**1. Introduction**

Titanium dioxide (TiO2) is one of the main food additives used for its coloring and opacifying properties to improve the appearance and taste of processed foods. Food-grade TiO2 is found in over 900 food products such as pastries, sauces, ice-creams, candies, chocolates, and chewing gum. In foods, TiO2 is commonly reported as E171. It is also referred to as CI 77891 when used in cosmetics and toothpaste as a white colorant [1]. E171 consists of a wide range of particle TiO2 sizes and can contain up to 36% nanosized TiO2 particles, i.e., less than 100 nm in diameter [2,3]. Compared with their macroscopic counterparts, nanoparticles (NPs) can easier pass through the body's cells and then into the bloodstream and internal organs such as liver, kidney, and lung tissues. Daily, the human

**Citation:** Rinninella, E.; Cintoni, M.; Raoul, P.; Mora, V.; Gasbarrini, A.; Mele, M.C. Impact of Food Additive Titanium Dioxide on Gut Microbiota Composition, Microbiota-Associated Functions, and Gut Barrier: A Systematic Review of In Vivo Animal Studies. *Int. J. Environ. Res. Public Health* **2021**, *18*, 2008. https:// doi.org/10.3390/ijerph18042008

Academic Editors: Diana María Cardona Mena and Pablo Roman

Received: 26 December 2020 Accepted: 13 February 2021 Published: 19 February 2021

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dietary exposure dose of TiO2 NPs can reach one to four micrograms per kilogram body weight per day (μg per kg bw per day) [3]. In 1966, the Food and Drug Administration (FDA) approved the use of food-grade TiO2 referred to as INS171, specifying that the quantity of TiO2 must not exceed one percent by weight of the food [4]. In Europe, in 2006, the European Food Safety Authority (EFSA) authorized the use of E171 in food concluding that E171 is safe for consumers, having margins of safety (MoS) of 2.25 mg per kg bw per day [5,6]. However, TiO2 NPs raise health concerns among the scientific community and governments given their potential to cross the gu<sup>t</sup> barrier and distribute to other organs eliciting immunological response. In June 2018, the EFSA evaluated four new in vivo and in vitro studies [7–10] assessing potential toxicities and reaffirmed the safety of E171 [11]. In April 2019, the French Agency for Food, Environmental and Occupational Health and Safety (ANSES) published a review suggesting a genotoxic and carcinogenic potential even if further in vivo mammalian studies are warranted to confirm or rule out these hypotheses [12]. As requested by the European Commission, EFSA provided urgen<sup>t</sup> scientific and technical review regarding the opinion issued by ANSES [13]. The EFSA concluded that the latest ANSES opinion does not identify any major new findings that would overrule the conclusions made in the previous two scientific opinions in 2016 and 2018. The latest ANSES opinion reiterated the previously identified uncertainties and emphasized that there was still not enough data available to carry out a proper assessment of the risks associated with the food use of E171. EFSA considered this recommendation should be revisited once the ongoing work on the physicochemical characterization of E171 will be completed. In January 2020, France has adopted a decree to ban the use of E171 in foods as a precautionary measure to protect consumers' health.

In a scientific context of "microbiota revolution", potential health risks of TiO2 NPs and their impact on the intestinal tract and the gu<sup>t</sup> microbiota are increasingly being studied. Gut microbiota is composed of millions of bacterial species that bi-directionally interact with the host in the intestinal tract, regulating the development of immune cells. Alterations in the abundance and composition of intestinal microbiota, known as dysbiosis, are associated with host health such as brain function, lipid metabolism, immune responses, and development of diseases [14]. Recent studies reported adverse effects of in vitro exposure of intestinal epithelial cells to E171 [9,15,16]. Indeed, TiO2 NPs could damage microvilli structure and alter epithelial integrity [17,18]. TiO2 NPs can be internalized and can cross the epithelial barrier to enter the bloodstream and potentially affect the function of distant organs, such as the liver [19]. Moreover, in vitro, NPs have the potential to negatively affect intestinal functions and gu<sup>t</sup> homeostasis associated with gu<sup>t</sup> microbiota [20]. New evidence from numerous recent animal studies has emerged highlighting the effects of various physiological doses of TiO2 NPs on gu<sup>t</sup> microbiota composition and gu<sup>t</sup> homeostasis. Such evidence has not ye<sup>t</sup> been systematically reviewed. Hence, we sought to systematically review current evidence from in vivo animal models to disentangle the TiO2 effects on the gu<sup>t</sup> microbiome composition and functions.
