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Nanomaterial engineering provides an important technological advance that offers substantial benefits for appli- cations not only in the production and processing, but also in the packaging and storage of food. An expanding commercialization of nanomaterials as part of the modern diet will substantially increase their oral intake worldwide. While the risk of particle inhalation received much attention, gaps of knowledge exist regarding possible adverse health effects due to gastrointestinal exposure. This problem is highlighted by pigment-grade titanium dioxide ( TiO ), which confers a white color and increased opacity with an optimal particle diameter of 200–300 nm. However, size distribution analyses showed that batches of food-grade TiO always comprise a nano-sized fraction as inevitable byproduct of the manufacturing processes. Submicron-sized TiO particles, in Europe listed as E 171, are widely used as a food additive although the relevant risk assessment has never been satisfactorily completed. For example, it is not possible to derive a safe daily intake of TiO from the available long-term feeding studies in rodents. Also, the use of TiO particles in the food sector leads to highest exposures in children, but only few studies address the vulnerability of this particular age group. Extrapolation of animal studies to humans is also problematic due to knowledge gaps as to local gastrointestinal effects of TiO particles, primarily on the mucosa and the gut-associated lymphoid system. Tissue distributions after oral administration of TiO differ from other exposure routes, thus limiting the relevance of data obtained from inhalation or parenteral injections. Such difficulties and uncertainties emerging in the retrospec- tive assessment of TiO particles exemplify the need for a fit-to-purpose data requirement for the future evaluation of novel nano-sized or submicron-sized particles added deliberately to food. Keywords: Anatase, Cancer, Corona, Dendritic cells, Food additives, Food safety, Food toxicology, Innate immunity, Lymphoid tissue, Rutile Background and anemia, affecting nearly 2 billion people worldwide Potential applications of recent nanomaterial develop- [1–3]. Nano-sized materials may further provide mark- ments in the food sector include, for example, nano- ers of food freshness and quality, or allow for traceabil- sized coatings of packaging materials to protect from ity and the detection of pathogens or contaminants [4, mechanical damage or microbial contamination, thereby 5]. In contrast to these novel developments, submicron- extending the shelf life. Nano-sized additives may also sized particles of titanium dioxide (TiO ) have been used be deliberately incorporated in food to optimize prop- in the food sector for more than 50 years as a pigment erties such as taste, sensation, color, texture or consist- to enhance the white color and opacity of foods like cof- ency. Nanomaterials may be employed to supplement fee creamer, sauces, spreads, pastries, candies and edible food with vitamins in a highly bioavailable form and ices. Also, TiO confers brightness to toothpaste and is could contribute to prevent nutritional iron deficiency added to enhance the flavor of non-white foods (pro - cessed fish, fruits, meat, vegetables, breakfast cereals, fermented soybean, soups and mustard) and to clear bev- erages (beer, cider and wine) [6–9]. *Correspondence: email@example.com Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Currently, the annual consumption volume of T iO par- Winterthurerstrasse 260, 8057 Zurich, Switzerland ticles reaches four million tons, which makes it the most Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Winkler et al. J Nanobiotechnol (2018) 16:51 Page 2 of 19 widely used pigment globally . In the United States on Cancer (IARC) concluded that there is inadequate (US), the Food and Drug Administration allows up to 1% evidence from epidemiological studies to assess whether by weight of T iO particles as a food colorant . In the TiO dust causes cancer in humans, but that there is suf- 2 2 European Union (EU), TiO is an authorized food addi- ficient evidence for carcinogenicity in experimental ani - tive (listed as E 171) at quantum satis, meaning that no mals, based on the induction of respiratory tract tumors maximum level is imposed as long as the additive is used in rats after prolonged inhalation [24, 25]. Therefore, in accordance with good manufacturing practice, i.e., at IARC classified TiO as a Group 2B carcinogen . a level not higher than necessary to achieve the intended Considering the widespread food-related uses, there is a scope . A comparison of use levels reported by the pressing need to review the suitability of studies support- food industry show that the highest TiO concentrations ing the risk assessment of TiO particles as food additive 2 2 are expected in chewing gum (up to 16,000 mg/kg), food . Comprehensive reviews on this topic have been supplements delivered in a solid form (up to 12,000 mg/ provided inter alia by Shi et al. , Heringa et al.  kg), processed nuts (up to 7000 mg/kg) and ready-to-use and the Scientific Panel on Food Additives and Nutrient salads and sandwich spreads (up to 3000 mg/kg) . Sources added to Food (ANS Panel) . The purpose TiO particles can, therefore, be viewed as a paradig- of our contribution is to focus on data gaps and uncer- matic case for the safety assessment of inorganic particles tainties in relevant risk assessment studies covering the employed as food additive and comprising a nano-scale dietary uptake of TiO particles. fraction. The standard risk assessment procedure with TiO particle manufacture and their physicochemical risk = hazard × exposure, which includes hazard identifi - properties cation, hazard characterization, exposure assessment and Although Ti is the ninth most abundant element in the risk characterization, is also applicable to small inorganic earth’s crust, it never appears in a metallic state in nature. particles in food. The prefix “nano” does not make a sub - TiO , an odorless powder with a molecular weight of stance automatically harmful and possible adverse effects 79.9 g/mol, also known as Ti(IV) oxide, constitutes the should be tested case-by-case. However, reductions in naturally occurring oxide [30, 31]. TiO minerals contain size may change the material characteristics as compared impurities such as iron, chromium, vanadium or zirco- to larger particles or the same substance in solution. nium that confer a spectrum of different colors. Manu - Nano-sized particles display an increased surface-to-factured TiO is, instead, a white powder commonly mass ratio that enhances their reactivity [14, 15]. Also, used as a pigment in ceramics, paints, coatings, plastics nanoparticles display an increased propensity to pene- and paper due to its high refractive index. Pure T iO trate through cell membranes thus conferring the poten- assembles in three crystal structures, i.e., anatase, rutile tial for trafficking across biological barriers including the (with tetragonal coordination of Ti atoms) and brookite intestinal mucosa [16–18]. In principle, a nanomaterial (with rhombohedral coordination of Ti atoms), but only exists in different forms, i.e., with one dimension in the anatase/rutile or mixtures of these two polymorphs are nano-scale (for example nano-films), two dimensions in employed in food . In addition, as a fourth form, the nano-scale (for example nano-rods) or, as for nano-amorphous TiO has been described . The surface of particles, all three dimensions in the nano-scale range. A anatase crystals is considered to be more reactive than European Commission Recommendation defines nano - that of rutile counterparts, as indicated by their ability materials as natural, incidental or manufactured materi- to generate reactive oxygen species in aqueous solutions als, containing 50% or more of the particles, determined when irradiated with ultraviolet (UV) light . Also, in a number-based size distribution, with at least one anatase nanoparticles display a stronger adjuvant activ- external dimension not exceeding 100 nm . However, ity than rutile nanoparticles in an allergy model based on there is no scientific ground to defend such a strict size the intranasal sensitization of mice with ovalbumin . boundary in the identification of possible hazards, as one Nonetheless, the anatase form is the most frequently would rather expect a gradient in the capacity of eliciting used in the food sector [8, 36, 37]. adverse effects with changing particle dimensions. In any Food-grade TiO is manufactured from Ti minerals case, a final answer to the question of when a material by either a sulfuric acid-based process, which can yield becomes nano-sized has not been provided  and the anatase, rutile or a mixture of both polymorphs depend- above Recommendation is not yet adopted for regulatory ing on the reaction conditions, or a chlorine-based pro- purposes. cess yielding only the rutile form . Specifications for Until now, the health effects of TiO particles have food use include a minimum purity of 99.0%, thus allow- been studied mainly with regard to their uptake by inha- ing some contamination with arsenic, cadmium and mer- lation [21–23]. The International Agency for Research cury (up to 1 mg/kg), antimony (up to 2 mg/kg) or lead Winkler et al. J Nanobiotechnol (2018) 16:51 Page 3 of 19 (up to 10 mg/kg). Also, food-grade T iO may be coated even when the mean diameter reaches 200–300 nm. In with a small proportion (no more than 2% in total) of this respect, a frequently cited size distribution is the one alumina and silica to enhance technological properties, determined by Weir et al.  using transmission electron for example to improve dispersion in host matrices [32, microscopy (TEM), whereby 36% of particles by num- 38]. All TiO particles are insoluble in water, organic sol- ber were below the threshold of 100 nm. This data was vents, hydrochloric acid and dilute sulfuric acid. They are derived from a single determination with one lot of E 171 highly stable to heat and remain unaffected by food pro - and, hence, is not representative for all T iO on the mar- cessing. Also, they are not or only minimally degraded ket. In a follow-up distribution analysis of five different or dissolved under conditions, including low pH, which food-grade TiO samples by TEM, nano-sized particles mimic the gastrointestinal milieu [39, 40]. Such indigest- occured with a frequency between 17 and 35% by num- ible particles, once released from the food matrix during ber . Studies by scanning electron microscopy (SEM) their gastrointestinal transit, reach the intestinal mucosa suggested that commercial E 171 materials contain ~ 10% raising the question of whether they might be prone to of particles with dimensions below 100 nm . Clearly, absorption and systemic distribution. the outcome of particle size determinations varies with Optimal light scattering is needed to achieve the the method of measurement, whereby smaller diameters desired whitening effect. Therefore, food-grade TiO are generally reported from TEM measurements com- ideally displays a primary particle size of approximately pared for example to laser diffraction [13, 45]. Another half the wavelength of the light to be scattered , i.e., relevant aspect is that, as illustrated in Fig. 1, suspended half of the 400–700-nm of the visible range. Accordingly, TiO particles tend to aggregate/agglomerate to form scattering of visible light is maximized in fine particles larger clusters, although a majority of the individual that are 200–300 nm in diameter. Ultrafine products particles may display a primary diameter < 100 nm. The are instead not suited for this purpose as they become term “aggregate” designates an assembly of particles held transparent when their size remains below the 100-nm together by covalent or metallic bonds. Instead, “agglom- threshold . Such nano-sized T iO particles are often erates” result from weak forces like van der Waals inter- included at concentrations of up to 25% in cosmetic prep- actions, hydrogen bonding, electrostatic attractions arations, including lip balms and sunscreens to protect or adhesion by surface tensions. It is important not to from solar light by reflecting UV radiation away from the equate the nanoparticle fraction measured by num- skin [43, 44]. As a consequence of the production process ber with the same value by mass. The ANS Panel at the there is inevitably a broad size distribution that com- European Food Safety Authority (EFSA) proposes to use prises nanoparticles with a primary size below 100 nm a proportion of 3.2% by mass to estimate the nano-sized Fig. 1 Example of food-grade TiO particles (E 171). a A sample of food-grade anatase dispersed in H O was deposited on a copper grid coated 2 2 with glow-discharged parlodion and analyzed by TEM as described [146, 147]. Scale bar, 100 nm. b Size distribution of the imaged food-grade TiO particles. The diameter measured as longest distance across particles is 100 ± 24 nm (mean ± standard deviation) and 54% by number of the particles have a diameter < 100 nm Winkler et al. J Nanobiotechnol (2018) 16:51 Page 4 of 19 Determinants of intestinal uptake fraction of E 171 for risk assessment considerations . After oral exposure, foreign particles released by diges- TEM analyses of T iO particles in the coatings of chew- tion from the food matrix encounter a layer of entero- ing gums revealed a nano-sized mass fraction of 4.2% on cytes, lining the intestinal tract, that are responsible for the average [45, 46]. This review only includes studies nutrient absorption. This digestive epithelium surface, where TiO test materials have been characterized with in humans estimated to 30 m , presents a structural respect to their size preferably with indications on the barrier to foreign materials that also secretes a protec- primary particle diameter. tive layer of mucus. Indigestible particles like the ones consisting of TiO may nevertheless gain entry into Human exposure the underlying lamina propria by penetration across In the US, the dietary intake of TiO was estimated or between intact enterocytes. However, the digestive at 1–2 mg/kg body weight per day for children under mucosa is additionally defended by the gut-associated the age of 10 years and 0.2–0.7 mg/kg/day for other lymphoid tissue (GALT), which is arranged into lym- age groups. This dietary exposure in the United King - phoid follicles that, in the small intestine, aggregate to dom (UK) population was estimated to be 2–3 mg/kg/ form Peyer’s patches [50–52]. The epithelium cover - day for children and around 1 mg/kg/day for the other ing this intestinal lymphoid tissue displays phagocytic age groups . The corresponding exposure values esti - microfold cells (M-cells), whose specialized function is mated for the German population are between 0.5 and to absorb particulates from the intestinal lumen to be 1 mg/kg/day in adults but reach ~ 2 mg/kg/day in chil- forwarded to the innate immune system including den- dren . To obtain dietary exposures across Europe, the dritic cells and macrophages . Intestinal dendritic ANS Panel at EFSA selected food categories for which cells also reach out their membrane projections across the use of TiO is authorized, and assumed that 60–80% the epithelial barrier into the gut lumen to take up par- by weight of these food items actually contain TiO as an ticulates directly . Thus, TiO particle can be incor- additive. Next, the ANS Panel used the EFSA Compre- porated by cells of the innate immune system, where they hensive European Food Consumption Database and the persist without being substantially degraded or dissolved typical TiO inclusion levels reported by industry (see . The local accumulation of such particles appears as background section above), as well as reported analyti- pigments in the lymphoid tissue of the intestinal mucosa cal results, to calculate chronic dietary exposures to TiO [51, 55–57]. for different age groups. The highest values were found for children of 3–9 years where, depending on the dietary Oral bioavailability in rodents habits, the mean exposures were 0.9–8.8 mg/kg body Inhalation studies in animals converge on the finding that weight per day with 95th percentiles of 2.4–30.2 mg/kg nano-sized TiO particles can enter, in small amounts, per day. Some relevant food categories (for example edi- the systemic circulation from the alveolar epithelium and ble cheese rind) are not displayed in the Consumption disseminate into other organs [21, 23, 58, 59]. Instead, Database and, as a consequence, could not be taken into dermal exposure studies indicated that TiO particles of account for the exposure estimate. Also, the contribution any size do not penetrate the stratum corneum of the skin of the accidental swallowing of toothpaste or lip balms [60–63]. Less certain is the extent of intestinal absorp- was not included in these calculations, possibly resulting tion, but an elegant vanadium (V) radiotracer study in an underestimation of oral T iO intake. Another study established that the vast majority of ingested TiO nan- employed the Dutch National Food Consumption Survey oparticle is directly excreted in the feces. Briefly, com - and the T iO concentrations in food products reported mercial anatase particles were irradiated with a proton by industry . Again, the highest exposure (median beam to generate a radiolabeled [ V]TiO fraction with of 1.4 mg/kg body weight per day and 95th percentile of a mean particle diameter of 50 nm. After demonstrating 4.9 mg/kg) was found in children 2–6 years old. A parallel that most V ions remain associated with TiO parti- study also employed the Dutch National Food Consump- cles, a single dose (30–80 µg/kg body weight) of this test tion Survey but used Ti and/or TiO concentrations in material was administered by intraesophageal instillation food products and toothpaste as reported in the literature (oral gavage) to Wistar-Kyoto rats . Groups of ani- . The calculations confirmed that the highest intake mals were sacrificed 1, 4, 24 h and 7 days after gavage to (median of 0.59 mg/kg body weight per day and 95th per- assess the transfer of radioactivity from the gastrointesti- centile of 1.29 mg/kg) is found in children 2–6 years old. nal tract into the cardiovascular system and various tis- The generally elevated exposure of children is attributed sues. This time course revealed that a small proportion of to their lower body mass and disproportionately higher the applied radioactivity (only ~ 0.6%) was detected in the consumption of TiO -containing products like pastries blood and internal organs like liver, spleen and kidneys, and candies [6, 13]. Winkler et al. J Nanobiotechnol (2018) 16:51 Page 5 of 19 at 1 h after gavage. This overall proportion of systemically administration and no Ti translocation from the gastro- distributed TiO particles gradually dropped to ~ 0.05% intestinal tract into blood or urine was observed. The ani - after 7 days. mals were sacrificed 4 days after administration for tissue Table 1 presents an overview of relevant oral expo- analysis, but Ti concentrations in liver, spleen and kidney sure studies in rodents found in the literature. A high- remained at control levels. In a subchronic study, T iO dose biokinetic/acute toxicity study was carried out in particles consisting of 80% anatase and 20% rutile (mean CD-1 mice after a single administration by oral gavage size of 26 nm) were administered orally to Sprague–Daw- of differentially sized TiO particles (crystal structure ley rats at daily doses of up to 1042 mg/kg body weight not specified) administered at 5000 mg/kg body weight for 90 days . Upon analysis by ICP-mass spectrom- . The mean diameter of the three tested particles was etry, no increased Ti levels were detected in liver, spleen, 25 nm, 80 nm and 155 nm. Ti concentrations in tissues kidney and brain tissues even in the group of animals were determined by inductively coupled plasma (ICP)- receiving the highest dose, thus indicating a very low oral mass spectrometry 2 weeks after treatment. In animals bioavailability. In the blood taken at necropsy, the high that received the fine particles of 155 nm in diameter, background Ti concentration of ~ 0.4 µg/g was minimally increased Ti concentrations (~ 500 ng/g) over control lev- increased, but only in males of the 521 and 1042-mg/kg els (< 100 ng/g) were found only in the spleen. For com- groups. parison, the highest Ti level (~ 4000 ng/g) was detected Similar results were obtained from a study performed in the liver of animals that received the 80-nm particles. with differentially sized TiO particles provided by the A major deficiency of this and other bioavailability stud - Joint Research Center (JRC) Nanomaterials Repository. ies is that distribution measurements rely on a chemical These reference materials consist of anatase or rutile with Ti analysis and, therefore, it is not clear whether the Ti mean particle sizes ranging from 6 to 90 nm. Wistar rats detected in fluids and tissues is due to translocation of were administered these particles by oral gavage at a daily TiO from the gastrointestinal tract in the form of par- dose of around ~ 10 mg/kg body weight for 5 consecutive ticles or as solubilized material. A subsequent experi- days . The ICP-mass spectrometry analysis of liver, ment in CD-1 mice involved the oral administration by spleen and mesenteric lymph nodes performed 24 h after gavage of anatase particles with mean diameters of 18 the last exposure revealed low Ti levels exceeding the and 120 nm. The dose was 64 mg/kg body weight . limit of detection of 30 ng/g only occasionally. In a few Increased Ti levels, measured by ICP-optical emission TiO -exposed animals, there was a detectable but very spectrometry, were detected in the blood, liver and pan- slight increment of Ti in liver, spleen or the mesenteric creas, but only in animals administered the 18-nm par- lymph nodes. On the basis of these findings, the fraction ticles. A peak Ti blood concentration of ~ 0.15 µg/ml of TiO particles absorbed after repeated oral adminis- (against a background of ~ 0.05 µg/ml) was detected 1 h tration was estimated to be maximally 0.02% by weight. after administration. Upon intravenous application, the same particles were In an earlier study, Sprague–Dawley rats were treated predominantly retained in the liver, and the subsequent with rutile particles (mean size of 500 nm) by oral gav- analysis of animals sacrificed at different times after age at a dose of 12.5 mg/kg body weight per day . injection reveled long half-lives of up to 650 days in the SEM analysis and histologic examination of tissues after liver and spleen. It is therefore possible that even a lim- 10 days of dosing revealed the presence of TiO parti- ited systemic absorption from the gastrointestinal tract cles in the GALT and mesenteric lymph nodes and even in combination with slow elimination might potentially demonstrated some translocation to sinusoidal cells of result in tissue accumulation. the liver. The Ti content of the aforementioned tissues In a short-term exposure test, anatase particles (pri- was demonstrated by ICP-atomic emission spectros- mary size of 20–60 nm) were administered by oral gavage copy and quantitative estimates suggested that approxi- to Sprague–Dawley rats at doses of up to 2 mg/kg body mately 6.5% of T iO particles were absorbed. However, weight per day for five consecutive days . ICP-mass the authors did not consider the background Ti content spectrometry measurements revealed a slight but statisti- in their calculations, likely resulting in an overestimation cally significant increase of Ti concentrations relative to of systemic retention. In another study, Sprague–Dawley untreated controls not only in the spleen but surprisingly rats fed with Ti-free diet received by gavage a single oral also in the ovaries of animals exposed at the higher dose. dose of TiO (5 mg/kg) in the form of nano- or micron- The penetration of TiO into the spleen (but not into 2 2 sized particles with mean diameters ranging from 40 nm the ovaries) was confirmed by single-particle ICP-mass to 5 µm . These particles consisted of anatase or spectrometry and SEM analysis of tissue homogenates, rutile. Ti levels were measured by ICP-mass spectrom- thus demonstrating the presence of particle aggregates/ etry in the feces, blood and urine at different times after agglomerates. Winkler et al. J Nanobiotechnol (2018) 16:51 Page 6 of 19 Table 1 Overview of oral toxicokinetic and toxicodynamic studies in rodents Study type Oral dose Particle structure (mean Internal exposure Main reported effects Source size) Acute toxicity in mice 5000 mg/kg Structure not specified (25, ~ 4 µg/g Ti in liver Histopathologic findings  80 and 155 nm) in brain, liver and kidney Acute toxicity in rats Up to 5000 mg/kg Coated rutile/anatase Not examined None  (73 nm) Bioavailability in rats Up to 80 µg/kg Radiolabeled anatase Oral particle bioavailability None  (50 nm) of ~ 0.6% Bioavailability in rats 5 mg/kg Anatase and rutile None detected None  (40 nm–5 µm) Toxicokinetics in rats ~ 10 mg/kg/day for 5 days Anatase and rutile Oral particle bioavailability None  (6–90 nm) of ~ 0.02% Toxicokinetics in rats Up to 2 mg/kg/day for Anatase (20–60 nm) Increased Ti concentra- Altered testosterone  5 days tions in spleen and levels, histopathologic ovaries findings in thyroids Toxicokinetics in rats Up to 30 mg/kg/day for Anatase and rutile (pri- None detected None  7 days mary sizes not specified) Toxicokinetics in rats 12.5 mg/kg/day for Rutile (500 nm) Detection of particles in None  10 days GALT, lymph nodes and liver Subacute toxicity in mice Up to 500 mg/kg/day for Anatase/rutile (46 nm) Not examined Histopathologic findings  5 days in gut mucosa Subacute toxicity in mice Up to 100 mg/kg/day for Anatase (20–50 nm) Not examined Histopathologic findings  14 days in liver Subacute toxicity in mice 150 mg/kg/day for 14 days Anatase (21 nm) Not examined Histopathologic findings  in liver Subacute toxicity in rats 300 mg/kg/day for 14 days Structure not specified Not examined Histopathologic findings  (50–100 nm) in liver Subacute toxicity in rats 24,000 mg/kg/day for Rutile (173 nm) Detection of particles in None  28 days GALT Subacute toxicity in rats Up to 200 mg/kg/day for Anatase (75 nm) Not examined Histopathologic findings  30 days in liver Subchronic toxicity in Up to 250 mg/kg/day for Anatase (25 nm) Not examined Increased sperm abnor-  mice 42 days malities Subchronic toxicity in 64 mg/kg/day for 196 days Anatase (18 and 120 nm) ~ 0.15 µg/ml Ti in whole Histopathologic findings  mice blood in liver, kidney, spleen and pancreas Subchronic toxicity in rats Up to 1000 mg/kg/day for Coated rutile (145 nm) Detection of particles in None  90 days GALT Subchronic toxicity in rats Up to 1042 mg/kg/day for Anatase/rutile (26 nm) Marginally higher Ti blood None  90 days levels in males Subchronic toxicity in rats Up to 50 mg/kg/day for 30 Anatase (24 nm) None Altered serum enzyme  and 90 days levels Carcinogenicity in mice Up to 8350 mg/kg/day for Anatase (pigment-grade) Not examined Lower survival, hepatocel-  2 years lular carcinomas Carcinogenicity in rats Up to 2900 mg/kg/day for Anatase (pigment-grade) Not examined Hyperplastic bile ducts,  2 years thyroid carcinomas Reproductive toxicity in Up to 1000 mg/kg/day in Anatase and/or rutile Not examined None  rats gestation (43–213 nm) Reproductive toxicity in 100 mg/kg/day in gesta- Anatase (10 nm) Increased Ti content in Impaired learning and  rats tion hippocampus memory Acute colitis model in Up to 500 mg/kg/day for Rutile (30–50 nm) Not examined Histopathologic findings  mice 7 days in gut mucosa Colon cancer model in rats Up to 10 mg/kg/day for Anatase/rutile (22 and Detection of particles in Histopathologic findings  up to 100 days 118 nm) GALT and liver in gut mucosa Winkler et al. J Nanobiotechnol (2018) 16:51 Page 7 of 19 Taken together, the above reports indicate a size- was detected at 6 h after ingestion of the test item. Thus, dependent biokinetic behavior with low systemic absorp- this report presented by Pele et al.  not only confirms tion of orally administered fine-sized TiO particles the earlier finding of Böckmann et al.  in revealing a displaying primary sizes > 100 nm. This conclusion is con - peak of Ti in the blood after oral intake of TiO , but also firmed by a toxicokinetic study in Sprague–Dawley rats demonstrates that at least part of this Ti appears in the carried out according to the OECD test guideline 417, blood as whole particles. There is an intriguing difference which failed to detect any systemic uptake of pigment- between the background Ti concentration of whole blood grade rutile and anatase particles . Although some in most rodent studies (0.05 µg/ml or higher) and the penetration may take place across and between entero- lower levels observed in human blood (0.007–0.02 µg/ cytes, the observed intestinal uptake of nano-sized TiO ml). particles occurs primarily through the GALT as the port of entry [16, 53, 69]. Thus, a unique feature of the gas - Impact of the particle corona trointestinal exposure is that a fraction of TiO particles A critical aspect that has not yet been investigated with is retained in the GALT from where the particles may regard to bioavailability and systemic distribution is the reach the blood presumably through the lymphatic tho- effect of bound biomolecules that alter surface properties racic duct. There is finally only slow elimination of these [73–75]. In fact, small particles avidly and rapidly adsorb particles from internal organs (with estimated half-lives on their surface macromolecules including proteins that of up 650 days), indicating the potential for persistence modify key characteristics like their overall size, aggrega- and accumulation after repeated uptake. tion state, bioavailability, tissue distribution and bioaccu- mulation. The term “corona” was introduced to describe Oral bioavailability in humans the simultaneous attachment of multiple macromol- Studies in adult human subjects highlight a low but ecules from a physiologic environment to the surface of detectable oral bioavailability. Male volunteers ingested nanoparticles [76, 77]. For example, T iO particles incu- anatase particles at doses of 23 and 46 mg in gelatin cap- bated in a simulated intestinal digestion juice form a sules (mean particle size of 160 nm) or as a powder (mean corona of bile acids and proteins . Also, TiO nano- particle size of 380 nm) . Pretreatment blood Ti lev- particles incubated in blood plasma are readily decorated els, measured by ICP-atomic emission spectroscopy, with a layer of proteins like apolipoprotein A-1, comple- ranged between 0.007 and 0.02 µg/ml. After T iO admin- ment factors and immunoglobulins [73–75]. Thus, each istration, blood was obtained at different times over 24 h. biologic compartment has its own set of macromolecules Around 8–12 h after the intake of 160-nm anatase at the that interact with outer particle surfaces. Although not dose of 23 mg (~ 0.4 mg/kg body weight), peak Ti concen- explicitly tested, it can be assumed that TiO adopts dis- trations in the blood reached 0.04–0.05 µg/ml in the five tinctive corona compositions in the context of each dif- volunteers. The highest Ti concentration of 109.9 µg/l ferent food matrix in which this material is incorporated was detected in the blood of one volunteer 8 h after as additive . Also, any given corona configuration is ingesting 46 mg (~ 0.75 mg/kg body weight) of 160-nm expected to progressively change upon oral ingestion, as anatase. Administration of 380-nm anatase in the same the particles and surrounding food constituents move amounts yielded lower blood concentrations. In another through saliva in the mouth to the gastric and intestinal human study, nine volunteers received a 5-mg/kg single fluids . Presumably, the corona composition further oral dose of different TiO particles, i.e., anatase with a changes if the particles move from the intestinal lumen size of 15 nm, rutile with a size of ~ 100 nm and another to the central blood compartment and internal organs. rutile in the micron-scale range, dispersed in water . A potential effect of this continuously changing corona The ICP-mass spectromety analysis of blood collected is to modify key surface properties, which could medi- over a 4-day period, starting 24 h before dosing and end- ate particle transfer across biological barriers and their ing 3 days post-dose, revealed that essentially none of the uptake into cells including for example macrophages, administered particles were systemically absorbed. The dendritic cells or hepatocytes, thus influencing bio - background Ti concentration in the blood was 0.014 µg/ availability and tissue permeability . Importantly, ml. A further study involved seven volunteers who the extent of macromolecular interactions and composi- ingested a single 100-mg dose of anatase (particle size of tion of the resulting corona depends on both the surface 260 nm) in the form of gelatin capsules . The parti - chemistry of the particles and their exact diameter. At cles were subsequently identified in the blood of by dark the outer interface of nano-sized particles, for example, field microscopy and the presence of Ti was confirmed a highly curved surface increases the deflection angle by ICP-mass spectrometry. A peak Ti blood concentra- between absorbed macromolecules, possibly leading to a tion of ~ 10 µg/ml (against a background of ~ 1.5 µg/ml) higher density of such macromolecules in the corona of Winkler et al. J Nanobiotechnol (2018) 16:51 Page 8 of 19 smaller nanoparticles compared to the corona of larger exposure study was carried out in male albino mice with particles [73–75]. Therefore, nanomaterial characteristics anatase particles displaying a mean diameter of 21 nm like surface chemistry and outer curvature determine the . The particles were administered daily at 150 mg/kg corona composition and these considerations imply that body weight, leading to statistically significantly higher uncoated versus coated or small versus large particles liver weights as well as significantly increased serum lev - exert fundamentally different biological effects as a con - els of the liver enzymes alanine aminotransferase (ALT) sequence of their distinct corona composition. and aspartate aminotransferase (AST). The authors also reported histopathologic changes in the liver (focal Acute toxicity degeneration of hepatocytes with mononuclear cell infil - An overview of available toxicity studies is shown in tration) supporting the hypothesis that the tested nano- Table 1. With the exception of genotoxicity tests, only particles cause liver injury. Swelling and vacuolization the oral route of exposure is considered relevant for the of hepatocytes as well as infiltration of inflammatory risk assessment of TiO as food additive. No mortality cells were additionally detected in the liver of Wistar rats or adverse signs resulted from an acute exposure by sin- treated daily for 14 consecutive days by oral gavage with gle oral gavage administrations of TiO particles (crys-300 mg/kg TiO particles (composition not specified, pri - 2 2 tal structure not specified) according to the OECD test mary size of 50–100 nm) . These adverse hepatocel - guideline 420. A suspension of the test material (mean lular effects were supported by a statistically significant particle size of 155 nm) was administered to male and increase of ALT, AST and alkaline phosphatase serum female CD-1 mice at the dose of 5000 mg per kg body activity in treated animals compared to vehicle controls. weight . The same dose of nano-sized TiO particles A 28-day study was carried out in line with OECD (diameters of 25 and 80 nm) resulted in an increased liver test guideline 407 using rutile particles with a mean size weight. There were also histopathologic findings in the of 173 nm. This material was administered by oral gav - brain (fatty degeneration of hippocampal regions), in the age to 8-week old male Cr: CD(SD) rats at daily doses of liver (hydropic degeneration around the central vein) and 24,000 mg/kg body weight . One rat each from the kidney (glomerular swelling). These organ damages were control and test group died during the dosing period due reportedly more serious in the animals treated with the to accidental perforation of the esophagus. There were, 80-nm particles, which is consistent with higher Ti tissue however, no test item-related effects on mortality, food levels achieved by administration of these medium-sized intake, body weight, clinical signs, hematology, serum test items compared to the 25 and 155-nm counterparts clinical chemistry, hematology, organ weights, gross (see section on oral bioavailability above). No statisti- pathology or histopathology. Brown granular aggregates cal analysis supported the causal relationship between or clumps, likely indicative for the presence of TiO , were histopathologic findings and TiO treatments. For an seen upon hematoxylin and eosin staining in sections of acute oral toxicity study in Crl: CD(SD) rats according to the intestinal mucosa and draining lymphoid tissue, but OECD guideline 425, alumina- and silica-coated particles without overt cellular reactions. These microscopic find - with a primary size of 73 nm and a crystalline composi- ings related to the presence of test particles in the GALT tion of 79% rutile/21% anatase were administered by gav- were not considered to be adverse. age at doses of up to 5000 mg/kg body weight . These An oral subchronic (90-day) toxicity study was per- treatments with surface-coated particles failed to elicit formed in line with OECD test guideline 408 using rutile mortality, biologically relevant body weight changes, particles with a mean diameter of 145 nm. The particle clinical signs (except grey-colored feces) or gross organ surface was alumina-coated. This material was adminis - lesions. tered to groups of 8-week old Cr: CD(SD) rats by daily gavage doses of up to 1000 mg/kg body weight . Subacute and subchronic toxicity There were no treatment-related effects on survival, food A subacute exposure was carried out in male albino intake, body weight, clinical signs, hematology, clinical mice with anatase particles displaying a primary size of chemistry, hematology, organ weights, gross pathology 20–50 nm (see Table 1 for an overview of oral toxicity or histopathology. Test material-related findings were studies). Daily doses of 10, 50 and 100 mg/kg body weight limited to microscopic observations consistent with the were applied for 14 consecutive days . At the high- oral route of uptake. In particular, granular aggregates est dose, this treatment induced a statistically significant or clumps, indicating the presence of TiO , were seen in increase of liver weight and histologic changes includ- the intestinal mucosa and the draining lymphoid tissue, ing a recruitment of mononuclear cells to the vicinity of without tissue reactions. Again, these findings related to sinusoids accompanied by angiectasis (dilated sinusoi- the presence of TiO particles in the GALT were not con- dal spaces filled with blood cells). Another 14-day oral sidered adverse. There is also a 28-week study with CD-1 Winkler et al. J Nanobiotechnol (2018) 16:51 Page 9 of 19 mice exposed orally at 64 mg/kg/day to anatase particles serum testosterone levels, decreased layers of spermato- with mean diameters of 18 and 120 nm . The authors genic cells and an increased appearance of vacuoles in reported histopathologic findings such as tissue fractures the seminiferous tubules. No statistical evaluation of in the liver, glomerular atrophy in kidneys and islet hya- these histologic findings was given. The testosterone linization in the pancreas induced by 18-nm particles but reduction described in the young mice of this report is not upon treatment with 120-nm particles, consistent contrasted by another study using 9–10-week old rats, with the missing systemic retention of the latter (see sec- described in more detail in the section on reproductive tion on oral bioavailability above). toxicity below, where anatase particles (primary size of In summary, these oral toxicity studies in rodents 20–60 nm) given by the oral route were associated with reveal major uncertainties limiting their predictive value increased serum testosterone levels in males, whereas for the risk assessment of human dietary exposure. Many the concentration of this same hormone was reduced in reports are based on a small number of animals per treat- females . Another study focused on cardiac toxicity ment group, involve an unusual or inadequate design or in young rats (4 weeks old at the beginning of the study) lack statistical analyses. Some studies used insufficiently following 30 and 90 days of an oral exposure to anatase characterized particles with regard to composition, pos- particles (mean size of 24 nm) at 2, 10 and 50 mg/kg sible contaminants, impurities or physico-chemical body weight per day . The authors report changes in properties, and most reports failed to monitor particle some biochemical endpoints like decreased serum lac- size distributions. Single-dose  or repeated-dose oral tate dehydrogenase, hydroxybutyrate dehydrogenase and exposures [83–85] point to liver toxicity as a common creatine kinase activity in the high-dose group, but this endpoint following gastrointestinal absorption of nano- study did not reveal any toxicologically relevant effects. sized TiO particles with mean diameters below 100 nm. A comparison between rats aged 4 weeks and rats aged This endpoint involving liver toxicity is not seen after 9 weeks at the start of the study was carried out to test oral administration of TiO particles with mean diame- the susceptibility to anatase particles with a mean size ter above 100 nm . Further adverse outcomes in oral of 75 nm . Sprague–Dawley rats were administered toxicity studies were reported by the Medical College daily doses at 10, 50 or 200 mg/kg body weight for 30 of Soochow University (Suzhou, China). These studies consecutive days. Histologic examinations of the organs tested TiO nanomaterials synthesized by technical pro- after the treatment period revealed changes in the liver cedures that are not consistent with commercial prod- described by the authors as hepatic cord disarray, peri- ucts in the food sector. Three of these studies indicating lobular cell swelling, vacuolization and hydropic degen- toxicity in animals were withdrawn by journal editors due eration in both the 50 and 200-mg/kg dose groups, but to inadequate statistical analyses. As already pointed out only in young animals. These alterations were accompa - [13, 29], the same methodological deficits are also found nied by a statistically significant rise of serum bilirubin in other publications from the same group such that their at the anatase dose of 200 mg/kg. In adult rats, less seri- reports were not further considered. ous infiltrations of inflammatory cells in the liver paren - chyma were seen at 10 and 50 mg/kg (but not at 200 mg/ kg) and considered to represent background liver lesions Oral toxicity in young animals frequently observed in rats. In view of the higher exposure of children relative to In summary, these few studies on the reaction of dif- adults (see section on human exposure), it is also appro- ferentially aged rodents suggest the possibility that young priate to screen the literature for oral toxicity studies car- animals may be more susceptible than adults to develop- ried out in young laboratory animals. A seminal report ing adverse effects upon oral exposure to TiO particles. involved pubertal male mice aged 4 weeks at the begin- ning of exposure. Anatase particles with a mean diam- eter of 25 nm were administered orally at daily doses of Genotoxicity 10, 50 and 250 mg/kg body weight for 42 consecutive TiO particles with varying composition (anatase, rutile days . The analysis of epididymal sperm at the end or mixtures of these two polymorphs) and different sizes of the exposure period revealed a statistically significant were probed for mutagenicity in the canonical reverse and dose-dependent increment of morphologic abnor- mutation assay with bacteria (Ames test), usually at con- malities. Although no changes in sperm number were centrations of up to 5–10 mg per standard plate. In all detected, the fraction of sperm cells displaying abnor- cases, the tested particles failed to elicit mutations in the malities increased from ~ 13% in controls to ~ 23% in absence or in the presence of rat liver microsomes medi- the 50-mg/kg group and ~ 29% in the 250-mg/kg group. ating metabolic activation (see for example [89–93]). These effects on spermatogenesis were associated, in However, the Ames test is not considered suitable for this the medium- and high-dose groups, with a reduction of Winkler et al. J Nanobiotechnol (2018) 16:51 Page 10 of 19 purpose due to the presumed inability of bacterial cells, of 45 nm. The daily doses were 500–2000 mg/kg body conferred by their rigid cell wall, to take up the particles. weight applied for 5 consecutive days . The animals Conflicting findings were reported from in vitro tests were sacrificed 24 h after the last treatment and geno - carried out in rodent or human cells and aimed at the toxic effects were evaluated by counting the frequency detection of DNA strand breaks, point mutations, dele- of micronuclei in polychromated erythrocytes of the tions, chromosomal aberrations, micronuclei or sister bone marrow, and by monitoring the appearance of DNA chromatid exchanges. Mammalian cell-based genotox- strand breaks when subjecting bone marrow, brain and icity assays yielded both positive and negative outcomes liver cells to comet assays. These experiments revealed when used to test T iO particles. These methods include, a statistically significant and dose-dependent increase in in particular, single-cell electrophoresis (comet assays) as micronuclei and DNA strand breaks in response to the well as reporter gene, micronuclei, chromosomal aberra- TiO treatment. However, the report lacks an assessment tion and sister chromatid exchange assays (see for exam- of cytotoxicity in the tested tissues raising the possibility ple [83, 91, 92, 94–107]). The interpretation of findings is that the genotoxic effects might be secondary to particle- uncertain because the effects, if any, may be secondary induced cell death. The above considerations lead to the to cytotoxic or apoptotic reactions elicited by incubation conclusion that there is no solid evidence for anatase/ of rodent or human cells with high concentrations of the rutile particles being genotoxic. test material. For the evaluation of in vivo genotoxicity, it is impor- Carcinogenicity tant to consider that, in view of the low absorption of Long-term (2-year) carcinogenicity studies were carried TiO particles after oral uptake (see section on oral bio- out in both B6C3F1 mice and Fisher 344 rats using, as the availability), sufficient systemic exposure can only be test material, pigment-grade anatase particles designated achieved by parenteral administration. For example, Unitane 0–220 . The size of these test particles is B6C3F1 mice were subjected to daily intravenous injec- not specified but it can be assumed from their optical tions of anatase particles (mean size of 10 nm) at doses properties that they have a mean diameter in the 200– ranging from 0.5 to 50 mg/kg body weight, for 3 consecu- 300 nm range conferring a white color. The test mate - tive days. Genotoxicity was assessed by determining the rial was included in the diet of mice (groups of 50 males micronuclei in reticulocytes and the frequency of muta- and 50 females) at daily doses of 3250 and 6500 mg/kg tions in the X chromosome-linked phosphatidylinositol body weight (for male animals) and 4175 and 8350 mg/ glycan complementation group A (Pig-A) gene in periph- kg (for females). The particle-supplemented feed was eral blood cells . This study concluded that anatase not examined for possible nutritional imbalances. With nanoparticles reaching the bone marrow cause consider- the exception of white feces, there were no clinical signs able cytotoxicity but without inducing direct genotoxic related to T iO administrations. The test item did not effects. Transgenic C57Bl/6 mice harboring the bacte - affect the survival of male mice but, in the females of the rial lacZ reporter gene were injected intravenously with high-dose group, only 66% survival was reported until anatase particles (mean size of 22 nm) obtained from the end of the 104-week study compared to 90% survival the JRC Nanomaterials Repository, at daily doses of up in controls. No accompanying effects were reported that to 30 mg/kg body weight on 2 consecutive days . would explain this gender-specific difference. All sur - No genotoxic effects were detected by scoring the fre - viving animals were sacrificed after 103 weeks for com - quency of micronuclei in reticulocytes of the peripheral prehensive macroscopic and microscopic inspection for blood and by monitoring lacZ mutations as well as DNA neoplasms. There was a dose-dependent increase in the strand breaks (by comet assay) in liver and spleen cells. incidence of hepatocellular carcinomas in males (17% of Anatase/rutile particles (primary size of 21 nm) were animals in the control group, 19% in the low-dose group injected intravenously to Wistar rats at a single dose of and 29% in the high-dose group), although the test labo- 5 mg/kg body weight . No genotoxicity was subse- ratory noted that this higher occurrence of liver cancer quently detected by analyzing bone marrow cells in the remained within the range of historical reference con- comet assay. A threefold increase in micronucleated trols. The study authors, therefore, concluded that the cells was detected 1 h after the injection when polychro-oral TiO administration is not carcinogenic in the tested matic erythrocytes were stained with the conventional mice strain. May-Grunwald-Giemsa reagents, but not in concomi- In rats, the same U nitane 0–220 particles were tant stains with acridine orange. Although not recom- tested by dietary administration to groups of 50 males mended by the relevant OECD test guideline 474, other and 50 females at daily doses of 1125 and 2250 mg/kg authors selected the intraperitoneal route to expose rats body weight (for males) and 1450 and 2900 mg/kg (for with anatase/rutile particles displaying a primary size females). Again, the particle-supplemented feed was Winkler et al. J Nanobiotechnol (2018) 16:51 Page 11 of 19 not examined for possible nutritional imbalances. With not reveal any reproductive or developmental toxicity the exception of white feces, there were no clinical signs . related to TiO exposure. The test substance had no effect Other reports ascribe adverse reproductive effects to on survival. After 103 weeks, the organs of surviving ani- orally administered T iO particles but, unfortunately, mals were subjected to macro- and microscopic analyses. these further experiments in rodents were not carried There was an increased frequency of hyperplastic bile out following standardized procedures. Undoubtedly, ducts in males of both the low- and high-dose groups. these studies raise uncertainties but their impact on Tumor incidences in the treatment groups were report- hazard identification and characterization is still limited edly not higher than in controls. However, in females the in view of the small number of animals per treatment combined incidence of c-cell adenomas and carcinomas group, inappropriate study design or insufficient statisti - of the thyroid was substantially increased from 2% in cal analyses. In a study already mentioned in the preced- controls to 14% in the high-dose group. No such adeno- ing sections, anatase particles (primary size of 20–60 nm) mas or carcinomas were detected in the low-dose group. were given to Sprague–Dawley rats by the oral route at Although statistically significant by the Cochran–Armit - doses of up to 2 mg/kg body weight per day for 5 con- age test (P = 0.013) and the Fisher exact test (P = 0.042), secutive days . This oral exposure was associated with this increased neoplastic incidence was dismissed by increased serum testosterone levels in males, whereas the introducing a Bonferroni correction for multiple com- concentration of this hormone was reduced in females. parisons. Irrespective of such statistical considerations, The authors also reported histologic changes in the thy - the emergence of thyroid tumors in rats needs careful roid (desquamation into follicular lumen, follicles with considerations due to the questionable relevance of this irregular shape, smaller colloid space) after oral treat- finding for humans . ments at 1 or 2 mg/kg body weight per day for 5 days. Neurodevelopmental consequences of a T iO exposure Reproductive toxicity were suggested by a study in which pregnant Wistar rats A pivotal prenatal developmental study evaluated three were treated by oral gavage with anatase particles (pri- pigment-grade (pg-1, pg-2 and pg-3) and three ultrafine mary size of 10 nm) at 100 mg/kg body weight . This (uf-1, uf-2 and uf-3) anatase and/or rutile particles given dose was applied daily from gestation day 2 to gestation to pregnant rats following the OECD test guideline day 21. Two male pups from each litter were sacrificed 414. The primary particle size was 153–213 nm for the for the examination of brains immediately after birth. pigment-grade material and 43–47 nm for the ultrafine The Ti content in the hippocampus of the pups in the test material. These test substances were administered to group was increased to a statistically significant degree. Crl:CD(SD) rats by oral gavage on gestation days 6–20. Concomitantly, the authors observed that expression Also, pregnant Wistar rats were exposed to TiO parti- of the cell proliferation marker Ki67 is reduced in that cles (uf-2, pg-2 and pg-3) by oral gavage on gestation days brain region of treated animals relative to controls. On 5–19. The dose levels in both rat strains were 100, 300 post-natal day 60, the learning and memory capacity was and 1000 mg/kg body weight per day . At the end of tested in randomly selected male pups by means of the each exposure period, the rats were sacrificed for caesar - passive avoidance and Morris water maze test, and was ean sections and examination of the dam and fetuses. As found to be impaired in the treatment group relative to the only finding, at 1000 mg/kg per day, the uf-1 particles controls. led to an increased fetal sex ratio (males/females) from 0.46 in the controls to 0.60 in the treatment group. The Local effects in the gastrointestinal tract range of the test facility historical control for this param- Depending on the identified hazards, the usual risk eter was 0.43–0.53. Because the sex ratio is determined characterization may need the assessment of additional by events that occur around conception well before the endpoints that are not routinely inspected in the toxi- start of the treatment on gestation day 6, the authors con- cological evaluation of most chemicals. One question is cluded that this finding was not test item-related. These whether TiO particles may influence directly the bacte - results are confirmed by a reproduction study according rial community in the gut lumen. When tested in vitro to OECD test guideline 421, which involved Sprague– using anaerobic reactors, which provide an intestinal Dawley rats dosed with daily oral gavages of pigment- microbiome surrogate, food-grade anatase has been grade TiO at 1000 mg/kg body weight. The males were shown to cause marginal shifts in bacterial populations, administered the test substance for 40 days (beginning for example by slightly reducing the abundance of Bacte- from 2 weeks before the mating period) and females were roides ovatus in favor of a Clostridium species as seen at treated for 2 weeks before mating, during the gestation particle concentrations of 100 µg/ml or higher [117, 118]. period and until day 4 after delivery. Also this study did The biological relevance of these observations needs to Winkler et al. J Nanobiotechnol (2018) 16:51 Page 12 of 19 be ascertained but it appears so far that TiO particles do animals were sacrificed 24 h, 1 or 2 weeks after the last not exert major effects on the human gut microbiota at treatment. Analysis of the gastric epithelium by ICP- realistic concentrations. mass spectrometry revealed a dose-dependent increase Another potential target of nanomaterials is the intes- of Ti levels associated with histopathologic effects like tinal surface under the surveillance of dendritic cells that submucosal edema, necrosis of epithelial cells and act as first-line sentinels of foreign materials by filtering ulcerations . The severity of these histopathologic out a volume of up to 1500 µm , which equals their own findings increased with the dose. Another approach to cell volume, per hour . Unlike other antigen-pre- this problem made use of a colorectal cancer model to senting cells, dendritic cells constitutively express class II test the ability of food-grade T iO particles to acceler- major histocompatibility complexes and, in response to ate intestinal tumor progression. This murine model pathogen recognition, display co-stimulatory surface gly- involves the intraperitoneal injection of the tumor ini- coproteins and secrete inflammatory cytokines. By these tiator azoxymethane (AOM) combined with repeated combined features, dendritic cells constitute key activa- exposures to the pro-inflammatory agent DSS in drink - tors of both the innate and adaptive immune system [120, ing water. BALB/c mice were treated with the AOM/DSS 121]. It is not surprising to find that, based on their func - protocol, with intragastric gavage of TiO particles at tion in sampling their environment for intruding insults, 5 mg/kg body weight per day alone, or with a combina- dendritic cells are also able to capture nanoparticles tion of AOM/DSS and T iO particles . The primary in an efficient manner . It was shown in vitro that size of these particles is not specified but TEM analyses endotoxin-activated dendritic cells release the potent revealed aggregates/agglomerates with diameters rang- pro-inflammatory cytokine interleukin-1β (IL-1β) upon ing from 50 to 600 nm. The particle administrations took incubation with nano-sized anatase/rutile particles . place during 5 days per week for 10 weeks. Necropsies By activation of the inflammasome, leading to IL-1β carried out after 11 weeks revealed that the combination secretion, gavage applications of rutile particles (30– of AOM/DSS with TiO particles increased the expres- 50 nm) at a daily dose of 50 mg/kg body weight aggravate sion of markers of tumor progression (COX2, Ki67 and macro- and microscopic signs of acute colitis induced in β-catenin) in the epithelium of the colon, whereas TiO C57BL/6 mice by repeated exposures to dextran sulfate alone did not induce such expression changes. Moreo- sodium (DSS) given in the drinking water . In addi- ver, the authors observed that the tested food-grade TiO tion, a nano-Trojan horse hypothesis has been proposed particles, even in the absence of any tumor initiator/pro- due to the enhanced adsorptive surface property of nano- moter, reduced the density of protective mucin-produc- particles and, hence, their potential to carry harmful sub- ing goblet cells detected by alcian blue staining. stances as their cargo . A relevant pro-inflammatory The consequences of an oral exposure to food-grade cargo of dietary inorganic particles like those containing TiO on the intestinal mucosa was further investigated TiO consists of bacterial fragments such as lipopolysac- in male Wistar rats. Two types of anatase/rutile materi- charides (LPS) or peptidoglycan [126–128]. In this con- als were tested: commercial E 171 (primary particle size text, pigment-grade T iO particles have been shown to of 118 nm) and, as a reference, nanoparticles from the stimulate secretion of IL-1β from macrophages isolated JRC Nanomaterials Repository (primary size of 22 nm). from mice carrying a mutation in the nucleotide-binding In a first experiment, these test materials were given by oligomerisation domain-containing 2 (NOD2) gene , daily gavage administrations at 10 mg/kg body weight a mutation that confers an increased risk for inflamma - for 7 days in the absence of any additional treatment. tory bowel disease (IBD) [130, 131]. It is, therefore, pos- In a second experiment, the animals were pretreated sible that the binding of luminal antigens or adjuvants with the tumor initiator 1,2-dimethylhydrazine (DMH) to TiO particles could aid their delivery to inflamma - followed by exposure to E 171 at daily doses of 0.2 and tory cells of the gastrointestinal tract and contribute to 10 mg/kg body weight for 100 days through the drinking the pathogenesis of IBD in susceptible individuals. Con- water. Another experimental group was exposed to E 171 versely, dietary nanoparticles by adsorptive sequestration for 100 days without DMH pretreatment . Interest- on their surface may negatively influence the bioavailabil - ingly, exposure to E 171 in these experiments led to the ity of iron, zinc and fatty acids [132, 133]. internalization of light-diffracting particles not only in Reactions of the gastric mucosa were examined after cells of the Peyer’s patches but, to a minor extent, also in oral treatment of Swiss Webster mice with nanopar- the colonic mucosa and liver, particularly in proximity ticles (mean size of 46 nm) composed of rutile (77%) of the portal vein sinus. An accumulation of both E 171 and anatase (22%). The TiO nanoparticles were dis-and TiO nanoparticles in the Peyer’s patches of treated 2 2 persed in water and administered at daily doses of 5, 50 rats was confirmed by secondary ion mass spectrometry. and 500 mg/kg body weight for 5 consecutive days. The Effects on the lymphoid tissue included an increased Winkler et al. J Nanobiotechnol (2018) 16:51 Page 13 of 19 number of dendritic cells in Peyer’s patches and a factor of 200 (to adjust for inter-species as well as higher capacity of spleen cells to secrete cytokines like inter-individual variations in sensitivity, and for the interferon-γ and IL-17, possibly dysregulating immune extrapolation from subchronic to chronic exposure responses. In the carcinogenesis model, exposure to E ), this NOEL of 1000 mg/kg/day translates to 171 at 10 mg/kg per day led to a statistically significant a tentative safe upper level for the lifetime intake of increase of aberrant crypt foci (defined as abnormal tube- TiO particles of 5 mg/kg body weight per day. How- like glands in the lining of the colonic mucosa) regard- ever, the alumina-coated rutile under scrutiny is not less of whether the animals were pretreated with DMH at all representative for the full range of TiO used or not. Based on these findings, the authors concluded in the food sector, which includes anatase or mixed that food-grade TiO particles induce a low-grade local anatase/rutile polymorphs, particularly also in an inflammation in the mucosa that has the potential to uncoated form. initiate preneoplastic lesions in the colonic mucosa. An • Acute , subacute [83–85, 88], subchronic  and important caveat in this interpretation of the reported chronic toxicity studies  converge on the liver as findings is that the relevance of abnormal crypt foci as a possible target organ for adverse effects after oral an early precursor of colorectal cancer is controversially TiO intake (Table 1). The carcinogenicity study in discussed. mice revealed an increased incidence of hepatocellu- lar carcinomas in males at 6500 mg/kg/day (but not at 3250 mg/kg/day) compared to controls. The lack Conclusions of genotoxicity of TiO particles allows to assume a The Joint FAO/WHO Expert Committee on Food Addi - thresholded mode of liver cancer promotion and, by tives considered unnecessary the establishment of an application of a default uncertainty factor of 100 (to acceptable daily intake (ADI) for TiO additives in food adjust for inter-species and inter-individual varia- . This decision was taken on the basis of the low tions in sensitivity ), the resulting no observed solubility, poor absorption into internal organs like liver adverse effect level (NOAEL) of 3250 mg/kg/day and the absence of acute toxic effects. Many reports pre - translates to a tentative safe upper level for the life- sented in the context of this review suggest that, contrary time intake of 3.25 mg/kg body weight per day. The to the assumptions made 50 years ago, food-grade TiO subacute 30-day study by Wang et al.  identified particles are not totally inert upon oral intake. The obser - a NOAEL for anatase nanoparticles in young animals vation that TiO particles cause at least some adverse of 10 mg/kg body weight per day. Assuming a nano- reactions in experimental animals is disturbing because particle fraction of 4.2% by weight , this value of this material, including its unavoidable nano-scale 10 mg/kg corresponds, in terms of food-grade TiO , byproducts, has been in use as food additive since 1966. to a NOAEL of 238.1 mg/kg body weight per day. By The now available literature reveals data gaps and uncer - applying a default uncertainty factor of 600 (to adjust tainties that should be addressed before declaring food- for inter-species as well as inter-individual variations grade TiO particles as generally safe. A non-exhaustive in sensitivity, and for the extrapolation from 30 days list of such data gaps und uncertainties comprises the to chronic exposure as proposed by Heringa et al. following. ), the NOAEL of 238.1 mg/kg/day yields a tenta- tive safe upper level for the intake of TiO particles of • Although there is only limited absorption from the 0.40 mg/kg body weight per day. gastrointestinal tract, toxicokinetic experiments • One study suggests effects on spermatogenesis in rats revealed very long tissue half-lives for TiO resulting in sperm abnormalities upon intragastric nanoparticles in internal organs . This observa - gavage administration of anatase nanoparticles for tion indicates that there is the potential for a slow but 42 days . This study was carried out in pubertal continued accumulation of particles upon lifelong mice and is, therefore, relevant to address the pos- exposure. sibly higher vulnerability of children. The NOAEL, • The public literature offers only one subchronic oral as a nanoparticle dose, was 10 mg/kg body weight toxicity study in rodents carried out following inter- per day. Assuming as above a nanoparticle fraction nationally recognized OECD test guidelines . of 4.2% by weight, this 10-mg/kg value corresponds This study was based on the daily administration of again, in terms of food-grade TiO , to a NOAEL of food-grade TiO consisting of coated rutile particles. 238.1 mg/kg body weight per day. By applying a There were no test item-related adverse effects, yield - default uncertainty factor of 400 (to adjust for inter- ing a no observed effect level (NOEL) of 1000 mg/ species as well as inter-individual variations in sen- kg body weight per day, which corresponds to the sitivity, and for the extrapolation from 42 days to highest dose tested. By applying a default uncertainty Winkler et al. J Nanobiotechnol (2018) 16:51 Page 14 of 19 chronic exposure as proposed by Heringa et al. ), not only of nano-sized but also of novel submicron-sized this NOAEL of 238.1 mg/kg/day yields a tentative particles to be used as food additives. In particular, this safe upper level for the intake of T iO particles of review identified the following five main issues related 0.40 mg/kg body weight per day. Another study indi- to particles that resist rapid degradation or dissolution cates a penetration of T iO nanoparticles into the under digestive tract conditions: ovaries from the gastrointestinal tract of rats . A further report suggests neurodevelopmental distur- • To become eligible for the safety assessment, novel bances upon exposure to T iO nanoparticles during particles should first undergo a detailed characteri - pregnancy in rats . These findings, if confirmed, zation to provide unambiguous information on their would raise additional concerns regarding the repro- constituents, structure and shape, surface character- ductive safety. istics and coating, average size and size distribution, • There is finally only poor understanding of the con - impurities, aggregation or agglomeration states. All sequences of the proven uptake of TiO by reac- these parameters should be specified with accompa - tive GALT cells, possibly triggering inflammatory nying analytical methods. responses that could favor chronic conditions like • Particle characteristics like size, size distribu- IBD, and perhaps leading to initiation, promotion tion, crystalline form, shape and coating are critical and/or progression of neoplasms in the mucosa of determinants of intestinal uptake and adverse reac- the gastrointestinal tract [18, 134, 135]. Food-borne tions (see for example the above sections on particle inorganic particles have been shown to accumulate corona and local gastrointestinal effects). Therefore, during lifelong exposure in “pigment cells” of the there is little opportunity for read-across proce- GALT where the earliest signs of IBD are noted [126, dures to assess simultaneously multiple variants of 138, 139]. This is a group of chronic conditions rang - the same particulate material. Instead, we advocate ing from Crohn’s disease (affecting all segments of a case-by-case testing of particles with clearly estab- the digestive tract) to ulcerative colitis (restricted to lished specifications. the large bowel) . IBD has a multi-factorial etiol- • The potential genotoxicity of nano- and submicron- ogy with genetic susceptibility, gut microflora com - size particles should be ruled out with mamma- position and a dysfunctional local immune reaction lian cell models and, pending on the findings, using as main drivers . Dietary factors have also been in vivo genotoxicity tests with proven systemic expo- implicated in IBD and several authors raised the con- sure, following generally recognized OECD guide- cern that inorganic particles in food may contribute lines. to initiating this chronic inflammatory condition by • In the absence of any evidence of genotoxicity, the inappropriate stimulation of the innate immune sys- safety testing should proceed with an extended tem [55, 56, 142, 143]. 90-day oral toxicity assay in rodents following OECD test guideline 408 , but with additional param- In conclusion, the existing toxicity studies cannot com- eters for the detection of endocrine disruptors as pletely exclude human health risks from the long-term listed in test guideline 407 . This subchronic ingestion of T iO particles. The above hypothetical upper study should also include the analysis of tissues safe levels for dietary intake (between 0.4 and 5 mg/kg (including the GALT, mesenteric lymph node, spleen body weight per day) calculated from rodent studies are and liver) to examine the degree of systemic particle in no way conclusive and are only meant to illustrate in uptake. Such a toxicokinetic analysis should compare, quantitative terms the wide range of uncertainty in the if necessary using satellite groups of animals, the tis- current risk assessment of this ubiquitous food addi- sue level of particles at an early and late time of the tive. Especially the estimated consumption by children study to exclude a possible accumulation. suggests that, in any case, the dietary exposure to TiO • Additionally, more research is needed to under- particles should be reduced to remain, even in a worst- stand the consequences of interactions of indigest- case exposure scenario, below this proposed lowest safety ible nano- and submicron-sized particles with the threshold of 0.4 mg/kg daily. Further studies are needed GALT and, in particular, with dendritic cells residing to reduce existing uncertainties. in the intestinal mucosa. As outlined in the section on local effects in the gastrointestinal tract, dendritic Recommendations cells are the first-line sentinels of foreign materials as The uncertainty emerging in the retrospective assess - well as key activators of both the innate and adap- ment of T iO particles demonstrates the need for a fit- tive immune system and, as such, potential triggers to-purpose data requirement for the future evaluation of particle-induced chronic inflammatory conditions. Winkler et al. J Nanobiotechnol (2018) 16:51 Page 15 of 19 Abbreviations the food sector. Food Addit Contam. 2008;25:241–58. https ://doi. ANS: Scientific Panel on Food Additives and Nutrient Sources added to Food; org/10.1080/02652 03070 17445 38. AOM: azoxymethane; DMH: dimethylhydrazine; DSS: dextran sulfate sodium; 5. Puddu M, Paunescu D, Stark WJ, Grass RN. Magnetically recover- EFSA: European Food Safety Authority; EU: European Union; GALT: gut-associ- able, thermostable, hydrophobic DNA/silica encapsulates and their ated lymphoid tissue; IARC : International Agency for Research on Cancer; IBD: application as invisible oil tags. ACS Nano. 2014;8:2677–85. https ://doi. inflammatory bowel disease; ICP: inductively coupled plasma; Ig: immuno -org/10.1021/nn406 3853. globulin; IL: interleukin; JRC: Joint Research Centre; LOD: limit of detection; 6. Weir A, Westerhoff P, Fabricius L, Hristovski K, von Goetz N. Titanium LPS: lipopolysaccharides; M-cell: microfold cell; NOAEL: no observed adverse dioxide nanoparticles in food and personal care products. Environ Sci effect level; NOEL: no observed effect level; OECD: Organization for Economic Technol. 2012;46:2242–50. https ://doi.org/10.1021/es204 168d. Co-operation and Development; ROS: reactive oxygen species; SEM: scanning 7. Peters RJB, van Bemmel G, Herrera-Rivera Z, Helsper HPFG, Marvin HJP, electron microscopy; TEM: transmission electron microscopy; Ti: titanium; TiO : Weigel S, Tromp PC, Oomen AG, Rietveld AG, Bouwmeester H. Charac- titanium dioxide; UV: ultraviolet; V: vanadium. terization of titanium dioxide nanoparticles in food products: analytical methods to define nanoparticles. J Agric Food Chem. 2014;62:6285–93. Authors’ contributionshttps ://doi.org/10.1021/jf501 1885. HCW, TN, UM and HN wrote the manuscript. All authors read and approved 8. Yang Y, Doudrick K, Bi XY, Hristovski K, Herckes P, Westerhoff P, Kaegi the final manuscript. R. Characterization of food-grade titanium dioxide: the presence of nanosized particles. Environ Sci Technol. 2014;48:6391–400. https ://doi. Author detailsorg/10.1021/es500 436x. Institute of Food, Nutrition and Health, Department of Health Sciences 9. 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Brun E, Barreau F, Veronesi G, Fayard B, Sorieul S, Chaneac C, Carapito Ethics approval and consent to participate C, Rabilloud T, Mabondzo A, Herlin-Boime N, Carriere M. Titanium Not applicable. dioxide nanoparticle impact and translocation through ex vivo, in vivo and in vitro gut epithelia. Part Fibre Toxicol. 2014;11:13. https ://doi. Funding org/10.1186/1743-8977-11-13. Research in the authors’ laboratory was supported by the National Research 17. Gitrowski C, Al-Jubory AR, Handy RD. Uptake of different crystal Program “Opportunities and Risks of Nanomaterials” grant 406440-141619 and structures of TiO nanoparticles by Caco-2 intestinal cells. Toxicol Lett. by grant FK-15-053 from the “Forschungskredit” of the University of Zurich. 2014;226:264–76. https ://doi.org/10.1016/j.toxle t.2014.02.014. 18. 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