Annex B - Statement on the safety of Titanium Dioxide (E171) as a Food Additive

Reference
 

TiO2 characterisation 

Quality of study e.g., OECD/GLP 

Method and duration of dosing 

Study methodology to include species, numbers, controls, 

Results 

Bettini et al., 2017

1) E 171, anatase, 20–340 nm (118 nm) (TEM); 44.7% particles < 100 nm;

2) TiO2 NPs (NM-105), anatase/rutile, 15–24 nm.

OECD

Series One Dosage: 200 μ L with TiO2 NM-105, E171 (10 mg/kg of BW/day) or water for 7 days by gavage.

Series Two Dosage: E-171 at 200 μ g or 10 mg/kg of BW/day via drinking water for 100 days (with or without DMH treatment).

Series Three Dosage: No treatment followed by a single dose of 10 mg/kg E-171.

Series One: rats (n = 10 rats/group) dosed daily by intragastric gavage (200 μ L) with TiO2 NM-105, E171 (10 mg/kg of BW/day) or water for 7 days.

Tissue imaging, flow cytometry and cytokine assays, tissue inflammation

and gut permeability measurements were conducted.

Series Two: rats (n = 11 to 12 per group) were treated or not with 1,2-dimethylhydrazine (DMH) to induce colon carcinogenesis and exposed to E-171 at 200 μ g or 10 mg/kg of BW/day via drinking water for 100 days. Control animals (n = 12) received water only.

Flow cytometry and cytokine assays were assessed for gut inflammation and ACF.

Series Three: untreated rats (n = 4) were evaluated for ex-vivo cytotoxicity and proliferative assays on isolated immune cells.

E-171 particle agglomeration was assessed in the luminal contents of the jejunum and colon collected from 4 rats 4 h after a single dose of 10 mg/kg was delivered.

Titanium was detected in the immune cells of Peyer's patches. Dendritic cell percentage were increased, observed days after exposure but no effect at 100 days.

No effects in the spleen.

Regulatory T cells and T-helper cells were significantly decreased days after exposure and at 100 days for rats exposed to E 171.

Stimulation in vitro of immune cells isolated from Peyer's patches had a decrease in T-helper (Th)-1 IFN-γ secretion and splenic Th1/Th17 inflammatory responses increased.

With exposure to TiO2 NP there was an observed increase in the percentage of dendritic cells in Peyer's patches with no decrease in the percentage of Tregs.

E 171 exposure (1 week) did not initiate intestinal inflammation, but E 171 treatment (100-days) showed colon microinflammation - significantly increased IL-1β, IL-8 and TNF-α expression in the colon and increased IL-10.

 Talamini et al., 2019

E171 (35% nano determined by TEM), 99.3% pure anatase, 201.2 ± 8.5 nm in suspension (NTA). 

No sonification or deagglomeration to simulate realistic conditions.

This work was reviewed by the Institute for Pharmacological Research Mario Negri IRCCS Animal Care and Used Committee (IACUC) and then approved by the Italian National Institute of Health (code:42/2016-PR).

Treatments were given 3 days per week for 3 weeks for a total of 9 treatments in 21 days. Average daily dose of ~2 mg/kg bw.

Treatments were dripped slowly into the mice’s mouths, allowing each drop to be swallowed. 

NFR male mice (22/group) were administered either water (control) or 5 mg/kg bw E171 suspended in water.

Ti concentrations in tissues were determined by single particle ICP-MS analysis.

Ti concentrations in the liver (0.94 ± 0.57 µg/g tissue) and large intestine (1.07 ± 0.38 µg/g tissue) were significantly higher in treated mice compared to controls.

Ti concentrations in the brain, kidney, and testes were below the quantification limit (<0.03 µg/g).

Ti concentrations in lungs, spleen, stomach, and small intestine were not statistically significant between treated and control mice.

Riedle et al., 2020

E171, anatase, 119 nm.

N/A

Mice were exposed to 0, 1, 10, or 100 mg/kg bw/d E171 via the diet for 6, 12 and 18 weeks.

Mice were divided into 4 groups of 18 and given 0, 6.25, 62.5, or 625 mg/kg diet (equivalent to approximately 0, 1, 10, or 100 mg/kg bw). Then 6 mice per group were euthanized at 6, 12 and 18 weeks.

No evidence of gross alteration of immune-cell physiology or inflammation at doses up to 100 mg/kg bw/d via the diet.

Authors demonstrate E171 uptake by Peyer’s patches, validating the delivery model.

Presence of E171 particles detected by reflectance confocal microscopy (no quantification of particles completed).

Weak signals observed at the base of Peyer’s patches at low and mid-doses. Higher signals observed at highest dose, indicating evidence of dose-response.

Comera et al. 2020

Food grade TiO2 (E171) 95% anatase.

European legislation (Council Directive 2010/63/UE) and French Decree 2013–118-compliant.

Mice (4 per group).

Dosage: single dose (200 μl) of either

E171 at 40 mg/kg of body weight (BW) or 200 μl of vehicle (water) by intragastric gavage.

In addition, in some experiments, the gavage solution from sonicated E171 particles was equilibrated in 30% corm oil and vortexed before oral delivery.

Adult C57BL/6 mice (12–18 weeks).

Animals were terminated at 2, 4, 8, and 24 hours to recover the intestine.

Small intestine: TiO2 absorption peaked at 4 h in jejunal and ileal villi and returned to basal levels at 8 h and undetectable at 4 h but was present at 8 h in the jejunal Peyer’s patches.

Colon: Low absorption.

Blood: TiO2 particles were detected at 4- and 8-hours post-treatment.

30 minutes post-exposure to TiO2 in the presence and absence of pharmacological inhibitors of paracellular tight junction (TJ) permeability, TiO2 absorption by the jejunal villi was decreased by 66% (p < 0.001 vs. control) in the presence of triaminopyrimidine.

Other inhibitors had no significant effect.

Absorption via a goblet cell (GC)-associated pathway was also detected.

Dudefoi

2017b

Food-grade TiO2 (E171-1, 17% NPs and 100% anatase and E171-6a).

N/A

Dosage: 100–250 ppm.

Method: A defined model intestinal bacterial community.

At these low concentrations no impact on gas production and only a minor effect on fatty acids profiles was observed with limited effect on bacterial communities.

Proquin et al. 2018

E171 in combination with azoxymethane (AOM)/dextran sodium sulphate (DSS) vs E171 only.

N/A

Dosage: 5 mg/kg bw per day of E171 by gavage for 2, 7, 14, and 21 days.

BALB/c mice.

E171 induced a downregulation of genes involved in the immune system which were indicative of impairment.

Additionally signalling genes involved in a variety of types of cancer including colorectal cancer were modulated and effects were observed which indicated a  potential association with oxidative stress.

Jensen et al. 2019

Vegetable carbon (E153) and food-grade titanium dioxide (E171), mean TiO2 particle size of 270 nm.

N/A

Dosage: 10 weeks by oral gavage once a week.

Rats.

TiO2-only Results:

Decreased gene expression of protein TJP1 was observed in the rats only exposed to E171 (500 mg/kg/week) and shorter lung telomeres.

This study found no oxidative DNA damage in the liver or lung and no changes in the DNA repair activity of oxidative DNA damage in the lung.

Farrell TP and Magnuson B. (2017).

4 different food grade TiO2 test items containing a range of particle sizes and morphologies.

GLP-compliant, OECD TG 41.

Dosage: Four grades of TiO2 (200 ppm) or control (0 ppm) via the diet for 7 days followed by a control diet for 1, 24, or 72 hours.

Male and female Sprague-Dawley rats were given TiO2 by the diet equivalent to 30 mg/kg bw/day for 7 days.

Animals were then terminated post-feeding at 1, 24 and 72 hours.

Ti in kidney, liver and muscle were below LOD (<0.1-<0.2 mg/kg ww).

Ti in tissues, where above LOD, was 0.1-0.3 mg/kg w.

Ti in blood was <0.04 mg/L for all samples.

Ti in urine was equal to <2% daily dose/L and below LOQ.

Ti in faeces was found to be the main route of excretion.

No differences in absorption were found between the different grades of TiO2.

Reference

TiO2 characterisation 

Quality of study e.g., OECD/GLP 

Method and duration of dosing 

Study methodology to include species, numbers, controls, 

Results 

Warheit, Boatman and Brown, 2015

1)    anatase/rutile (89/11%) (uf-1), d50=43 nm d50=23 nm.

Methods: XSDC and TEM respectively Shape: Irregular.

2)    anatase (100% nano) (uf-2) d50= 42 nm d50=19 nm.

Methods: XSDC and TEM respectively.

Shape: Irregular.

3)    rutile (100% nano) (uf-3), d50=47 nm d50=22 nm Methods: XSDC and TEM respectively.

Shape: rod-like.

4)    anatase (27% nano) (pg-1), d50=153 nm d50=120 nm Methods: XSDC and TEM respectively.

Shape: irregular.

5)    rutile (11% nano) (pg-2), d50=195 nm d50=165 nm Methods: XSDC and TEM respectively.

Shape: irregular.

OECD Guideline 414.

Sterile water-based TiO2 sample formulations were administered by oral gavage to time-mated rats from the time of approximate implantation until the day prior to expected parturition.

Dose levels: 0, 100, 300 or 1,000 mg/kg bw per day.

Dosage volume: 5 mL/kg bw per day.

Three studies (Group size n=22): Time-mated pregnant Sprague–Dawley rats, (Crl:CD(SD)) exposed to TiO2 (uf-1, uf-3 and pg-1) by gavage on Gestational Days 6–20.

Three additional studies (Group size n=22-23) pregnant Wistar rats exposed to TiO2 (uf-2 and pg-2) by gavage from Gestational Days 5 to 19.

Necropsy:

• Gross examination of the dam,
• Counting of corpora lutea,
• Implantation sites,
• Resorptions,
• Live and dead fetuses,
• Fetal sex,
• Fetal weight,
• Fetal pathological external, visceral and skeletal examinations for abnormalities.

At 1,000 mg uf-1/kg per day, mean fetal sex ratio and the means for male and female fetuses per litter were statistically significantly different from the control group means.

Mean male fetuses: 7.2.

Mean male fetuses control group: 5.5.

Test facility historical control group data range: 5.2 to 7.4.

Mean female fetuses: 4.8.

Mean female fetuses control group: 6.7.

Test facility historical control group data range: 5.8 to 8.3.

Mean fetal sex ratio of the 1,000 mguf-1/kg bw per day group: 60% (males/females).

Mean control group fetal sex ratio: 46%.

Test facility historical control group data range: 43% to 53%.

A few incidental changes in body weight and feed intake were noted, no other changes were observed in the dams or the fetuses.

Tassinari et al., 2014

TiO2 nanoparticles (anatase, primary size <25 nm, BET surface area 45-55 m²/g, purity 99%). 

All experiments on animals were performed according to the European Community Council Directive 86/609/EEC (EEC 1986). 

TiO2 nanoparticles were administered by oral gavage over 5 consecutive days at a dose of 0, 1, 2 mg/kg body weight per day. 

Sprague-Dawley rats were divided into 3 treatment groups (7 rats/sex/group).  

Treatment groups were high dose (2 mg/kg bw), low dose, (1 mg/kg bw), and controls (CTRL) (vehicle only (distilled water)). 

In the high dose treatment group, significant increases in total Ti tissue levels were found in spleen (0.036 ± 0.009 vs. 0.046 ± 0.008 µg/g fresh weight; p ≤ 0.05) and ovaries (0.28 ± 0.07 vs. 0.12 ± 0.04 µg/g fresh weight; p ≤ 0.01).  

Sex-related histological alterations were observed at both dose levels in thyroid, adrenal medulla, adrenal cortex (females) and ovarian granulosa, without general toxicity.  

Altered thyroid function was indicated by reduced T3 (males). Testosterone levels increased in high-dose males and decreased in females.  

In the spleen of treated animals TiO2 aggregates and increased white pulp (high-dose females) were detected, even though Ti tissue levels remained low reflecting the low doses and the short exposure time. 

Ammendolia et al. 2017 

Nano-sized titanium dioxide (anatase, primary size <25 nm, BET surface area 45–55 m²/g, purity 99%).

N/A

TiO2 NPs at 2 mg/kg bw per day for five days in male and female rats.

N/A

Nanoparticle deposition the intestinal tissue and increased serum testosterone levels. There was no evidence of oxidative stress or cellular alteration at low concentrations of TiO2 NPs however NP treatments associated with testosterone or Insulin-like Growth Factor-1 showed increased cell proliferation.

Geraets et al. 2014

TiO2 NPs (sizes NM-100, NM-101, NM-102, NM-103, and NM-104) with differing particle sizes and structure.

N/A

Dosage: Oral and intravenous administration of a single or five repeated doses.

TiO2 nanoparticle kinetics were investigated using intravenous injection and oral dosing in rats.

For orally dosed rats, liver, spleen and lymph nodes were targeted for analysis.

Following oral exposure, Ti levels in the liver and spleen were only occasionally above the LOD and was detected in lymph nodes at low levels.

Following intravenous exposure, Ti distribution was observed across all tissues (liver, kidney, lung, spleen, heart, brain, thymus and reproductive organs with the liver identified as the main target.

Recovery 24 hours post-exposure was 64-95% or 59-108% respectively for male and female rats.

The maximum relative decrease of TiO2 up to 90 days post-exposure was 26%.

Hendrickson et al. 2016

2 test items TiO2 NPs (5-10 nm and 20-25 nm respectively).

N/A

Dosage: Intragastric administration of TiO2 NPs (1 of 2 test items) for 28 days at a dose of 250 mg/kg of body weight per day.

Male rats.

GIT and secondary organ translocation were size-dependent.

Larger nanoparticle exposure showed deposition in liver, kidneys, spleen, and small intestine (0.01– 0.29 μg/g of organ).

Smaller nanoparticle exposure resulted in Ti deposits in brain, lungs, heart, liver, kidneys, spleen, small intestine, testicles, and blood (0.004–0.227 μg/g of organ).

Hendrickson et al. 2020

TiO2 NPs

N/A

Dosage: A single dose suspension of TiO2 NPs (250 mg/kg of body weight).

Model: A Physiological model designed to mimic the intestinal lumen of an experimental animal.

TiO2 NPs were found in the small intestine mucosa, liver, and spleen.

TiO2 NPs resulted in different changes in the cellular ultrastructures in the endoplasmic reticulum, mitochondria, extensions into the perinuclear spaces and caused myelin-like structures to appear.

The most sensitive organ was noted to be the spleen.

Kreyling et al. 2017a

TiO2 anatase NPs.

Median agglomerate size: 70 nm.

N/A

Dosage: 40–400 μg/kg bw single intravenous dose in aqueous suspension.

Female Wistar rats.

Clearance and biokinetics were observed from 1-hour post-dosage to 4 weeks.

Highest TiO2 NP accumulation occurred in the liver after one day (95.5%) then then spleen (2.5%), carcass (1%), skeleton (0.7%) and blood (0.4%). TiO2 NPs were detected in all other organs at levels lower than these.

TiO2 NPs in the blood decreased quickly 24 h after exposure.

Organs and tissue NP levels were stable until day-28.

Kreyling et al. 2017b

TiO2 NPs.

N/A

Dosage: Oral dosage of a single dose of an aqueous TiO2 NP suspension at 30–80 µg/kg bw.

Female Wistar-Kyoto rats.

Assessed 1 h, 4 h, 24 h and 7 days post-oral exposure.

0.6% of the administered dose passed the gastro-intestinal-barrier after 1 hour.

0.05% of the dose were still distributed in the body after 7 days distributed across the following organs:

liver (0.09 ng/g), lungs (0.10 ng/g), kidneys (0.29 ng/g), brain

(0.36 ng/g), spleen (0.45 ng/g), uterus (0.55 ng/g) and bone deposits (0.98 ng/g).

Faecal excretion was complete after 4-5 days.

Sadiq et al. 2012

TiO2 NPs.

N/A

Dosage: 1) Intravenous 0.5, 5.0, and 50 mg/kg TiO2 NPs.

2) Intravenous three daily doses of 50 mg/kg TiO2 NPs Ti levels in bone marrow measured after 4, 24, and 48 hours of the last treatment.

6–7-week-old male B6C3F1 mice.

In vivo micronucleus and Pig-a (phosphatidylinositol glycan, class A gene) mutation assays using TiO2 NPs to evaluate genotoxicity.

Blood: Samples taken one day before the treatment and on Day 4, and Weeks 1, 2, 4, and 6 after the beginning of the treatment.

Pig-a mutant frequencies were determined at Day −1 and Weeks 1, 2, 4 and 6, percent micronucleated-reticulocyte frequencies were measured only on Day 4.

Blood results:

No increase in Pig-a mutant frequency or %MN-RETs,

Tissue results: Ti NPs peaked at 4 hours post-exposure.

%RETs was reduced in treated animals on Day 4 (dose-dependent) which suggests cytotoxicity of TiO2-NPs in the bone marrow.

No evidence of genotoxicity.

Reference

TiO2 characterisation 

Quality of study e.g., OECD/GLP 

Method and duration of dosing 

Study methodology to include species, numbers, controls, 

Results 

Pele et al. 2015

Pharmaceutical/food

grade TiO2, anatase, 50-250nm.

This study was conducted based on ethical approval under EC01/037.

Dosage: A single dose of 100 mg TiO2.

Test subjects: Humans with normal intestinal permeability.

Blood samples were collected at between 0.5 to 10 h post-oral exposure.

Blood samples were analysed for visual TiO2 reflective particles using dark field microscopy.

Early absorption occurred by 2 hours with a peak maximum at 6 hours.

Following oral dosing. This mirrors the results of a previous study by Bockmann et al (2000) which the authors were attempting to replicate.

Guillard et al. 2020

Basal Ti level in human placenta study.

TiO2 with 55% NPs, 20 to 440 nm.

N/A

Samples were taken of plancenta and meconium from human babies (n=22) and tested for TiO2 and other metals and trace elements.

TiO2 in human placentas was analysed by ICP-MS and STEM coupled to EDX spectroscopy.

All placenta contained TiO2 at 0.01 to 0.48 mg/kg of tissue with the majority below 100nm in size (over 50%).

Meconium samples also contained TiO2 between 0.02-1.5 mg/kg.

Heringa et al. 2018

Post-mortem analysis of human liver and spleen TiO2 analysis.

N/A

N/A

High resolution ICP-MS was used to detect TiO2 in the liver and spleen in 15 deceased humans (9 female and 6 male).

7 of the 15 livers sampled contained TiO2 and 13 of 15 of spleen samples contained the same.

Particle sizes respectively for liver and spleen:

85–550nm and 85–720nm with 24% <100 nm in size.

Particle mass concentration for liver and spleen respectively:

To 0.3 mg titanium/kg tissue and 0.01 to 0.4 mg titanium/kg tissue.

Average concentration in liver and spleen samples:

40 ng/g and 80 ng/g.

Peters et al. 2020

Postmortem tissue analysis of deceased persons for the presence of TiO2.

Detected particle sizes were in the range of 50–500 nm, with a mode of 100–160 nm.

15 humans sampled, 8 female and 7 male aged 64-98 years.

Postmortem liver, spleen, kidney, jejunum and ileum were sampled.

Findings of between <0.01 to 2.0 mg Total Ti/kg with median values (mg Ti/kg):

Liver = 0.02,

Spleen = 0.04,

Kidney = 0.05,

Jejunum = 0.13,

Ileum = 0.26.

Particulate TiO2 were observed from 0.01 to 1.8 mg Ti/kg with median values (mg Ti/kg):

Liver = 0.02,

Spleen = 0.02,

Kidney = 0.03,

Jejunum = 0.08,

Ileum = 0.25.

Particulate TiO2 accounted for 80% of the total-Ti concentration.

Reference

TiO2 characterisation 

Quality of study e.g., OECD/GLP 

Method and duration of dosing 

Study methodology to include species, numbers, controls, 

Results 

Blevins et al., 2019

E 171, anatase, 110–115 nm (SEM), 36% particles < 100 nm.

N/A

E-171 concentrations:

0 mg/kg* dose (22.3 ± 1.2, 23.7±1.8 ppm)

40 ppm dose (59.6 ±1.1, 61.0±2.6 ppm)

400 ppm dose (384 ± 8, 387±13 ppm)

5000 ppm dose (4310 ± 132, 4610±160 ppm).

Six-week-old male Wistar Han IGS (Crl:WI (Han)) rats.

Test material: Food grade sample E-171. DIfferent grades of commercially-available E-171 were averaged to produce the test material supplied. Test material was added to feed.

Two feed batches: batch one was fed throughout the 7-day study and through week 10 of the 100-day study. Batch two was fed post-week 10 of the 100-day study.

7-day study: 4 groups of 5 animals (randomised based on weight). Total food and water consumption were calculated at the end of the study. Body weights measured at the start and end of the 7-day exposure period.

100-day study: 8 groups of 16 animals.

Groups 1-4 were dosed with 180 mg/kg BW dimethylhydrazine dihydrochloride (DMH · 2HCl) in 1.5% EDTA-0.9% NaCl, pH 6.5 by intraperitoneal injection (the highest dose that showed no signs of toxicity in pilot studies).

Animals in groups 5-8 were given a dose of 1.5% EDTA-0.9% NaCl, pH 6.5.  by intraperitoneal injection.

Seven days post-injection, feeding of the test material was commenced at 0, 40, 400, or 5000 ppm E-171 in groups 1-4 and 5-8 for 100 days.

 E-171 consumption did not alter T-cell-mediated mechanisms of immune control.

Dietary E-171 did not induce inflammation peripherally or in the GI tract.

An increase was observed in the relative spleen weight in 22.4 mg E-171/kg bw per day + DMH compared to not initiated animals and an increase in IL-17A in colon (22.4 mg E 171/kg bw per day + DMH) and IL-12p70 in plasma (3.5 mg E 171/kg bw per day + DMH), with no dose-related effects.

No changes were observed in spleen cellularity.

No changes were observed in the percentage of CD103+ DC, CD4+ T helper cells or total or activated Treg in peripheral blood, spleen or Peyer's patches in animals exposed to E-171 + DMH compared to animals treated with only DMH.

No treatment related histopathological changes were observed in the duodenum, jejunum, ileum, spleen, liver, lung and testes in animals exposed only to E-171.

Rats treated with DMH only and those which received E-171 in the diet after the initiation displayed several histopathological abnormalities.

A significant surface area of the epithelial surface of the sampled colon (proximal, middle and distal) was obscured when observed by light microscopy therefore the entire surface of the colon samples for ACF and ABC could not be examined.

No change in the number of ACF and ABC were observed due to E-171 exposure alone.

Akagi et al., 2023 – 28 Day Study

6 nm TiO2 nanoparticles.

N/A

5 female and 5 male F344/DuCrlCrlj rats.

TiO2 NPs with a crystallite size of 6 nm were examined in male and female F344/DuCrlCrlj rats by repeated oral administration of 10, 100, and 1000 mg/kg bw/day (5/sex/group) for 28 days.

No mortality was observed in any group, and no treatment-related adverse effects were observed in body weight, urinalysis, haematology, serum biochemistry, or organ weight. Histopathological examination revealed TiO2 particles as depositions of yellowish-brown material. The particles observed in the gastrointestinal lumen were also found in the nasal cavity, epithelium, and stromal tissue in the 28-day study.

Overall, No effects were observed after repeated oral administration of TiO2 with a crystallite size of 6 nm at up to 1000 mg/kg bw/day regarding general toxicity, accumulation of titanium in the liver, kidneys, and spleen, abnormality of colonic crypts, and induction of DNA strand breaks and chromosomal aberrations.

Donner et al. 2016

One of three pigment-grade or one of three ultrafine /nanoscale anatase and/or rutile TiO2 test materials.

OECD 474 Guidelines.

Dosage: Single oral gavage doses of 500, 1000 or 2000 mg/kg body weight with negative (water) and positive controls (cyclophosphamide).

Male and female rats.

Blood samples were collected 48 and 72 h post-exposure.

There were no relevant increases in the micronucleated RET frequency in any TiO2-exposed group at either time point at any dose level.

All tests showed negative results for in vivo genotoxicity effects, no significant blood or liver TiO2 increases following exposure to the highest dose.

Reference
 

TiO2 characterisation 

Quality of study e.g., OECD/GLP 

Method and duration of dosing 

Study methodology to include species, numbers, controls, 

Results 

Leuschner, 2020 – Main Study

Test substance: Anatase E-171, median particle diameter 99.9 (± 2.0 nm), 51% of particles < 100 nm.

Dietary particle size: 109.2 (± 1.4) to 113.7 (± 4.9 nm), 31-43% of particles < 100 nm. 

OECD Test Guideline 443.

Dosages (by oral diet, dependent on endpoint for each cohort):

F0, F1-1A and 1B - 0, 100, 300, and 1000 mg/kg bw/day over 10 weeks (prior to mating and up to the end of weaning periods).

F1-2A and 2B: indirectly exposed to test item via milk of feed-dosed rats.

Additional background levels of TiO2 in feed were estimated to be approximately 1.4 mg/kg bw/day.

Diet mixtures were prepared weekly. Food intake was monitored and volumes of dosed feed was adjusted according to previous consumption weekly.

Administration of keyhole limpet hemocyanin (KLH) and cyclophosphamide KLH via intravenous bolus injection in F3 cohort.

96 male and 96 female CD® (Sprague Dawley) IGS Rat (Crl:CD(SD)).

Test Grouping Sizes:

F0 – 4 groups, 20 male, 20 female per group.

F1-1A and 1B – 20 male, 20 female.

F1-2A, 2B and 3– 4 groups, 10 male, 10 female.

Endpoint Groupings:

F1-1A and 1B - reproductive & developmental toxicity

F1-2A and 2B - developmental neurotoxicity.

F3 - developmental immunotoxicity.

Test Item Exposure:

F0 - Exposure to test item was from 10 wks prior to mating until F1 generation weaning. 

F1 – Exposure to test item was from weaning to PND 4/PND 8 of F2 generation.

F2 – Exposure from birth until PND 4 or PND 8 (via milk).

Results:

F0 - Dose-dependent marginal increase in TiO2 blood and urine concentration in rats dosed with 1000 mg/kg bw/day.

No test item-related effects on sexual function or fertility in males or females. No test item-related pre- or postnatal loss observed.

No test item-related thyroid hormone or haematological effects.

No test item-related differences in splenic lymphocyte subpopulation distribution.

No test item-related changes related to histopathology examinations including the testis and epididymides and intestinal examinations for ACF.

Reproductive & developmental toxicity F1-1A and 1B.

No test item-related effects on sexual function or fertility in males (F1-1A – sexual hormones and maturation and sperm) or females (F1-!B - sexual maturation and hormones, number and length of estrous cycles, follicle number or corporea lutea). No treatment-related pre- or postnatal loss. No external or internal abnormalities detected in pups.

No test item-related effects on growth or sexual development.

No test item-related differences in body weight or food consumption.

No test item-related thyroid hormone or haematological effects reported at any dose for F1-1A cohort.

No test item-related differences in weight or histopathology (spleen, thymus, lymph nodes, bone marrow, white blood cell count, and splenic lymphocyte subpopulation distribution in F1-1A.

No test item-related effects on pathology or histopathology.

There was a statistically significant decrease in the antigen specific IgM level was measured at the highest dose tested (1,000 mg/kg bw/d) in males only (–9%) and without an apparent dose-response.

No dose-dependent increase in TiO2 blood concentration in rats up to 1000 mg/kg bw/day.

Observation of pale faeces not of toxicological relevance.

Developmental neurotoxicity

F1-2A and 2B

No test item-related pre- or postnatal loss observed. No external or internal abnormalities detected in pups.

No test item-related effects on growth or sexual development.

No test item-related differences in body weight or food consumption.

No test item-related effects on neurofunctional endpoints (F1-2A).

Dose-dependent elevations of blood TiO2 levels were found exceeding 300 mg/kg.

No test item-related effects on pathology or histopathology.

Observation of pale faeces not of toxicological relevance.

Developmental immunotoxicity

F3

No test item-related differences in body weight or food consumption.

No mortality and no test item-related effects reported at any dose for any generation.

No ACF were found in the colons of animals in any dose group.

Observation of pale faeces not of toxicological relevance.

Leuschner, 2020 – Satellite study

Test substance: Anatase E-171, 51% of particles < 100 nm.

Dietary particle size: 31-43% of particles < 100 nm. 

OECD Test Guideline 443.

F0 satellite group: 0, 100, 300, and 1000 mg/kg bw/day over 10 weeks (prior to mating and up to the end of weaning periods).

CD® (Sprague Dawley) IGS Rat (Crl:CD(SD)).

F0 satellite group – 30 male, 30 female per group + additional 40 (20 male, 20 female) for use as an F1 generation of satellite animals to be used as the positive control group in the KLH-assay (?)

Endpoint: ACF.

No test item-related effects in behaviour or external appearance.

No test item-related thyroid hormone effects. 

No test item-related effects on body weight, food consumption and water consumption.

No test item-related effects on haematology and

biochemical parameters or urinalysis.

No test item-related effects on thyroid and sexual hormones or sperm.

No test item-related changes in bone marrow or organ weights.

No test item-related histopathological effects in the high dose group.

No test item-related induction of aberrant crypt foci (ACF) in rat colons.

Satellite animals of F1 used as the positive control for the KLH assay (at a different time and ages from F1-3 cohort). No additional controls were tested.

No test animals died prior to termination.

Observation of pale faeces not of toxicological relevance.

Lee et al., 2019

TiO2 NPs P25 (15–24 nm).

OECD Guideline 414 (Pre-natal Toxicity Study).

Test item:

Nanoparticles in deionised water.

80/20 anatase/rutile.

Mean diameter of approximately 21 nm (minimum of 100 particle sizes averaged) administered daily by oral gavage.

Dosage:

Test item was administered from Gestational Days 6 to 19 at dose levels of 0, 100,

300 and 1000 mg/kg with a dose volume of 10 mL/kg.

Sprague–Dawley rats (12 females per group).

Quantitative analysis in blood/tissues.

Four groups of twelve females per group in the toxicology

group (total test animals: 48) and four groups of four females in the tissue distribution group (total test animals: 16). 

No statistically significant differences in general clinical signs, body weight, organ weights (absolute and relative to body weight), macroscopic findings except a statistically significant decrease in food intake but no correlated decreased body weight or body weight gain during the study period of the females of the high-dose group.

No statistically significant differences in caesarean section parameters and fetal external and visceral examination.

A small but statistically significant increase (4%) was observed in the number of ossification centres in the metatarsals of both hindlimbs of the fetuses of 100 mg/kg bw per day group which may have been incidental.

Reference

TiO2 characterisation 

Quality of study e.g., OECD/GLP 

Method and duration of dosing 

Study methodology to include species, numbers, controls, 

Results 

Han et al., 2020

E171, anatase, 150 nm, 99.5% purity.

Study conducted according to OECD TG 408.

E171 suspended in distilled water, sonicated for at least 10 minutes.

E171 administered by oral gavage at doses of 0, 10, 100 or 1,000 mg/kg bw/d for 90 days.

Quantitative analysis in Sprague-Dawley rat’s tissues.

Sprague–Dawley rats (10/sex/group) were administered E171 by oral gavage at doses of 0, 10, 100 or 1,000 mg/kg bw/d for 90 days.

Ti concentrations were measured in the colons, kidneys, and spleens harvested from all rats at necropsy.

Statistically significant decreases in GM-CSF plasma levels (~30% in females) and plasma IgM (~12% in females and 9% in males) were observed at the highest dose compared to controls.

E171 accumulation in the stomach wall of several rats administered 1,000 mg/kg E171 for 90 days.

Ti concentration increased in the colons of both sexes administered 1,000 mg/kg E171 compared with the control, while colonic, superoxide dismutases (SOD)-1 (male and female) and SOD-2 (female) protein levels were down-regulated.

When exposed to AGS cells (human stomach epithelial cell line) for 24 h, E171 (40 μg/mL) was located in the perinuclear region. The E171 treatment affected expression of ER stress-related proteins but did not induce cell death up to the used maximum concentration (40 μg/mL). A gene profile analysis also showed that immune response-related microRNAs were most strongly affected by E171 exposure.

NCI, 1979 

TR-097: Titanium Dioxide (CASRN 13463-67-7) (nih.gov)

Titanium dioxide anatase.

Purity: 98%.

N/A

Groups of 50 rats of each sex and 50 mice of each sex were administered titanium dioxide in the diet at one of two doses, either 25,000 or 50,000 ppm, for 103 weeks and then observed for 1 additional week. Matched controls consisted of 50 untreated rats of each sex and 50 untreated mice of each sex. All surviving rats and mice were killed at 104 weeks.

Administration of the titanium dioxide had no appreciable effect on the mean body weights of rats or mice of either sex. With the exception of white feces, there was no other clinical sign that was judged to be related to the administration of titanium dioxide. Survival of the rats and the male mice at the end of the bioassay was not affected by the test chemical; mortality in female mice was dose related. Sufficient numbers of dosed and control rats and mice of each sex were at risk for development of late-appearing tumors.

In the male and female mice, no tumours occurred in dosed groups at incidences that were significantly higher than those for corresponding control groups. It is concluded that under the conditions of this bioassay, titanium dioxide was not carcinogenic by the oral route for Fischer 344 rats or B6C3F1 mice.

Akagi et al., 2023 – 28 Day Study

6 nm TiO2 nanoparticles.

N/A

5 female and 5 male F344/DuCrlCrlj rats.

TiO2 NPs with a crystallite size of 6 nm were examined in male and female F344/DuCrlCrlj rats by repeated oral administration of 10, 100, and 1000 mg/kg bw/day (5/sex/group) for 28 days.

No mortality was observed in any group, and no treatment-related adverse effects were observed in body weight, urinalysis, haematology, serum biochemistry, or organ weight. Histopathological examination revealed TiO2 particles as depositions of yellowish-brown material. The particles observed in the gastrointestinal lumen were also found in the nasal cavity, epithelium, and stromal tissue in the 28-day study.

Overall, no effects were observed after repeated oral administration of TiO2 with a crystallite size of 6 nm at up to 1000 mg/kg bw/day regarding general toxicity, accumulation of titanium in the liver, kidneys, and spleen, abnormality of colonic crypts, and induction of DNA strand breaks and chromosomal aberrations.

Akagi et al., 2023 – 90 Day Study

6 nm TiO2 nanoparticles.

N/A

10 female and 10 male F344/DuCrlCrlj rats.

TiO2 NPs with a crystallite size of 6 nm were examined in male and female F344/DuCrlCrlj rats by repeated oral administration of 100, 300, and 1000 mg/kg bw/day (10/sex/group) for 90 days.

No mortality was observed in any group, and no treatment-related adverse effects were observed in body weight, urinalysis, haematology, serum biochemistry, or organ weight. In addition, they were observed in Peyer’s patches in the ileum, cervical lymph nodes, mediastinal lymph nodes, bronchus-associated lymphoid tissue, and trachea in the 90-day study.

Overall, no effects were observed after repeated oral administration of TiO2 with a crystallite size of 6 nm at up to 1000 mg/kg bw/day regarding general toxicity, accumulation of titanium in the liver, kidneys, and spleen, abnormality of colonic crypts, and induction of DNA strand breaks and chromosomal aberrations.

Pinget et al., 2019

E171, anatase, 30-300 nm.

E171 was dispersed in drinking water using sonication.

N/A

Mice were exposure to E171 via drinking water for 4 weeks at doses of 0, 2, 10, 50 mg/kg bw/d. Dose is calculated based on water intake measured per cage.

Microbiota populations in fecal samples or the small intestine were examined through 16S rRNA sequencing.

Canonical correspondence analysis (CCA) constrained to the 4 distinct TiO2 concentrations used.

Male C67BL/6JAusb mice were exposed to E171 via drinking water at doses of either 0, 2, 10, or 50 mg TiO2/kg BW/day for 3 weeks to determine impact on colonic microbiota composition and on gut bacterial metabolites (10 mice/group).

Incubated commensal bacteria derived from mouse colons anaerobically for 5 days with dose of 0, 2, 10, 50 µg/ml of TiO2 biofilm formation (6 mice/group).

Impact of TiO2 on colonic epithelial function was determined by comparison of gene expression of key markers Muc2, Tjp1, Defb3, and Gzmb in colonic tissue of mice administered 0, 2, 10, or 50 mg TiO2/kg BW/day in drinking water (n = 5–8 mice per group).

To investigate impact of TiO2 on colonic inflammation, flow cytometric analysis, gene expression determined by qPCR and tissue staining were used on mice administered 0, 2, 10, or 50 mg TiO2/kg BW/day in drinking water (n = 8–10 mice per group).

TiO2 impact on adaptive immune cell infiltration into the colon was investigated using gene expression via qPCR and flow cytometric analysis on mice treated with 0, 2, 10, or 50 mg TiO2/kg BW/day in drinking water (5-8 mice/group).

At the highest dose tested, TiO2 had minimal impact on the composition of the gut microbiota. Alterations in bacterial metabolites were observed from 10 mg/kg bw/d.

Doses of 10 and 50 µg/ml TiO2 significantly promoted biofilm formation by commensal bacteria.

There was reduced expression of the colonic mucin 2 gene, a key component of the intestinal mucus layer, and increased expression of the beta defensin gene, indicating that TiO2 significantly impacts gut homeostasis. These changes were associated with colonic inflammation, as shown by decreased crypt length, infiltration of CD8+ T cells, increased macrophages as well as increased expression of inflammatory cytokines.

Reference

TiO2 characterisation 

Quality of study e.g., OECD/GLP 

Method and duration of dosing 

Study methodology to include species, numbers, controls, 

Results 

Sofranko et al., 2021

10 mg/g TiO2, 2 mg/g polyvinylpyrrolidone-coated Ag.

OECD 424 Neurotoxicity study in the rodents.

N/A

10 female and 10 male C57BL/6J mice.

The mice were fed ad libitum with food pellets dosed with 10 mg/g TiO2, 2 mg/g polyvinylpyrrolidone-coated Ag or control pellets for 28 days.

The neurotoxicity of TiO2 and Ag NMs, applied in food pellets, in male and female C57BL/6 J mice in a 28-day oral exposure study with or without a 14-day post-exposure recovery period.

No major neuropathological changes regarding neuroinflammation in biochemical and immunohistochemical analyses could be observed and behavioural changes in anxiety and cognition were absent. However, in the Ag NM exposed mice motor performance effects were observed by

the rotarod test that differed between sexes. The female mice that were exposed to Ag NM for 28 days, showed a consistent diminished motor coordination and increased cortical activity of specific tyrosine kinases.

Female mice that were exclusively investigated in a subsequent toxicokinetic study also revealed whole brain levels of Ag that steadily increased during the 28 days of exposure and persisted up to 4 weeks post-exposure. Our study demonstrates that subacute exposure to foodborne TiO2 and Ag NMs does not cause marked neurotoxicity in mice. However, our toxicokinetic and specific toxicodynamic findings with Ag NMs indicate that long-term oral exposures to this nanomaterial may cause adverse effects on the central nervous system in a sex dependent manner.

Grissa et al. (2016)

TiO2 NPs, anatase, 5–12 nm (TEM, XRD).

N/A

Internal exposure: quantitative in male Wistar rat tissues; methodology with important flaws.

N/A

There was a statistically significant dose-related increase in the level of NO in 100 and 200 mg/kg bw per day TiO2 NPs groups observed and a statistically significant dose-related increase in brain TNF-α in 200 mg/kg bw per day TiO2 NPs group.

Gerber et al., 2022

TiO2 NPs, average primary particle size of 26.2 ± 10.7 nm.

N/A

N/A

The aim of the study was to investigate the effects of two common types of NP,

Titanium dioxide NP (TiO2 NP) and silver NP (AgNP), on neuronal function following acute (0.5 h), sub-chronic (24 h and 48 h) and chronic (14 days) exposure in vitro rat cortical cells.

Acute and sub-chronic exposure to TiO2 NP is without effects, whereas chronic exposure only modestly reduces neuronal function without affecting morphology.

Ciu et al., 2021

TiO2 NPs.

N/A

36 male Sprague Dawley rats aged postnatal day 21 (PND 21) were injected intraperitoneally with TiO2 NPs (20 mg/kg) and/or BEO (200 mg/kg). 

N/A

TiO2 NPs exposure during the adolescent period induced anxiety‐like behaviour, cognitive impairment, neuroinflammation and oxidative damage in hippocampus, and BEO treatment could significantly ameliorate the neurotoxicity induced by TiO2 NPs exposure.

Naima et al., 2021

TiO2 NPs.

N/A

Rats were injected intravenously with a single dose of TiO2-NPs (20 mg/kg body weight) and were subjected to cognitive and emotional tests using Morris water maze and elevated plus maze.

N/A

Acute intravenous injection of TiO2-NPs impaired behaviour performances through brain biochemical and structural changes and precautions should be taken to their usage in food additive and medical applications.

Canli et al., 2020

TiO2 NPs

N/A

Oral administration of TiO2 for 14 days (0, 0.5, 5, and 50 mg/kg bw/day).

Female rats.

Results showed that brain AChE activity decreased at all treatment levels, and ATPase activities increased.

Intestine and kidney ATPase activities showed no significant change.

Levels of GSH showed no significant change but the TBARS level at the highest NP dose showed a significant decrease.

TiO2 NPs accumulated (dosage-dependent) in tissues.

The brain was found to be the most sensitive organ against the effects of TiO2 NPs.