# Food-grade titanium dioxide translocates across the buccal mucosa in pigs and induces genotoxicity in an in vitro model of human oral epithelium

Julien Vignard, Aurelie Pettes-Duler, Eric Gaultier, Christel Cartier, Laurent

Weingarten, Antje Biesemeier, Tatjana Taubitz, Philippe Pinton, Cecilia

Bebeacua, Laurent Devoille, et al.

► To cite this version:

Julien Vignard, Aurelie Pettes-Duler, Eric Gaultier, Christel Cartier, Laurent Weingarten, et al. Food-grade titanium dioxide translocates across the buccal mucosa in pigs and induces genotoxicity in an in vitro model of human oral epithelium. Nanotoxicology, inPress, pp.1-21. 10.1080/17435390.2023.2210664. hal-04104748

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# Food-grade titanium dioxide translocates across the buccal mucosa in pigs and induces genotoxicity in an *in vitro* model of human oral epithelium

Julien Vignard, Aurelie Pettes-Duler, Eric Gaultier, Christel Cartier, Laurent Weingarten, Antje Biesemeier, Tatjana Taubitz, Philippe Pinton, Cecilia Bebeacua, Laurent Devoille, Jacques Dupuy, Elisa Boutet-Robinet, Nicolas Feltin, Isabelle P. Oswald, Fabrice H. Pierre, Bruno Lamas, Gladys Mirey & Eric Houdeau

**To cite this article:** Julien Vignard, Aurelie Pettes-Duler, Eric Gaultier, Christel Cartier, Laurent Weingarten, Antje Biesemeier, Tatjana Taubitz, Philippe Pinton, Cecilia Bebeacua, Laurent Devoille, Jacques Dupuy, Elisa Boutet-Robinet, Nicolas Feltin, Isabelle P. Oswald, Fabrice H. Pierre, Bruno Lamas, Gladys Mirey & Eric Houdeau (2023): Food-grade titanium dioxide translocates across the buccal mucosa in pigs and induces genotoxicity in an *in vitro* model of human oral epithelium, Nanotoxicology, DOI: 10.1080/17435390.2023.2210664

**To link to this article:** https://doi.org/10.1080/17435390.2023.2210664

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# Food-grade titanium dioxide translocates across the buccal mucosa in pigs and induces genotoxicity in an in vitro model of human oral epithelium

Julien Vignard<sup>a</sup>, Aurelie Pettes-Duler<sup>a</sup>, Eric Gaultier<sup>a</sup>, Christel Cartier<sup>a</sup>, Laurent Weingarten<sup>b</sup>, Antje Biesemeier<sup>c</sup>, Tatjana Taubitz<sup>c</sup>, Philippe Pinton<sup>a</sup>, Cecilia Bebeacua<sup>d</sup>, Laurent Devoille<sup>e</sup>, Jacques Dupuy<sup>a</sup>, Elisa Boutet-Robinet<sup>a</sup>, Nicolas Feltin<sup>e</sup>, Isabelle P. Oswald<sup>a</sup>, Fabrice H. Pierre<sup>a</sup>, Bruno Lamas<sup>a</sup>, Gladys Mirey<sup>a</sup> and Eric Houdeau<sup>a</sup>

## ABSTRACT

The whitening and opacifying agent titanium dioxide (TiO₂) is used worldwide in various food-stuffs, toothpastes and pharmaceutical tablets. Its use as a food additive (E171 in EU) has raised concerns for human health. Although the buccal mucosa is the first area exposed, oral transmucosal passage of TiO₂ particles has not been documented. Here we analyzed E171 particle translocation *in vivo* through the pig buccal mucosa and *in vitro* on human buccal TR146 cells, and the effects on proliferating and differentiated TR146 cells. In the buccal floor of pigs, isolated TiO₂ particles and small aggregates were observed 30 min after sublingual deposition, and were recovered in the submandibular lymph nodes at 4 h. In TR146 cells, kinetic analyses showed high absorption capacities of TiO₂ particles. The cytotoxicity, genotoxicity and oxidative stress were investigated in TR146 cells exposed to E171 in comparison with two TiO₂ size standards of 115 and 21 nm in diameter. All TiO₂ samples were reported cytotoxic in proliferating cells but not following differentiation. Genotoxicity and slight oxidative stress were reported for the E171 and 115 nm TiO₂ particles. These data highlight the buccal mucosa as an absorption route for the systemic passage of food-grade TiO₂ particles. The greater toxicity on proliferating cells suggests potential impairment of oral epithelium renewal. In conclusion, this study emphasizes that buccal exposure should be considered during toxicokinetic studies and for risk assessment of TiO₂ in human when used as food additive, including in toothpastes and pharmaceutical formulations.

## ARTICLE HISTORY

Received 18 January 2023
Revised 3 April 2023
Accepted 1 May 2023

## KEYWORDS

Oral mucosa; titanium dioxide; nanoparticles; cytotoxicity; genotoxicity

## Introduction

Due to the rapid expansion of nanotechnologies and the daily increasing use of nanomaterials in consumer products, there is a growing need to assess the toxicological risks of these materials on human health. Such concern increases when nano-particles (NPs) are found in food additives and coating substances or are included in food packaging, leading to chronic oral exposure to NPs for consumers. Among these agents, food-grade titanium dioxide (TiO₂) is commonly used as a food additive worldwide, and is referred to as E171 in European

Union. It is used *ad quantum satis* as a whitening and brightening agent in a variety of food products (confectionary and bakery commodities, white sauces and icing), as beverage whiteners and in personal care products such as toothpaste but also in pharmaceutical tablets (Bischoff et al. 2020; European Medicines Agency 2021; Palugan et al. 2022). For these uses, large amounts of TiO₂ powders are produced and are composed of particles of various sizes ranging from 20 to 400 nm, and up to 55% of them by number are NPs (diameter <100 nm) (Weir et al. 2012; Bettini et al. 2017;

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<footnote><sup>a</sup>Toxalim UMR1331 (Research Centre in Food Toxicology), Toulouse University, INRAE, ENVT, INP-Purpan, UPS, Toulouse, France;</footnote>

<footnote><sup>b</sup>Centre de MicroCaractérisation Raimond Castaing, UAR 3623, Toulouse, France; <sup>c</sup>Luxembourg Institute of Science and Technology (LIST), Materials Research and Technology (MRT), Advanced Instrumentation for Ion Nano-Analytics (AINA), Esch-sur-Alzette, Luxembourg; <sup>d</sup>ETH Zurich, Zurich, Switzerland; <sup>e</sup>Department of materials, LNE, Trappes, France</footnote>

<footnote>CONTACT Julien Vignard julien.vignard@inrae.fr; Eric Houdeau eric.houdeau@inrae.fr</footnote>

<footnote>Supplemental data for this article can be accessed online at https://doi.org/10.1080/17435390.2023.2210664.</footnote>
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Dorier et al. 2017; Guillard et al. 2020). Focusing on only food origin, depending on the exposure scenario and population groups, the mean dietary intake in humans has been estimated to range from 0.03 mg of TiO₂/kg of body weight (bw)/day (d) in infants to 11.5 mg/kg bw/d in children under 10 years of age and up to 6.7 mg/kg bw/d for older groups (Younes et al. 2021). Concerning TiO₂ fate and organ toxicity, chronic exposure to TiO₂ has been reported to result in particle accumulation in human tissues, including the intestine, liver, spleen and kidney (Heringa et al. 2018; Peters et al. 2020) as well as in the placenta (Guillard et al. 2020). Investigations in rodent models and cell lines have raised concerns regarding genotoxicity, inflammation and oxidative stress (Bischoff et al. 2020) as well as the potential for E171 to initiate and promote preneoplastic lesions in the rat colon (Bettini et al. 2017; Medina-Reyes et al. 2020). In mice, daily exposure to food-grade TiO₂ in a colitis-associated colorectal cancer model also exacerbated tumor formation in the colon (Urrutia-Ortega et al. 2016).

Because of these potential hazards to humans, a ban on the use of E171 in foods has been implemented in the EU in 2022 (Commission Regulation (EU))) 2022), while TiO₂ remains approved in the pharmaceutical industry for oral formulations among other applications (cosmetics, toothpaste), and is still allowed in the food chain outside the EU. To date, risk assessments of TiO₂ by food and consumer product safety authorities has been mainly based on the assumption that orally ingested TiO₂-NPs are mainly absorbed by the intestine (EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS))) 2016; Heringa et al. 2016; Younes et al. 2021). However, oral toxicokinetic studies have estimated that only 0.02% to 0.6% of the administered TiO₂ dose is absorbed at the intestinal level including in humans (Cho et al. 2013; Jones et al. 2015; EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS))) 2016; Kreyling et al. 2017). When considering the oral uptake of xenobiotics, the buccal cavity represents the first area of exposure and thus the first possible systemic delivery portal. In the context of food additives, the cellular uptake and toxicity potential of food-grade TiO₂ has not been addressed in a buccal model, although the mouth should be considered to be the body region

exposed to a higher load of TiO₂-NPs once they are released from the food matrix. Indeed, with the example of chewing gum, among other sweets in which TiO₂ is used as a surface coloring agent (Chen et al. 2013; Fiordaliso et al. 2018), TiO₂ particles may be easily released from the gum (Chen et al. 2013; Dudefoi et al. 2018), dispersed in the saliva, and rapidly come into contact with the buccal epithelium. Similar scenarios can be drawn in other food categories where TiO₂ is added to a liquid or semiliquid matrix including ice cream, sauces and drinks (Younes et al. 2021), or when used as an opacifier in pharmaceutical tablets coating formulations (Palugan et al. 2022).

Given the lack of information on the food additive E171, the potential of toxicity of TiO₂ at the mouth level has been addressed in few studies using non-food NP models of known sizes. In a porcine ex vivo model of the buccal cavity exposed to nanomodels, five TiO₂-NPs with distinct physicochemical properties were shown to permeate the mucosa layer and penetrate the oral epithelium (Teubl et al. 2015a; Teubl et al. 2015b). Mucosal penetration and intracellular outcome depend on particle size and surface hydrophilicity/hydrophobicity. Indeed, TiO₂-NPs penetrated the entire buccal epithelium and the connective tissue, except for the nanomaterials with the smallest particle size (i.e., <30 nm), which were unable to reach the lower epithelium (Teubl et al. 2015a). Such size-dependent permeation into the deeper part of the buccal mucosa has already been observed for the penetration of neutral polystyrene NPs (Teubl et al. 2013). Moreover, hydrophilic TiO₂-NPs appeared to be freely distributed in the cytoplasm as small aggregates whereas their hydrophobic counterparts were encapsulated into vesicle structures. Regardless of their cellular distribution, none of the tested TiO₂-NPs were shown to affect cell viability or membrane integrity in the TR146 human buccal cell line, while certain triggered the production of reactive oxygen species (Teubl et al. 2015a; Teubl et al. 2015b). Nonetheless, this evaluation remains to be investigated with the food form of TiO₂ for risk assessment purposes given the mixed composition of nano- and submicron-sized particles in commercial E171 batches.

The median turnover of the buccal mucosa is 14 days (Teubl et al. 2015a), implying active stem
cell division to ensure epithelium renewal. Therefore, in the context of oral exposure, it is important to take into account the role of the cell cycle when assessing particle toxicity. To gain insight into the possible toxic effects of food-grade TiO₂ at the mouth level, the translocation of TiO₂ particles from the food additive E171 was first assessed *in vivo* in piglet mouths, for which the histomorphology of the buccal mucosa is comparable to that of humans. Second, we used the human TR146 cell line, either in cycling or noncycling differentiated cells, as a model of the buccal mucosa composed of cells with different proliferation stages. The kinetic of the cellular permeability to foodborne TiO₂ particles, as well as cytotoxicity, genotoxicity and oxidative stress in TR146 cells exposed to the food additive were evaluated. Due to the wide particle size distributions in food-grade TiO₂ powders, a comparative toxicity study was also performed with two TiO₂ particle models with distinct primary sizes, below and above 100 nm in diameter.

# Materials and methods

## Chemicals and particle preparation

Food-grade TiO₂ (E171) was purchased as a powder from the website of a French commercial supplier of food coloring agents and was previously characterized as a representative E171 sample in the analysis crystal form that has been placed on the EU market (Guillard et al. 2020). Two other (anatase) TiO₂ test materials with distinct primary particle sizes were used in this study, namely 21 nm TiO₂-NP (Sigma-Aldrich, Saint-Quentin-Fallavier, France) and 115 nm NM-102, referenced to JRCNM10200a by the European Joint Research Center Nanomaterials Repository (JRC, Ispra, Italy). TiO₂ materials were sonicated in ultrapure water (1 mg/ml) placed in an ice bath for 1 min at 40% amplitude (VCX 750-230 V, Sonics Materials) to obtain a stable dispersion of TiO₂ particles for 15 days at 4°C. Dynamic light scattering (DLS; Zetasizer Nano ZS; Malvern Instruments Ltd.) measurements were performed on each TiO₂ material in ultrapure water (pH = 7.75) and in TR146 cell culture medium (Ham's F12, pH 7.54; Life Technologies, Illkirch, France). Ten microlitres of

E171, NM-102 or TiO₂-NP suspensions were diluted in 2 mL of ultrapure water or Ham's F12 medium, and the hydrodynamic diameter (Z-average), polydispersity index and zeta potential were measured. In addition, some E171 samples were prepared without dispersion protocol for *in vivo* experiments with pigs.

## Animals and study design for *in vivo* buccal exposure

Five 4-week-old weaned castrated male piglets (Pic 410) weighing 10–12 kg were obtained from a local swine supplier (Gaec de Calvignac, Saint-Vincent d'Autejac, France). All animal studies were carried out in accordance with the European Guidelines for the Care and Use of Animals for Research Purposes (Directive 2010/63/EU) and validated by the Ethics Committee for Animal Experiments Toxcomethique n° 86 (TOXCOM/121/LGU). Pigs were acclimatized for 1 week in the animal facility of the INRAE Research Center in Food Toxicology (Toxalim, Toulouse, France) and fed *ad libitum* with free access to water. One pig served as a control, being administered water free from the food additive E171, and the other 4 pigs were exposed to TiO₂ (E171) water suspension, dispersed (n=2) or not (n=2) by sonication. During a short restraint operated by an animal technician, a volume of 200 µl of food-grade TiO₂ (E171) water suspension (50 µg/ml) was gently deposited once at T0 in the mouth under the tongue using a syringe equipped with a flexible catheter to avoid any injury in the mouth. The same procedure was repeated at T0+1, 2 and 3 h, and animals were euthanized at T0+30 min (n=2) or at T0+4 h, i.e., one hour after the last sublingual deposit (n=3, including the control pig). During the exposure period, all piglets were allowed to move freely in the barn without access to water or feed to avoid dilution in the mouth. At sacrifice, tissue samples from the buccal cavity under the tongue (buccal floor) as well as the submandibular lymph nodes located underneath the tongue were quickly withdrawn and prepared for TEM analysis.

## Buccal tissue preparation for TEM-EDX analysis

Tissue samples from piglets were fixed in 2.5% paraffin-aldehyde-2.5% glutaraldehyde in 0.1 M caodylate buffer (pH 7.4) overnight at 4°C. After
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several rinses with cacodylate buffer, the samples were postfixed in 1% OsO₄ (Osmium (VIII) oxide) for 1 h (4°C) and then rinsed again with cacodylate buffer before being dehydrated using a graded series of ethanol. The sections were impregnated in low viscosity epoxy resin (EMS) under vacuum and then polymerized at 60°C for 48–72 h. Ultrathin sections (80 nm, Ultracut UCT, Leica) were collected on copper grids and stained with a UAR-EMS (uranyl acetate replacement) solution followed by a 0.4% lead citrate solution. Five to 6 tissue sections from each sample were observed under a JEOL JEM-1400 electron microscope (MeTi facility, Toulouse, France) operated at 200 kV for TEM observations of electron dense particles and analyzed by energy-dispersive X-ray spectroscopy (EDX) on a JEOL 2100 F (Raymond Castaing facility, Toulouse, France) for chemical elemental analysis. Measurements of minimum and maximum Feret diameters were performed from bright-field TEM images by using the image processing open-source software ImageJ (NIH, United States).

## Cell culture and TiO₂ treatments

Human TR146 buccal epithelial cells (Sigma-Aldrich) were cultured in Ham's F12 nutrient mix (Life Technologies) supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies) and 0.1% penicillin/streptomycin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂ and subcultured every 2–3 days. To differentiate the TR146 cells, 10 000 cells were seeded in 0.33 cm² Transwell inserts (Corning) for 30 days, and the culture medium was changed every 2–3 days. Cells were exposed for 2 h to different concentrations of food-grade TiO₂ (E171) (5, 50 or 100 µg/ml) or to the test materials TiO₂ (NM-102 and TiO₂-NP) in Ham's F12 without FBS and when indicated, washed twice with PBS before being incubated in fresh complete culture medium.

## Confocal microscopy, TEM and SIMS imaging on TR146 cells

To study the kinetics of food-grade TiO₂ particle absorption, human epithelial TR146 cells were exposed for 1 h, 2 h, 5 h and 24 h to a 50 µl/ml suspension of TiO₂ (E171) in Ham's F12. Control

cells were exposed to Ham's F12 only. Supplemental TR146 cells exposed to E171 for 2 h were rinsed with Ham's F12 before being cultivated for a 5 h wash-out period in the same culture medium free of TiO₂ particles. For confocal microscopy, TR146 cells were fixed in 4% buffered formalin, embedded in paraffin wax, and sectioned to a thickness of five microns. Sections were first incubated with WGA-Alexa 594 for 1 h in the dark and then washed before being mounted with DAPI (4',6-diamidino-2-phenylindole; Life Technologies, France)-containing ProLong Gold antifade mounting medium for fluorescence microscopy. Tissue sections were viewed under a Leica SP8 confocal microscope with a 40× immersion objective and examined at 488/BP 488–494 nm to detect laser reflection by the metal particles as previously described (Coméra et al. 2020).

For TEM, TR146 cell monolayers were treated as previously described for buccal tissues. Since EDX might not be sensitive enough to allow investigation of the uptake of single NPs in cell preparations, correlative high-resolution imaging and secondary ion mass spectrometry (SIMS) were performed using a customized Zeiss Orion NanoFab helium ion microscope called the 'npSCOPE' instrument (De Castro et al. 2021) developed in the framework of EU HORIZON 2020 project no. 720964. TR146 cells exposed for 24 h to food-grade TiO₂ (E171) were fixed and embedded as for electron microscopy. Unstained 60 nm thick sections were cut, placed on an EM grid and investigated on the npSCOPE. Secondary electron (SE) and scanning transmission ion microscopy (STIM) images were recorded with a He⁺ primary beam at 30 keV at a working distance of 7.5 mm. Acquisition conditions were as follows: beam current, 3.4 pA, and dwell time of 5 µs, with an average of 4 frames for the SE images; beam current, 0.1 pA, and dwell time of 600 µs for the STIM images. SIMS was performed with a Ne⁺ primary beam at 20 keV, beam current of 8–10 pA and a working distance of 18.7 mm on the same area of interest. Positive mode SIMS was acquired at a magnetic field of 364 mT with a dwell time of 2 ms, while negative SIMS was acquired at a magnetic field of 300 mT with a dwell time of 8 ms.
## Cell viability

The AlamarBlue® (Life Technologies) assay was used to evaluate the proliferation and viability of proliferating TR146 cells grown on 96-well plates, or to evaluate the viability of differentiated cells grown on Transwell inserts. For proliferating cells, 2000 cells were seeded per well and incubated for 24 h. The cells were exposed to 25 µM of etoposide as a positive control, or different concentrations (5, 50 or 100 µg/ml) of food-grade TiO₂ (E171) and TiO₂ test materials for 2 h. Cells were then washed twice with PBS and incubated in fresh culture medium for 72 h to allow at least two rounds of cell division for proliferating cells. Viability and proliferation were assessed using the AlamarBlue® assay according to the manufacturer's instructions. The fluorescence was measured at an excitation of 570 nm and emission of 610 nm using a SPARK spectrophotometer. At least three independent experiments were performed.

## Transepithelial electrical resistance

The transepithelial electrical resistance (TEER) of differentiated TR146 cells was monitored using a Millicell-ERS voltohmmeter (Millipore, Saint-Quentin-en-Yvelines, France). Cells were treated with food-grade TiO₂ or the TiO₂ test materials for 2 h, washed twice with PBS and incubated in fresh culture medium. The TEER was measured immediately and then measured again at the indicated times for 48 h. The TEER values were normalized to that of the untreated condition. At least three independent experiments were performed.

## Immunofluorescence and oxidative stress analyses

Proliferating TR146 cells were grown on glass coverslips. After at least 24 h of culture, cells were exposed to 50 µM of etoposide or menadione as positive controls for genotoxic and oxidative stresses, respectively, or to food-grade TiO₂ (E171) and the TiO₂ test materials for the indicated times and concentrations before being fixed with 4% paraformaldehyde. For the oxidative stress assays, 30 minutes before fixation, 5 µM CellRox® Green Reagent (Life Technologies) was added to the cells for incubation at 37°C in the dark. For immunofluorescence assays, cells were permeabilized with

0.5% Triton X-100, blocked with 3% BSA and 0.05% IGEPAL, and stained with primary antibodies (γH2AX antibody (05-636), Sigma-Aldrich; 53BP1 antibody (NB100-304), Bio-Techne, Noyal-Châtillonsur-Seiche, France) overnight at 4° C in blocking solution (all solutions were prepared in PBS). Cells were washed three times with PBS 0.05% IGEPAL and incubated with the secondary antibodies (Alexa Fluor 488 or 594 goat anti-mouse or Alexa Fluor 594 goat anti-rabbit; Life Technologies) for 1 h at room temperature. DNA was stained with 30 nM of DAPI. Coverslips or membranes cut out of the Transwell inserts were mounted onto slides with PBS-glycerol (90%) containing 1 mg/ml paraphenyle nediamine and observed at 20× magnification with a Nikon 50i fluorescence microscope equipped with a Luca S camera. Upon oxidation, CellRox® Green Reagent exhibits strong fluorescence and binds to DNA. Therefore, as for γH2AX, the signal intensity of CellRox® Reagent was automatically determined by an ImageJ macro in each nucleus. The γH2AX or CellRox® Reagent signal intensity of the whole cell population was averaged for each condition, and these results were normalized to 1 for the untreated samples. For each experiment, 200-250 cells were counted, and at least three independent experiments were performed.

## Comet assay

Proliferating TR146 cells were exposed to 50 µM of etoposide as a positive control or to food-grade TiO₂ (E171) and the two TiO₂ test materials for the indicated times at the indicated concentrations. The comet assay was performed under alkaline conditions using a Comet SCGE assay kit (Enzo Life Sciences, Villeurbanne, France) according to the manufacturer's instructions. Briefly, 800 cells embedded in low-melting agarose were spread in each sample area of the comet slide. Electrophoresis was performed in alkaline solution (0.3 N NaOH, 1 mM EDTA) at 4° C for 30 min at 35 V in a large electrophoresis tank (35 cm between electrodes). After staining with CYGREEN® Nucleic Acid Dye, slides were observed at 20× magnification using a Nikon 50i fluorescence microscope equipped with a Luca S camera. At least 60 cells were analyzed per sample using OpenComet
<figure><img src="image_17.png" /></figure>

software. At least three independent experiments
were performed.

Micronucleus assay

Proliferating TR146 cells were grown on glass cover-slips. After at least 24 h of culture, cells were exposed to 50 µM of etoposide as positive control or to food-grade TiO₂ (E171) and the TiO₂ test materials for 2 hours before to be released in fresh medium for 22 hours. Cells were then fixed with 4% paraformaldehyde and treated as for immunofluor-escence analyzes. Cells were observed at 40× magnification with a Nikon 50i fluorescence microscope equipped with a Luca S camera. The frequency of micronucleated cells was scored on at least 100 cells per sample. At least three independent experiments were performed.

Statistical analysis

The results are expressed as the mean ± SD of at
least 3 independent experiments. Statistical analysis
was performed with Prism 8 software (GraphPad
Software Inc., San Diego, CA, USA). Differential
effects were analyzed by one-way or two-way ana-
lysis of variance (ANOVA) followed by the appropri-
ate post hoc test (Dunnett or Sidak). A p value <
0.05 was considered significant (*p* < 0.05;
**p* < 0.01; ***p* < 0.001; ****p* < 0.0001).

Results

Physico-chemical characteristics of the TiO2
particles

The commercial E171 batch of food-grade TiO2
used herein was previously characterized for its par-
ticle size distribution by SEM analysis. It was shown
that 55% of the NPs by number were 20 to 440 nm
for a mean size of 105 ± 45 nm (Guillard et al. 2020).
DLS was carried out to determine the hydro-
dynamic diameter and zeta potential in ultrapure
water, and the sample was also analyzed by BET for
specific surface area (Guillard et al. 2020).
Correlative secondary electron and SIMS imaging
with the npSCOPE recently confirmed the SEM data,
also providing chemical information with < 20 nm
resolution (De Castro et al. 2021). Using TEM imag-
ing, the TiO2 particles from the E171 food additive

dispersed into ultrapure water were mostly recov-
ered as isolated particles mixed with small aggre-
gates and agglomerates of particles of various sizes
(Figure 1). Additional DLS analyses of the food-
grade TiO2 particles in the present study showed a
slight increase in hydrodynamic diameter after
resuspension in Ham's F12/TR146 cell culture
medium compared to that in water suspension
(Table 1). Similar observations were found for NM-
102, while the TiO2-NP material exhibited a larger
agglomeration state in the culture medium com-
pared to the water suspension (Table 1).

In vivo translocation of food-grade TiO₂ through pig oral mucosa

The concentration chosen for *in vivo* buccal expos-
ure to pigs (50 µg/ml) was considered realistic for
human exposure given the current estimate of TiO₂
concentrations for example from chewing gum
coated with E171 (range 0.35–15.25 mg TiO₂/gum)
(Fiordaliso et al. 2018), local loading of TiO₂ in the
mouth during chewing (Supplementary Figure S1)
and mean oral volumes of saliva of 1 and 0.5 ml in
adults and children, respectively (Lagerlöf and
Dawes 1984; Watanabe et al. 2021). In buccal tis-
sues, TEM-EDX was used to investigate the transmu-
cosal passage of TiO₂ particles from the food
additive deposited under the tongue once every
hour for 3 h. Translocated particles were recovered
deep into the buccal tissues in all E171-exposed
pigs (n = 4), as illustrated in Figure 2. Indeed,
30 minutes after the first sublingual deposit of E171,
the TEM observations clearly showed the presence
of electron-dense particles that had translocated
into the mucosa (Figure 2(A)). As shown in Figure
2(A), EDX analysis clearly revealed the presence of
titanium (Ti) on a particle of 104 nm (smaller diam-
eter) recovered from the buccal floor, while no Ti
particles was observed in the submandibular lymph
nodes at 30 min.

At 4 h (i.e., 1 h after the last E171 deposit), elec-
tron-dense particles were observed in the mucosa
of buccal floor (Figure 2(B1,C1)) as well as in the
lumen of blood capillaries (Supplementary Figure
S2). TEM-EDX analysis of 6 tissue sections sampled
from the buccal floor showed that most particles
recovered in the mucosa (i.e., 15 of 17) were Ti-
positive (Figure 2(B2,C2), and Table 2). They
<figure><img src="image_18.png" /><figcaption>**Figure 1.** TEM images of food-grade TiO₂ (E171) particles. E171 powder after dispersion in ultrapure water at low (A) and high (B) magnification showing morphology of isolated and aggregated TiO₂ particles.</figcaption></figure>

<table><caption>**Table 1.** TiO₂ sample characterization by DLS.</caption><thead><tr><th rowspan="2">TiO<sub>2</sub> sample</th><th colspan="3">Ultrapure water (pH = 7.75)</th><th colspan="3">Ham's F12 medium (pH = 7.54)</th></tr><tr><th>Zeta potential (mV)</th><th>H.diam. (nm)</th><th>PdI</th><th>Zeta potential (mV)</th><th>H.diam. (nm)</th><th>PdI</th></tr></thead><tbody><tr><td>E171</td><td>-32.1 ± 0.79</td><td>297.9 ± 5.1</td><td>0.21</td><td>-8.97 ± 0.54</td><td>318.7 ± 14.9</td><td>0.40</td></tr><tr><td>NM-102</td><td>-34.3 ± 0.66</td><td>301.6 ± 0.4</td><td>0.17</td><td>-8.19 ± 0.59</td><td>337.0 ± 20.1</td><td>0.35</td></tr><tr><td>TiO<sub>2</sub>-NPs</td><td>-11.1 ± 2.44</td><td>173.9 ± 1.7</td><td>0.16</td><td>-8.99 ± 1.96</td><td>226.4 ± 3.1</td><td>0.39</td></tr></tbody></table>

All data are presented as the mean ± SD. H. diam.: hydrodynamic diameter; PdI : polydispersity index.

appeared as isolated particles (n = 6) (Figure 2(B1)) or as small aggregates (n = 9), the latter being composed of 2 to 11 particles fused together (see Figure 2(C1)). Analysis of minimum Feret diameters showed isolated particles ranging from 72 to 199 nm, and aggregates from 117 to 392 nm (Table 2), and up to 550 nm in maximum Feret diameter for aggregates (Table 2). In addition, at 4 h, Ti was also found in isolated particles (n = 1) and aggregates (n = 7) recovered from tissue sections sampled from the submandibular lymph nodes located underneath the tongue (Figure 2(D)), and were similar in size range to those observed in the buccal floor (Table 2). In the control pig exposed to water only, no Ti signal was observed over 9 electron-dense objects found in the buccal mucosa, while only one Ti particle of 10 was recovered in the submandibular lymph nodes, showing irregular shape and mix composition with Si and Al elements (Supplementary Figure S3) not observed with E171 buccal exposure.

When pigs were treated for 4 h with the food additive E171 without sonication (i.e., not dispersed), TEM-EDX analysis also showed Ti-positive particles in the buccal floor (7 particles/aggregates

of 26 analyzed) and in the submandibular lymph nodes (3 of 17) (Supplementary Figure S4 and Supplementary Table S1). This showed transmucosal passage of food-grade TiO₂ as raw powder in water suspension, where translocated particles and aggregates exhibited sizes similar to those recovered using dispersed E171 preparation as described above (Table 2 and Supplementary Table S1).

## Kinetic of TR146 cell permeability to TiO₂ food additive

To assess the absorption kinetics of food-grade TiO₂ particles by human oral epithelial cells, TR146 cells were first observed by confocal microscopy after 1, 2 and 5 h of exposure to the food additive E171 (50 µg/ml) or after 2 h of exposure followed by 5 h of incubation in fresh culture medium free of E171 (i.e., wash-out). As shown in Figure 3, the laser-diffracting TiO₂ particles appeared as a bright green signal upon more or less agglomerated particles once absorbed by the cells (Figure 3(A–D)), as previously described (Coméra et al. 2020; Guillard et al. 2020). In 1 h-exposed cells, some laser-diffracting particles were recovered in the cytoplasm, some of
<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /><figcaption>**Figure 2.** TEM imaging and EDX analysis of ultrathin sections of the buccal mucosa and submandibular lymph nodes from pigs exposed to food-grade TiO₂ particles (E171). (A) TEM images (A1-2) and the corresponding EDX analysis for elemental analysis (A3) of the Ti(O₂) particles translocated into the buccal floor 30 min after a single E171 sublingual deposit. Note in the EDX spectrum (A3) additional Al and Si signals as main elements over an adjacent particulate deposit appearing as a chapelet (A1). Copper (Cu) and lead (Pb) are from the sample grid and lead citrate staining, respectively. (B,C) TEM images (B1-C1) and the corresponding EDX spectra (B2-C2) of the Ti(O₂) particles in the buccal mucosa at 4 h, i.e., one hour after the last E171 sublingual deposit. Note in (B1) the presence of an elongated Fe particle in the same microscopic field. (D) TEM image (D1) and the corresponding EDX analysis (D2) of the Ti(O₂) particles translocated into a submandibular lymph node at the same time point.</figcaption></figure>
<figure><img src="image_21.png" /><figcaption>**Table 2.** TiO₂ particles in pig buccal mucosa and submandibular lymph nodes after repeated sublingual deposition for 4 h of E171 suspension dispersed in water.</figcaption></figure>

<table><thead><tr><th rowspan="2">Tissue sample</th><th rowspan="2">Number of analyzed<br>electron-dense particles</th><th colspan="2">Ti-positive particles<br>(D<sub>MinFeret</sub>/D<sub>MaxFeret</sub> ±SD in nm)</th></tr><tr><th>isolated</th><th>aggregates</th></tr></thead><tbody><tr><td>Buccal floor</td><td>17</td><td>6 (143 ± 49/191 ± 88)</td><td>9 (257 ± 99/393 ± 116)</td></tr><tr><td>Submandibular lymph nodes</td><td>14</td><td>1 (129/140)</td><td>7 (257 ± 97/390 ± 120)</td></tr></tbody></table>

$D_{MinFeret}/D_{MaxFeret}$: minimum and maximum Feret Diameters.

which were in close contact with the nucleus (Figure 3(A)). The number of laser-diffracting particles progressively increased after 2 and 5 h of treatment (Figure 3(B,C)). No laser-reflective particulate matter was observed in the nucleus regardless of the time point. In cells exposed to the food additive for 2 h followed by 5 h of wash-out, large agglomerates of laser-diffracting particles were still present in the cytoplasm (Figure 3(D)), suggesting that TiO₂ could penetrate the buccal epithelium and that these particles would not be cleared from the cells even several hours after the end of exposure *in vitro*.

To achieve better resolution, a second set of experiments was carried out with TEM observations, which confirmed the large capacity of the TiO₂ particles to permeate TR146 cells over time. Electron-dense (TiO₂) particles were isolated or recovered as small aggregates and then larger agglomerates of submicron-sized particles mixed with NPs into the cytoplasm (Figure 3(E)). Again, absorbed TiO₂ were still observed 2 h after E171 treatment following wash-out (Figure 3(F)).

Cell areas containing electron-dense particles were further investigated by npSCOPE analyses combining SE, STIM and SIMS imaging for unprecedented TiO₂ identification within the cell matrix. As illustrated in Figure 4, single NPs as well as small and large clusters of electron-dense TiO₂ were found embedded in the cytoplasm of TR146 cells after 24 h of treatment with the food additive E171.

### Comparative cytotoxicity of the TiO₂ test materials in human buccal cells

To gain insight into the cytotoxic effects of food-grade TiO₂ compared with the two TiO₂ standards with different nominal sizes (NM-102 and TiO₂-NPs: 115 and 21 nm, respectively), proliferating or differentiated TR146 cells were exposed to different TiO₂

concentrations (5, 50, 100 µg/ml) for 2 h. The TR146 cells were then allowed to recover in fresh culture medium for 72 h before cell viability assessment, allowing at least two rounds of cell division in proliferating cells. In proliferating cells, the DNA damaging agent etoposide (used as positive control) and the three tested TiO₂ samples induced a significant cytotoxic activity at all tested concentrations compared to nontreated cells, except with 5 µg/ml for E171, with a dose-response tendency (Figure 5(A)). In contrast, after cell differentiation, no viability drop was observed in any tested condition (Figure 5(A)). This suggests that etoposide and TiO₂ particles may not directly affect cell viability but rather impede cell proliferation.

In addition, the TEER of differentiated TR146 cells was measured after 2 h of exposure to each TiO₂ sample to assess their respective impacts on epithelial integrity. Regardless of the time point tested during the 48 h after treatment, no alterations were detected at any dose, suggesting that epithelial barrier permeability and monolayer integrity were not affected regardless of the TiO₂ product or particle size (Figure 5(B)). Taken together, these data indicate that the epithelium formed by TR146 cells is not noticeably altered by exposure to foodborne TiO₂ but that cycling cells could be sensitized.

### Comparative genotoxicity of the TiO₂ test materials in human buccal cells

Next, we assessed the genotoxic potential of food-grade TiO₂ (E171) and standard TiO₂ products on buccal cells by immunofluorescent analyses using antibodies directed against γH2AX and 53BP1, two well-established DNA damage biomarkers (Vignard, Mirey, and Salles 2013). Proliferating or differentiated TR146 cells were exposed for 2 h to the three different TiO₂ materials at 5, 50 or 100 µg/ml. We first analyzed the phosphorylation of H2AX at
<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /><figcaption>**Figure 3.** Absorption kinetics of buccal TR146 cells exposed to food-grade TiO₂ (E171) particles. (A-D) Confocal images of TR146 cell sections treated with 50 µg/ml E171 for 1 h, 2 h and 5 h, or 2 h plus a wash-out (WO) of 5 h. The laser-reflecting (metal) particles appear green, the WGA-labelled glycoproteins appear red, and cell nuclei appear blue. (E-F) TEM images of TR146 cell sections treated with 50 µg/ml E171 for 5 h (E) and 2 h followed by a 5 h wash-out (F).</figcaption></figure>

Ser139 (referred to as γH2AX), which occurs at DNA double-strand breaks. While only a few cells presented a γH2AX signal in the control, cells exposed

to E171 or NM-102 accumulated γH2AX foci (Figure 6(A)). In contrast, the pure nanopowder of TiO₂-NPs (21 nm) did not increase γH2AX staining (Figure
<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /><figcaption>**Figure 4.** Correlative secondary electron (SE) imaging, scanning transmission ion microscopy (STIM) and secondary ion mass spectrometric (SIMS) elemental mapping of ultrathin sections of buccal TR146 cells exposed to food-grade TiO₂ (E171) particles for 24 hours. In contrast to the TEM images presented in figure 2, SE imaging obtained with a helium ion microscope (here, npSCOPE) reveals predominantly topographical information. The thin sections therefore show only limited contrast of the cell structures and the nanoparticles are easily recognized. For TEM-like imaging, the STIM detector attached to the npSCOPE prototype device allows investigation of the transmitted beam information and highlights the NP in relation to the cellular ultrastructure. The image shows the engulfment of electron-dense particles into the cell cytoplasm. The SIMS image obtained on the same area highlights cellular information when considering the ¹²C¹⁴N cluster ion and clearly identifies individual TiO₂ nanoparticles and clusters (lateral resolution down to a particle size of 15 nm). The integrated Ti "Σ"-map represents the signals obtained by summing the peaks of all Ti isotopes and all TiO cluster peaks.</figcaption></figure>

6(A)). We then observed the localization of 53BP1, which is a diffuse nuclear protein that displays a singular localization pattern as large nuclear speckles in unchallenged G1 cells, named 53BP1 nuclear bodies (Fernandez-Vidal, Vignard, and Mirey 2017). These structures represent the major staining found in the control or after exposure to TiO₂-NPs with a nominal size of 21 nm (Figure 6(A)). However, in the presence of DNA double-strand breaks, 53BP1 is recruited to the damaged site and forms foci. Interestingly, food-grade E171 and NM-102 induced 53BP1 foci formation in a subset of TR146 cells, mainly colocalizing with the γH2AX signal (Figure 6(A)). This staining was observed in proliferating as well as in differentiated cells. Hence, these data showed that food-grade and NM-102 TiO₂ but not TiO₂-NPs activate the DNA damage biomarkers

γH2AX and 53BP1 after 2 h of treatment, strongly supporting the formation of DNA double-strand breaks.

To confirm the genotoxic potential of E171, we performed an alkaline comet assay in proliferating cells, to detect DNA strand breaks and alkali-labile sites (that induce DNA relaxation under alkaline conditions) (Collins et al. 2023). Under our conditions, etoposide, E171 and NM-102 significantly increased the amount of DNA strand breaks compared to untreated cells, as revealed by an increase in the % tail DNA, whereas no difference was observed after treatment with TiO₂-NPs (Supplementary Figure S5). Taken together, these observations indicated that exposure to E171 or NM-102 induced the formation of DNA strand
<figure><img src="image_26.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_29.png" /></figure>

**Figure 5.** Cytotoxicity of the TiO₂ particles in TR146 cells. Proliferating or differentiated TR146 cells were exposed to 25 µM of etoposide or different concentrations (5, 50 or 100 µg/ml) of E171, NM-102 or TiO₂-NPs. (A) Cell viability was assessed using the AlamarBlue® assay. The graphs represent the viability normalized to that of untreated cells. The results are presented as the mean ± SD of at least three independent experiments. Statistics were calculated by two-way ANOVA followed by Dunnett's multiple comparison test. (B) The TEER was determined in differentiated TR146 cells at different time points after exposure to the TiO₂ materials. The results are presented as the mean ± SD of three independent experiments.

breaks that were signaled by the DNA damage response factors γH2AX and 53BP1 in TR146 cells.

We next assessed the level of DNA damage in TR146 cells at two different timing after exposure to the TiO₂ samples through γH2AX signal quantification. A dose-dependent increase in γH2AX staining was observed after 2 h of exposure to both E171 and NM-102 in proliferating cells (Figure 6(B)), to a lesser extent compared to the positive control etoposide. In differentiated cells exposed

under the same conditions, a significant increase in the γH2AX level was also detected with E171 and NM-102, but only at a concentration of 100 µg/ml (Supplementary Figure S6). When proliferating TR146 cells were allowed to recover and placed in fresh medium for 22 h, an approximately two-fold decrease in γH2AX signal was observed for etoposide, supporting that some of the DNA lesions were repaired after the recovery period (Figure 6(C)). Proliferating TR146 cells exposed to 100 µg/ml of E171 did no longer exhibit significant difference in γH2AX signal compared to untreated cells after the 22 h recovery time, highlighting the repair of at least part of the DNA damage. However, the fold increase in γH2AX signal was still significant for 50 µg/ml of E171 and for 50 µg/ml or 100 µg/ml of NM-102, showing that some DNA lesions were still present (Figure 6(C)). On the contrary, the γH2AX signals in the differentiated cells exposed to E171, NM-102 and etoposide returned to basal levels after the recovery time (Supplementary Figure S6). In the TiO₂-NP model, the γH2AX signal remained at the same level as the control under all tested conditions (Figure 6(B,C) and Supplementary Figure S6). In conclusion, TR146 cells exposed to food-grade (E171) TiO₂ or NM-102 generated DNA damage, some of which lingered in cycling cells after a period of recovery, suggesting that cell proliferation aggravates the genotoxic activity induced by TiO₂.

Finally, we asked whether E171 could impact the genomic stability of TR146 cells treated as in Figure 6(C), by performing a micronucleus assay. Under our experimental conditions, no clear difference was observed between untreated cells and cells exposed to the different TiO₂ samples, whereas the positive control etoposide induced a significant increase of cells with micronuclei (Supplementary Figure S7).

## E171-induced oxidative stress in human buccal cells

As many previous studies have reported that TiO₂-related genotoxicity mainly resulted from oxidative stress, we monitored the production of reactive oxygen species (ROS) using the fluorogenic probe CellROX® Green Reagent in proliferating TR146 cells exposed to the food additive E171 and two TiO₂ test materials for 2 h. CellROX® Green Reagent
<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /><figcaption>**Figure 6.** Genotoxicity of the TiO₂ particles in TR146 cells. (A) TR146 cells were left untreated (NT) or exposed to 50 µg/ml E171, NM-102 or TiO₂-NPs for 2 h and analyzed by immunofluorescence microscopy with antibodies against γH2AX and 53BP1. The images on the right represent magnification of the cells delineated by squares with white dotted lines (γH2AX and 53BP1 signals). (B, C) Proliferating TR146 cells were treated with 50 µM of etoposide or different concentrations of TiO₂ (5, 50 or 100 µg/ml), and the γH2AX signal was quantified immediately (B) or after 22 h of recovery in fresh culture medium (C). The results are presented as the mean ± SD of at least three independent experiments. Statistics were calculated by one-way ANOVA followed by Dunnett's multiple comparison test.</figcaption></figure>

emits green fluorescence upon oxidation by ROS and subsequently binds to DNA. Compared to untreated cells and TiO₂-NP treatments, cells exposed to the food-grade E171 and NM-102 exhibited green nuclear fluorescence after incubation with CellROX Green Reagent (Figure 7(A)), indicative of ROS production. Of note, these signals were slight compared to the positive control menadione. With the food additive E171 and contrary to the results with NM-102 or menadione, quantification

did not allow us to conclude that there was significant difference compared to untreated cells (Figure 7(B)). However, when the cells were allowed to recover in fresh culture medium for 22 h, CellROX® Green signal persisted and showed a significant increase after exposure to both E171 and NM-102 at 100 µg/ml (Figure 7(C)). Therefore, a short-term exposure to food-grade TiO₂ can induce weak ROS production in TR146 cells, which is maintained for at least 22 h following cell absorption.
<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /><figcaption>**Figure 7.** Oxidative stress induced by TiO₂ particles in TR146 cells. (A, B) TR146 cells were left untreated (NT) or exposed to 50 µM of menadione or different concentrations (5, 50 or 100 µg/ml) of E171, NM-102 or TiO₂-NPs for 2 h, and the presence of reactive oxygen species was quantified by using CellROX® Green Reagent. Representative images (A) and quantification (B) are shown. The results are presented as the mean ± SD of three independent experiments. Statistics were calculated by one-way ANOVA followed by Dunnett's multiple comparison test. (C) TR146 cells were treated as in A and B with 100 µg/ml TiO₂ agents and analyzed after 22 h of recovery in fresh culture medium. The results are presented as the mean ± SD of three independent experiments. Statistics were calculated by one-way ANOVA followed by Dunnett's multiple comparison test.</figcaption></figure>
Discussion

Due to the worldwide usage of TiO₂ as coloring agent in common foodstuffs, including drinks and ice cream, or in pharmaceuticals as a coating agent, these different TiO₂-containing products are viewed as the main source of body contamination by TiO₂-NPs in humans. However, in the context of oral route, the contribution of the buccal mucosa for TiO₂ uptake remains poorly documented. The buccal epithelium represents the first surface that is exposed to foodborne xenobiotics. Even though the potential for compound absorption is high in this tissue, as reported for various drug delivery systems including immediate release tablets (Madhav et al. 2009), the buccal epithelium is still not taken into account for risk assessments of the food additive TiO₂ containing a nanosized particle fraction. In the present study, using an *in vitro* model of the human oral epithelium, we report that buccal cells are highly permeable to the TiO₂ particles present in a commercial food-grade (E171) sample, including its NP fraction. To ensure that such passage occurs *in vivo*, we further show that TiO₂ particles rapidly cross the oral epithelium in piglets and are recovered deeper in the buccal mucosa and submandibular lymph nodes after repeated sublingual deposition. In human TR146 cells, we report cytotoxicity and genotoxicity in proliferating cells exposed to E171. The genotoxic effects were still observed after cell differentiation, suggesting the long-lasting impact of food-grade TiO₂ on the human oral epithelium, which should be considered for risk assessment purposes with this food additive.

Using fresh *ex vivo* porcine buccal mucosa as a model for the permeation assessment of NPs into the mouth, a previous study using non-food TiO₂-NP models of various sizes (namely JRC NM100, NM101 and NM105) highlighted the ability of nanosized TiO₂ particles to penetrate the oral cavity tissues (Teubl et al. 2015a). The authors concluded that smaller the NPs (i.e., NM101, 28 nm in Feret minimum diameter) exhibit less depth translocation and remained in the cytoplasm of the surface epithelial cells. In contrast, larger TiO₂ particles, such as NM 100 (displaying two fractions of 34 to 148 nm) or NM 105 (36 nm), also penetrated, but their penetration was deeper into the porcine

mucosa. This is in line with our *in vivo* observation in piglets that isolated TiO₂ particles of the E171 additive ranging from 70 to 200 nm in diameter were recovered deep in the oral mucosa. Interestingly, this corresponds to the larger fraction of particle size of food-grade TiO₂ in the E171 batch used in the current study, as previously characterized for primary size distribution by number (Guillard et al. 2020). Notably, such particle translocation occurred rapidly, since it was observed from thirty minutes after a single deposition of the E171 water suspension under the tongue. In addition, *in vitro*, using a human oral cell line (H376) exposed to carboxyl polystyrene particles with sizes of 20 and 200 nm, permeation of the buccal epithelium was reported to be dependent not only on primary particle size but also on agglomeration state in the culture medium (Roblegg et al. 2012). As more agglomeration occured outside the cells, less penetration was observed into the buccal cells. Based on these studies, a similar conclusion can be drawn from the present kinetic study focused on food-grade TiO₂. Although it was restricted to the sole epithelial layer with the TR146 cell line, this *in vitro* model is viewed as able to mimic the human buccal epithelium (Nielsen and Rassing 2000). Both nanosized and submicron-sized TiO₂ particles were found to be endocytosed as single particles or small aggregates, while the very large agglomerates that had previously formed in culture medium remained in contact with the external surface of the epithelial cells without apparent translocation under such large forms (not shown). The current evaluation of the absorption rate using confocal imaging at different time points completed with TEM and SIMS imaging data for high-resolution and elemental characterization, strengthens the idea that food-grade TiO₂ particles enter the cells as isolated particles or as very small aggregates regardless of their nominal size (i.e., NPs or submicron-sized). Such translocation started within 1 h, showing the passage of the particles that progressively accumulated over time into the cells, until 24 h of exposure in the current study, altogether showing high absorption capabilities for oral epithelium. Of note, the time-dependent agglomeration of TiO₂ particles in TR146 cells in our study is due to cell culture grown on filters as a monolayer, which prevented further passage of the particles beyond
<figure><img src="image_35.png" /></figure>

the cells and resulted in progressive accumulation in the cytoplasm. The kinetics for particle absorption determined that human buccal epithelial cells are highly permeable to TiO₂, in contrast to the low absorption rate by Caco-2 enterocytes used as an intestinal *in vitro* model (Brun et al. 2014). In buccal cells, no translocation to the nucleus was observed in the current study, which is in line with observations by Teubl et al. Teubl et al. (2015a) using different TiO₂ NP models from the JRC. The rapid absorption of food-grade TiO₂ NPs by the oral epithelium is also in accordance with their study showing that the NPs were internalized within 10 min following exposure. *In vivo* and as stated above, because the size range for isolated particles recovered in the pig oral mucosa corresponded to particle distribution in the commercial E171 powder (Guillard et al. 2020), we concluded the E171 sample the only source for orotransmucosal passage of TiO₂, and that most particles that composed a common commercial batch of E171 can be absorbed in the mouth. Of note, since TiO₂ particles were also found in the pig buccal mucosa with a E171 suspension without sonication for particle dispersion, it is concluded that an orotransmucosal passage also occurs from the food additive in its raw commercial form. These *in vivo* data using pig mouth for which the histomorphology of the buccal mucosa is comparable to that of humans confirmed that the food-grade TiO₂ particles rapidly pass through the surface epithelium in the mouth to reach mucosa underneath, thereby becoming systematically available. Because we herein report aggregate sizes up to 550 nm in the oral mucosa, this suggested that the buccal epithelium is unable to block the passage of such large inorganic structures *in vivo*. An unexpected result was the presence of TiO₂ particles of similar sizes and forms in the submandibular lymph nodes of exposed pigs, and whatever the initial preparation for E171 suspension (i.e., dispersed or not). As key players in the local immune system, lymph nodes act as the first line of defence against harmful agents from the oro pharyngeal region by filtering the lymphatic fluid of unwanted debris and antigens. Studies focused on dental prostheses and titanium implants have already reported Ti particle deposition in the submandibular lymph nodes due to microscopic disintegration of biomedical devices (Onodera, Ooya, and

Kawamura 1993; Weingart et al. 1994; Ng et al. 2021). The current study highlights that food-grade TiO₂ particles are also drained by the lymphatic fluid from the oral cavity and then transported to the local lymph nodes. However based solely on this, it is not possible to reach any conclusion regarding an inflammatory risk that requires chronic exposure to be evaluated. Finally, to conclude our kinetic study, and based on recent evaluations of TiO₂ intake from chewing gum that have estimated human exposure ranging from 0.1–84 billion TiO₂ NPs/kg bw/d (Fiordaliso et al. 2018), our study highlights the oral epithelium as a route for the direct systemic passage of food-grade TiO₂ (E171) NPs which has not been taken into account in previous toxicokinetic studies and human risk assessment.

We then explored the potential toxicity impacts of food-grade TiO₂ exposure in the mouth. Human TR146 cells were exposed to E171 for 2 h to ensure particle uptake without accumulation in the cells, as noted above. Experiments were conducted over a range of doses (i.e., 5, 50, 100 µg/ml) for a realistic scenario of the buccal epithelium coming in contact with the food-grade pigment. We first reported cytotoxic activity of E171 only in proliferating cells. In contrast, no defects were observed in TR146 cells once differentiated, a state that implies cell cycle withdrawal (Ruijtenberg and van den Heuvel 2016). As cell cytotoxicity assays, such as Alamar Blue® assay used in this study, depend on cell viability and number, it is unlikely that food-grade TiO₂ particles from the E171 sample directly affects the cell viability of cycling cells but should rather stop their proliferation, similar to the TiO₂ test materials and etoposide. In two previous studies using other TiO₂ nanomodels, no cytotoxicity was observed in proliferating TR146 cells (Teubl et al. 2015a; Teubl et al. 2015b). This discrepancy with the current study may be due to differences between the experimental designs, as viability was assessed 24 h posttreatment by these authors compared to 72 h posttreatment in our study, which allowed more time for cell division. In addition, our conclusion that food-grade TiO₂ mainly impacts cell proliferation is in agreement with a previous study using intestinal Caco-2 cells (enterocytes) exposed to TiO₂ particles (anatase NM100 from the JRC) of which mean size (104±39 nm) was close to that of the E171 sample (105±45 nm) or NM-102 (115 nm)
<figure><img src="image_36.png" /></figure>

used in the current study, and viability loss was
also reported for only undifferentiated intestinal
cells (Vila et al. 2018). Altogether, this corroborates
our hypothesis that buccal exposure to food-grade
TiO₂ could halt epithelial cell proliferation, suggest-
ing that TiO₂ particle absorption in the human
mouth primarily alters epithelium formation or
repair rather than directly affecting differentiated
epithelial cells. Because active cell division is neces-
sary to ensure the turnover of the buccal mucosa
every 14 days (Teubl et al. 2015a), our data raise
concerns about E171 exposure that could possibly
impact epithelial renewal in the mouth.

The genotoxicity of several sources of TiO₂ NPs,
including E171, has been analyzed mainly in intes-
tinal models in vitro, and have reported contradict-
ory results. Indeed, it is now well established that
NP physicochemical properties (size, shape, surface
properties, composition, solubility, aggregation/ag-
glomeration) and experimental conditions greatly
influence the cellular genotoxic response
(Magdolenova et al. 2014), hampering general con-
clusions on TiO₂ NP genotoxicity in vitro. Our data
reveal that E171 and NM-102 with similar particle
size distributions (i.e., mostly from 50 to 150 nm)
induce DNA damage and slight oxidative stress in
TR146 cells contrary to the pure TiO₂-NP particulate
model (21 nm). While E171 and TiO₂ from NM-102
contain nano- and submicron-sized particles, in con-
trast to the TiO₂-NP model, it was suggested that
the genotoxic potential of food-grade TiO₂ particles
mainly originated for particle with sizes generally
above 20 nm. These observations suggest that the
nanosized and submicron-sized TiO₂ fractions mixed
in the food additive E171 may exert distinct adverse
effects on buccal cells, as already reported on intes-
tinal cells (Proquin et al. 2017), and that submi-
cronic particles merit a specific attention when
assessing the genotoxicity of E171 in the buccal
cavity.

Interestingly, as observed during cytotoxicity
testing, we demonstrated that E171 genotoxic activ-
ity was higher during TR146 cell proliferation.
Similar observations have been reported in intes-
tinal cellular models. Indeed, treating differentiated
Caco-2 cells with E171 resulted in DNA base oxida-
tion but not DNA strand breaks after comet assay
in its alkaline and Fpg-modified versions (Dorier
et al. 2017). On the other hand, undifferentiated

proliferating Caco-2 cells exposed to E171 accumu-
lated DNA strand breaks, as assessed by comet
assay, but also micronuclei (Proquin et al. 2017). It
should be noticed that under our experimental con-
ditions in TR146 proliferating cells, E171 and NM-
102 exposure induced DNA strand breaks in alkaline
comet assay, but did not result in micronucleus for-
mation. In 2021, EFSA summarized that E171, and
more globally TiO₂ NPs, have the potential to
induce DNA damage based on *in vitro* and *in vivo*
comet assays (Younes et al. 2021). Conversely, EFSA
conclusions on the potential of TiO₂ NPs to induce
micronuclei were mainly based on *in vivo* assays, as
the majority of *in vitro* studies gave negative results.
The consequences of DNA damage are detrimental
for cycling cells because any DNA lesion may inter-
fere with S-phase progression by blocking the repli-
cation fork, eventually leading to fork collapse and
the formation of double-strand breaks (Zeman and
Cimprich 2014; Kondratick, Washington, and Spies
2020). It should be noted that γH2AX and 53BP1,
two markers of DNA double-strand breaks, were
activated in proliferating as well as in differentiated
TR146 cells, indicating that E171 exposure can pri-
marily induce this type of lesion independent of
DNA replication. However, we cannot exclude the
possibility that food-grade TiO₂ particle absorption
induces other types of lesions, such as DNA base
oxidation, as previously reported (Dorier et al.
2017). In TR146 cells exposed to E171, DNA double-
strand breaks were more efficiently repaired in dif-
ferentiated compared to proliferating cells, in which
a significant increase of γH2AX staining was main-
tained several hours after TiO₂ release from the cul-
ture medium. Double-strand breaks in noncycling
cells are processed by non-homologous end joining
which repairs the lesions in less than 1 h, whereas
DNA repair in proliferating cells involves different
pathways and should be delayed to overcome repli-
cation stress (Scully et al. 2019). Because E171-
induced CellROX® Green Reagent signal persisted
22 h after wash-out, it was suggested that the TiO₂
particles internalized into the buccal cells lead to
ROS formation, perhaps interfering with replication
progression and giving rise to late DNA double-
strand breaks. It has been proposed that the
carcinogenic properties of inhaled TiO₂ rely on gen-
otoxicity through oxidative stress. Animals exposed
to TiO₂ NPs *via* inhalation have demonstrated
<figure><img src="image_37.png" /></figure>

genotoxic effects in the lungs associated with ROS production, lipid peroxidation and anti-oxidases activation (Sun et al. 2013; Han et al. 2020). On the other hand, in vivo testing after TiO₂ ingestion failed to clearly conclude the presence of DNA damage in the intestinal tract (Bettini et al. 2017; Carriere, Arnal, and Douki 2020) despite the induction of oxidative stress (Abbasi-Oshaghi, Mirzaei, and Pourjafar 2019; Zhao et al. 2021). Our results suggest that the food-grade TiO₂ particles from the food additive E171 induce oxidative stress and possibly related DNA damage that at least contributes to cytotoxicity in proliferating cells of the buccal epithelium.

## Conclusion

The data presented here provide evidence that under realistic exposure conditions in terms of dose and duration of exposure, food-grade TiO₂ may translocate through the oral mucosa in an *in vivo* pig model of buccal mucosa that is close to the human mouth. We also report the high permeability of human buccal epithelial cells to TiO₂ particles *in vitro*. After these cells were exposed to the food additive for 2 h, TiO₂ particles generated oxidative and genotoxic stresses that were detrimental to proliferating cells mainly. This raises the issue of possible adverse consequences regarding the constant turnover of the buccal mucosa or during wound repair and regeneration. Thus, our study supports that buccal exposure should be considered for TiO₂ risk assessments when being used as a food additive in common foodstuffs, in oral care products such as toothpaste, or as a coating agent in various pharmaceutical drug delivery forms, including those for the sublingual route (European Medicines Agency 2021). To date, because most of the toxicokinetic studies on food-grade TiO₂ have been conducted by gastric gavage, i.e., direct administration into the gastrointestinal tract, the oral cavity is therefore bypassed. However, the buccal epithelium, in addition to the intestine (Teubl et al. 2015a), has to be considered as an additional route for the uptake of food-grade TiO₂, including its nanosized fraction, hence increasing the potential of absorption of foodborne TiO₂ NPs in humans.

## Acknowledgements

The authors thank Anne-Marie Cossalter and Mikael Albin for their assistance during animal experiments, and Matthieu Refrégiers for spectrofluorimetry analysis of human saliva.

## Author contributions

J.V., A.P.-D., E.G., C.C., P.P., E.B.R., I.-P.O., F.-H.-F.P., B.L., G.M. and E.H. designed the study; E.G., L.D. and N.F. performed physico-chemical characterization of TiO₂ samples; E.G. and P.P. conducted the animal experiments; C.C., C.B. and L.W. performed TEM and SEM-EDX analysis; A.B. and T.T. performed STIM/SIMS (npSCOPE) analysis; J.V., E.B.-R., J.D. and A.P.-D. performed *in vitro* toxicity testings; J.V., A.P.-D., E.G., C.C., A.B., T.T., L.D., J.D., E.B.R., N.F., I.-P.O., F.-H.-F.P., B.L., G.M. and E.H. analyzed the data; J.V., A.B., E.B.R., I.-P.O., F.-H.-F.P., B.L., G.M. and E.H. wrote the paper. All author(s) read and approved the final manuscript.

## Disclosure statement

No potential conflict of interest was reported by the author(s).

## Funding

The study was supported by INRAE (Institut national de recherche pour l'agriculture, l'alimentation et l'environnement). This work has received fundings from the European Union's Horizon 2020 Research and Innovation Programme under Grant agreement no. 720964 [npSCOPE], by the French National Research Agency (ANR) under Grant number ANR-16-CE34-0011-01 [PAIPITO], and by the Luxembourg National Research Fund via the project INTER/DFG/19/13992454.

## Data availability statement

All data generated or analyzed during this study are included in this published article and its supplementary information files.

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