanoparticles could affect the course of acute colitis in exacerbating the development of ulcerative colitis, prolonging the ulcerative colitis course and inhibiting ulcerative colitis recovery.”

Lithopone is rather nontoxic, due to the insolubility of its components. It has been used in medicine as a radiocontrast agent. Lithopone is allowed to be in contact with foodstuffs in the US and Europe.

Stability and darkening[edit]

Additionally, the committee noted that the available data did not provide convincing evidence of genotoxicity for titanium dioxide as a food additive, but recognized the limitations in current methodologies with respect to the testing of poorly soluble particulate materials. Although there were uncertainties in the genotoxicity data, the experts took into account the fact that the additive was not carcinogenic in adequately conducted two-year studies in mice and rats at doses of up to 7,500 mg/kg BW per day for mice, and 2,500 mg/kg BW per day for rats, the highest doses tested. There was also no evidence of reproductive or developmental toxicity in studies in rats at doses up to 1,000 mg/kg BW per day, the highest doses tested.