Possible Adverse Effects of Food Additive E171 (Titanium Dioxide) Related to Particle Specific Human Toxicity, Including the Immune System

In 2019, France made the decision to completely ban TiO2 in the nutraceutical sector from January 1, 2020. Now, the EFSA panel concluded, that E171 (TiO2) can no longer be considered as safe when used as a food additive in Europe on the basis of the following article. Read more about the possible consequences in the pharmaceutical industry in our special article about TiO2 here!

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.

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Background of TiO2 as a Food Additive

Titanium dioxide (TiO₂) is a widely used white pigment and opacifying agent, with applications in paints, pharmaceuticals, cosmetics, and food [1]. When used as a food additive in the European Union (EU), it is listed as E171 to refer to a specified food-grade form of TiO₂, which has no nutritional value and is used to attain a white color, shade other pigments, or in pharmaceuticals [2]. The whitening is best achieved with TiO₂ particles within a size range of 200–300 nm, due to their light scattering effects [3]. TiO₂ occurs in nature in three distinct crystal structures—anatase, rutile, and brookite, but only anatase and rutile are allowed as a food additive [4,5,6]. The European Union allows E171 (anatase and rutile in uncoated, no surface treatment forms) in quantum satis (without limitations), based on its low absorption and subsequent low toxicity, presumed inertness, and low solubility [5,7,8]. Its low toxicity and inertness, however, are being debated, as long-term inhalation studies over two years have shown the development of lung tumors in rats, following exposure to high concentrations of TiO₂ [9,10]. As a consequence of these findings, the International Agency for Research and Cancer (IARC) has classified TiO₂ as “possibly carcinogenic to humans after inhalation” [10]. In 2017 the Risk Assessment Committee (RAC) of the European Chemical Agency (ECHA) published an opinion that proposed the classification of TiO₂ as a category 2 carcinogen after inhalation, according to the criteria of the Classification, Labelling and Packaging (CLP) Regulation [11]. On the 18 February 2020, the EU took over ECHA’s opinion and published the classification of TiO₂ as a suspected carcinogen (category 2) by inhalation in powder form with at least 1% particles with aerodynamic diameter ≤ 10 μm, under the CLP Regulation (EC No 1272/2008). The classification will apply on 1 October 2021 after an 18-month transition period [12]. What the observed toxicity and hazard classification following inhalation mean for oral toxicity is of yet not clear.

Over the last years, an increasing number of studies investigated the behavior and effects of E171 and nano-sized TiO₂ after ingestion and discovered potential adverse effects, including the induction of inflammation, the formation of reactive oxygen species (ROS), and co-genotoxic effects [13]. Sub-acute and sub-chronic studies also revealed the induction of epithelial hyperplasia and preneoplastic lesions in the colon of rats and mice after the ingestion of E171, while other oral toxicological studies did not confirm such effects [14,15,16,17,18]. For the oral intake of food additive E171, the European Commission requested a re-assessment of TiO₂ by the European Food Safety Authority (EFSA), following the publication of studies by ANSES in 2017. EFSA concluded that the results of these studies did not merit a re-opening of the existing opinion but suggested to fill in the existing data gaps, reduce uncertainties and evaluate new findings carefully in regard to their adverse effects and physicochemical properties of the TiO₂ particles used [7,8,19,20,21]. The re-assessment of TiO₂ has recently been opened and was initiated in 2020 by the EFSA [22].

Parallel to the EFSA activities, the Office of Risk Assessment and Research (BuRO) at the Netherlands Food and Consumer Product Safety Authority (NVWA) organized a workshop that was held in July 2018, regarding the “potential health effects of the food additive titanium dioxide (E171)”, on which BuRO based its opinion that was published in 2019 [23].

In response to signals in the scientific literature about potentially harmful effects after ingestion of E171 in rodents and the widespread use of this substance in foods, BuRO identified the following questions in the process of risk assessment of E171 that have to be addressed:

Does oral exposure to E171 or nano-sized TiO₂ reveal a relevant toxicological hazard?
How reliable are these in-vitro and in-vivo studies?
Are the animal models, the exposure conditions, and the effects observed in these studies relevant to humans?
Can the data from in-vitro and in-vivo studies with TiO₂ be extrapolated to humans?
Are there epidemiological studies on the effects of E171 in humans after oral exposure?

Since this workshop in 2018, more studies have been published that investigated the concerns of adverse effects arising from E171 ingestion. This literature review integrates the main conclusions of the expert meeting initiated by BuRO, with recently published studies in order to present an overview of relevant findings regarding E171 toxicity after oral intake. The literature search on PubMed and EmBase was conducted from June 2020–September 2020 and included the search criteria “TiO2”, “titanium dioxide”, “E171” with publication dates from 2018–2020. Previous scientific papers in the field, as well as references in these publications were evaluated. The present literature review aims to shed light on the importance of complete particle characterization, on the effects of matrices, and highlight toxicological relevant pathways potentially involved in the induction of adverse health effects following E171 ingestion. Additionally, it provides approaches to decrease uncertainties concerning the health effects of E171 consumption, and finally formulates recommendations for future studies and follow-up actions regarding the risk assessment of E171.

Physicochemical Properties and Characterization of E171

Titanium is one of the most abundant elements in the earth’s crust, which occurs in nature only in its oxidized form as titanium dioxide or Ti(IV) oxide. Once processed, TiO₂ is a white, odorless powder that is poorly soluble in aqueous solutions [2,5]. The anatase form TiO₂ is most frequently used as a whitening agent in foodstuff, despite its high surface reactivity and ability to generate ROS in an aqueous solution after UV irradiation [2,24]. Food-grade TiO₂/E171 consists of micro-and nanoparticles with a primary particle size ranging from 60–300 nm [25]. Around 10–40% of the pristine TiO₂ particles in E171 are estimated to be smaller than 100 nm and can therefore be considered as nanoparticles [25,26,27,28]. However, according to the Commission’s recommendation in 2011 (2011/696/EU), a nanomaterial must contain over 50% of nanoparticles, which excludes E171 of this category [28,29]. Based on information reported in the literature the EFSA Panel on Food Additives and Nutrient Sources suggest that the food additive E171 mainly consists of micronized TiO₂ particles ranging from 104–166 nm and a percentage of particles < 100 nm ranging from 5.4–45.6% [21,30].

Recently published work by Verleysen et al. (2020) showed that 12 out of 15 pristine E171 materials purchased from manufacturers consist of more than 50% TiO₂ particles that are smaller than 100 nm and that commercially available anatase E171 materials constitute of 18–74% (TEM) or 32–64% (sp-ICP-MS) nanoparticles [30]. This examination assigns a larger fraction of TiO₂ particles present in pristine E171 to the nano-sized fractions than previously assumed. Analysis of food samples containing E171, via ICP-MS and Raman spectroscopy, showed anatase type TiO₂ particles in the range of 26.9–463.2 nm, with 21.3–53.7% of the particles in the nano-size fraction [31]. The determination of the nanoparticle fraction (Figure 1) within E171 is of importance since the size of particles is considered to be an important factor influencing toxicokinetics, toxicodynamics, and thus toxicity [8,21,32]. Nanoparticles display a higher surface to volume reactivity, translocation properties, bioavailability, and increased cellular interactions than larger particles [33].

Example of E171 particle characterization. Prior analysis the samples were dispersed according to the NanoGenotox dispersion protocol at a final concentration of 2.56 mg/mL in 0.05% BSA solution and probe sonicated on ice for 16 min (4 W). (A) Transmission Electron Microscope picture of E171. (B) Size distribution of E171 particles, measured by single-particle ICP-MS, with a median particle size of 79 nm and 72% of particles < 100 nm.

The shape, size, and state of agglomeration and aggregation are important properties regarding the effects of food-grade TiO₂. Generally, it is assumed that the round and spherical crystal forms of TiO₂ contribute to a lower extent to the induction of adverse effects, when ingested [34]. The size of food-grade TiO₂ particles, on the other hand, plays an important role regarding their toxicity. Nano-sized TiO₂ particles are suspected to induce more adverse effects, including ROS formation, cytotoxicity, and increased release of inflammatory cytokines, compared to micro-sized TiO₂ particles [32,35,36]. Proquin et al. (2018) demonstrated that a mixture of nano- and micro-sized TiO₂ particles, as they are present in E171, induce more adverse effects than the single fractions alone. This emphasizes the importance of testing food-grade TiO₂ particles as a whole, rather than its nano- and micro-sized fraction [16].

The interaction of E171 with its direct environment and colloidal stability are other factors that need to be considered during its characterization [37]. Suspended TiO₂ particles tend to agglomerate or aggregate, according to their isoelectric point and the pH of the milieu, leading to the formation of larger clusters. Aggregation describes the assembly of primary particles through covalent or metallic bindings, while agglomeration results from van-der-Waals interactions, hydrogen bonds, adhesion by surface tension, or electrostatic attraction [2,38]. The determination of agglomeration and aggregation status is crucial because it can significantly alter hydrodynamic diameter, size, and the stability of particle-complexes, thus affecting uptake, reactivity, and toxicity [39].

The high surface area, charge, and chemical properties of TiO₂ particles provide the possibility of many biomolecules to be adsorbed. The formation of a protein corona can change the physicochemical properties of TiO₂ particles, e.g., their reactivity and the interactions of these particles with their environment, including cellular uptake, accumulation, intracellular localization, distribution, and release [40]. The variability of protein coronas is dependent on the different molecules present at each location and can influence their interaction with cells [41]. The presence of transferrin in the protein corona, for example, can affect the clathrin-mediated endocytosis via the transferrin-receptor and result in significantly altered particle internalization [42].

The formation of protein coronas can also lead to conformational changes of the proteins themselves, resulting in irreversible changes to secondary protein structures and leading to protein dysfunction [43]. Additional interactions of TiO₂ nanoparticles with non-protein components might be harmful too. Bianchi et al. (2017) showed that the endotoxic effect of lipopolysaccharides (LPS) is increased when bound to TiO₂ nanoparticles, resulting in the potentiation of pro-inflammatory effects including induced expression of nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kB) and interferon regulatory factor 3 (IRF-3) dependent cytokines [44]. The consideration of TiO₂-protein-corona-complexes in the characterization and determination of physiochemical properties and adverse effects of food-grade TiO₂ is important for an adequate safety evaluation.

For this reason, it is important to carefully examine and analyze the physicochemical characteristics of TiO2 particles in its vehicle, as well as in its surrounding matrix as their final milieu, to guarantee a profound assessment of potential adverse health effects of E171 and to adequately compare different studies in the process of risk assessment.

Exposure to E171

E171 is used in products such as candy, coffee creamer, chewing gum, sauces, nutritional supplements, toothpaste, and pharmaceuticals. Although both the anatase and rutile forms of TiO₂ are authorized for foods, the characterization of European and American food samples showed that anatase is the predominant TiO₂ crystalline structure used as food additive E171 and thus the main source of exposure for the general population [27,45,46,47,48]. The intake of E171 varies between different age groups and countries, while children, in general are the most highly exposed group, due to their lower body mass and disproportionally higher consumption of E171-containing products [5,49]. Table 1 shows the estimated daily intake of E171 per kg body weight (bw) in different countries and age-groups [49,50].

Table 1. Mean and 95th percentile estimation of daily oral intake of TiO₂ from food products (E171), food supplements and toothpaste in different age groups and countries in mg/kg bw/day (n/a = data not available, * mg/person/day).

Author	Year	Country	Mean (mg/kg bw/Day)	95th Percentile (mg/kg bw/Day)
Wu [50]	2020	USA	0.15-3.9 * (PCP survey and usage patterns, no food included)	n/a
EFSA [21]	2016	Europe	<11 months: 0.2-0.8	<11 months: 0.7-3.9
			1-3 years: 0.6-4.6	1-3 years: 2.0-6.8
			3-9 years: 0.9-5.5	3-9 years: 2.4-14.8
			10-17 years: 0.4-4.1	10-17 years: 1.3-10.8
			18-64 years: 0.3-4.0	18-64 years: 1.1-9.7
			>65 years: 0.2-2.8	>65 years: 0.5-7.0
Rompelberg [49]	2016	NL	2-6 years: 0.66-0.70	2-6 years: 1.19-1.40
			7-69 years: 0.16-0.18	7-69 years: 0.47-0.54
			>69 years: 0.05-0.07	>69 years: 0.20-0.28
Bachler [51]	2015	DE	"Other Children": ~2	"Other Children": ~0.7-7.2
Bachler [51]	2015	DE	Toddlers, adolescents, adults, elderly: 0.5-1	Toddlers, adolescents, adults, elderly: ~0.1-4.2
Sprong [52]	2015	NL	2-6 years: 1.3-1.5	2-6 years: 4.5-5.6
			7-69 years: 0.6-0.7	7-69 years: 2.6-3.0
			>70 years: 0.5-0.6	>70 years: 1.7-2.2
Christensen [53]	2015	DK	Children: 2	n/a
Christensen [53]	2015	DK	Adults: 1	n/a
Weir [27]	2012	UK	<10 years 2-3	n/a
Weir [27]	2012	UK	>10 years: 1	n/a
Weir [27]	2012	US	<10 years: 1-2	n/a
Weir [27]	2012	US	>10 years: 0.2-0.7	n/a
Powell [54]	2010	UK	5 *	n/a

The highest concentrations of E171 are found in chewing gum, candies, and powder sugar toppings such as icings. Chewing gums contain between 1.1 mg (±0.3 mg) to 17.3 mg (±0.9 mg) TiO₂ particles per piece of gum with a mean average weight per piece of 1416 mg (±27 mg) to 2240 mg (±86 mg) [26]. TiO₂ nanoparticles account for up to 19% (±4) of all particles present in these gums [26]. The accidental ingestion of toothpaste, while brushing teeth is another major source of E171 intake, that can result in an exposure of 0.15 to 3.9 mg/day, when 10% of toothpaste is ingested [50]. Additional release of TiO₂ particles (70–200 nm) from food packaging materials or food-related products, such as frying pans, may also contribute to TiO₂ ingestion [55]. The focus of oral TiO₂ exposure estimation should potentially be extended from the food additive E171 to personal care products, packaging, and coating of household items [28,33,55]. Daily dietary intake of E171 can reach several hundred milligrams, of which at least 10–40% are in the form of TiO₂ nanoparticles. The long-term exposure to such quantities of nano- and micro-sized TiO₂ raises concerns about the risk of potential accumulation in organs and potentially harmful effects on human health [27].

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Article information: Bischoff, N.S.; de Kok, T.M.; Sijm, D.T.H.M.; van Breda, S.G.; Briedé, J.J.; Castenmiller, J.J.M.; Opperhuizen, A.; Chirino, Y.I.; Dirven, H.; Gott, D.; Houdeau, E.; Oomen, A.G.; Poulsen, M.; Rogler, G.; van Loveren, H. Possible Adverse Effects of Food Additive E171 (Titanium Dioxide) Related to Particle Specific Human Toxicity, Including the Immune System. Int. J. Mol. Sci. 2021, 22, 207. https://doi.org/10.3390/ijms22010207

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