Titanium dioxide (TiO₂) is used as a food additive (E171) and can be found in sauces, icings, and chewing gums, as well as in personal care products such as toothpaste and pharmaceutical tablets. Along with the ubiquitous presence of TiO₂ and recent insights into its potentially hazardous properties, there are concerns about its application in commercially available products. Especially the nano-sized particle fraction (<100 nm) of TiO₂ warrants a more detailed evaluation of potential adverse health effects after ingestion. A workshop organized by the Dutch Office for Risk Assessment and Research (BuRO) identified uncertainties and knowledge gaps regarding the gastrointestinal absorption of TiO₂, its distribution, the potential for accumulation, and induction of adverse health effects such as inflammation, DNA damage, and tumor promotion. This review aims to identify and evaluate recent toxicological studies on food-grade TiO₂ and nano-sized TiO₂ in ex-vivo, in-vitro, and in-vivo experiments along the gastrointestinal route, and to postulate an Adverse Outcome Pathway (AOP) following ingestion. Additionally, this review summarizes recommendations and outcomes of the expert meeting held by the BuRO in 2018, in order to contribute to the hazard identification and risk assessment process of ingested TiO₂.

Titanium dioxide (TiO₂) is present in many different food products as the food additive E171, which is currently scrutinized due to its potential adverse effects, including the stimulation of tumor formation in the gastrointestinal tract. We developed a transgenic mouse model to examine the effects of E171 on colorectal cancer (CRC), using the Cre-LoxP system to create an Apc-gene-knockout model which spontaneously develops colorectal tumors. A pilot study showed that E171 exposed mice developed colorectal adenocarcinomas, which were accompanied by enhanced hyperplasia in epithelial cells, and increased tumor size. In the main study, tumor formation was studied following the exposure to 5 mg/kg_bw/day of E171 for 9 weeks (Phase I). E171 exposure showed a statistically nonsignificant increase in the number of colorectal tumors in these transgenic mice, as well as a statistically nonsignificant increase in the average number of mice with tumors. Gene expression changes in the colon were analyzed after exposure to 1, 2, and 5 mg/kg_bw/day of E171 for 2, 7, 14, and 21 days (Phase II). Whole-genome mRNA analysis revealed the modulation of genes in pathways involved in the regulation of gene expression, cell cycle, post-translational modification, nuclear receptor signaling, and circadian rhythm. The processes associated with these genes might be involved in the enhanced tumor formation and suggest that E171 may contribute to tumor formation and progression by modulation of events related to inflammation, activation of immune responses, cell cycle, and cancer signaling.

Food-grade titanium dioxide (E171) is a widely used food additive with debated safety, particularly regarding its genotoxic effects. This study assessed the dose-dependent toxicity of E171 in human induced pluripotent stem cell (iPSC)-derived colon organoids. Organoids were exposed to E171 (0.1-1000 μg/mL) for 24 h, and effects on cell viability, reactive oxygen species (ROS) generation, DNA damage, and gene expression were evaluated. Results showed no impact on cell viability but a dose-dependent increase in ROS formation, peaking at 1000 μg/mL. Electrospin Resonance Spectroscopy (ESR) showed a dose-dependent increase in ROS, with increased E171 concentrations. The alkaline comet assay revealed significant DNA damage from 100 μg/mL, with oxidative DNA damage detected at 10 μg/mL using formamidopyrimidine DNA glycosylase (FPG). RNA sequencing identified differentially expressed genes (DEGs) at 100 and 250 μg/mL, linked to translational activity, signal transduction, and DNA damage repair. Gene set enrichment analysis (GSEA) indicated activation of ribosome, chemical carcinogenesis-ROS, and metabolic pathways (carbon metabolism, glycolysis), while key regulatory pathways (Wnt, MAPK, PI3K-Akt) were suppressed. These findings suggest that E171 induces oxidative stress and DNA damage, modulating transcriptomic pathways associated with metabolism, proliferation, and cancer. Further research is necessary to determine its long-term effects on human gastrointestinal health.

Food-grade titanium dioxide (E171) is widely used in food, feed, and pharmaceuticals for its opacifying and coloring properties. This study investigates the formation of reactive oxygen species (ROS) and the aggregation behavior of E171 using the TNO Gastrointestinal (GI) model, which simulates the stomach and small intestine. E171 was characterized using multiple techniques, including electron spin resonance spectroscopy, single-particle inductively coupled plasma–mass spectrometry, transmission electron microscopy, and dynamic light scattering. In an aqueous dispersion (E171-aq), E171 displayed a median particle size of 79 nm, with 73–75% of particles in the nano-size range (<100 nm), and significantly increased ROS production at concentrations of 0.22 and 20 mg/mL. In contrast, when E171 was mixed with yogurt (E171-yog), the particle size increased to 330 nm, with only 20% of nanoparticles, and ROS production was inhibited entirely. After GI digestion, the size of dE171-aq increased to 330 nm, while dE171-yog decreased to 290 nm, with both conditions showing a strongly reduced nanoparticle fraction. ROS formation was inhibited post-digestion in this cell-free environment, likely due to increased particle aggregation and protein corona formation. These findings highlight the innate potential of E171 to induce ROS and the need to consider GI digestion and food matrices in the hazard identification/characterization and risk assessment of E171.

Aims: Food-grade titanium dioxide (E171) is widely used as a food additive, yet concerns persist regarding potential gastrointestinal effects, possibly mediated by interactions with the gut microbiome. This study aimed to investigate the physicochemical behavior of E171 under different digestive contexts and to assess its effects on gut microbial composition and metabolic activity. Methods and results: The dynamic in vitro colon model TIM-2 was used to expose human fecal microbiota to E171 under fasted (aqueous suspension; E171-aq) and fed (yogurt matrix; E171-yog) conditions. Particle size distribution, reactive oxygen species formation, microbiome composition (16S rRNA gene sequencing), and short-chain fatty acid production were analyzed. Larger aggregates were observed under fasted conditions (mean diameter ∼210 nm), whereas digestion in yogurt produced smaller aggregates (mean diameter ∼167 nm) and a higher nanoparticle fraction, reaching up to 20%. No ROS production was detected following fermentation. Both E171-aq and E171-yog significantly increased butyrate levels, indicating altered microbial metabolic activity. Microbiome profiling revealed compositional shifts, including a decreased relative abundance of Blautia and an increased relative abundance of Lachnospiraceae, taxa associated with inflammatory and metabolic responses. Conclusions: E171 undergoes distinct physicochemical transformations depending on the digestive context, with enhanced nanoparticle formation under fed conditions. E171 exposure also modulates gut microbiome composition and function, notably by stimulating butyrate production.