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Toxicological information

Repeated dose toxicity: other routes

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Administrative data

Endpoint:
short-term repeated dose toxicity: other route
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
other: not rated acc. to Klimisch
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The references contained in this summary entry represent mechanistic investigations on the effect of titanium dioxide nanoparticles administered via unphysiological routes on on various organs with very limited value for risk assessment purposes. All references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The information contained therein were included for information purposes only.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Oxidative stress mediated cytotoxicity of TiO2 nano anatase in liver and kidney of Wistar rat.
Author:
Meena, R. & Paulraj, R.
Year:
2012
Bibliographic source:
Toxicolog. Environ. Chem. 94: 146-163.
Reference Type:
publication
Title:
In vivo biocompatibility studies of nano TiO2 materials.
Author:
Han, W. et al.
Year:
2009
Bibliographic source:
Ad. Mat. Res. 1: 79-82.
Reference Type:
publication
Title:
An increase in mouse tumor growth by an in vivo immunomodulating effect of titanium dioxide nanoparticles.
Author:
Moon, E.-Y. et al.
Year:
2011
Bibliographic source:
J. Immunotoxicol. 8(1): 56-67.
Reference Type:
publication
Title:
High sensitivity of testicular function to titanium nanoparticles
Author:
Miura, N. et al.
Year:
2017
Bibliographic source:
J. Toxicol. Sci. 42 (3): 359 - 366

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Meena, R. et al. (2012) (Rat): This study examined the adverse effects of TiO2 nanoparticle on the kidney and liver of Wistar rats. The test item was administered by intravenous injected at dose levels of 5, 25 or 50 mg/kg. The following parameters were investigated: clinical chemistry, body weights, organ weights, oxidative stress measurement, enzymatic activities, histopathology, analysis of titanium content and cell apoptosis.

Han, W. et al. (2009) (Mouse): During this study TiO2 nanomaterials with different dimensions, sizes, and crystal structures were confected to suspensions and evaluated in Balb-c mice. These mice were divided into various groups with suspension intraperitoneally injected. Treatment was conducted once daily during 7 days. Heart, lung, liver and kidney were collected and gross pathology and histopathology were conducted. Spectrophotometry was applied to study total antioxide capability and catalase activity of blood and tissues.

Moon, E.Y. et al. (2011) (Mouse): in this study TiO2 nanoparticles (<25 nm or <100 nm) were intraperitoneally injected in mice at a dose level of 10 mg particle/kg once a day for 7 days. The effect of the test items on immune cells was investigated.

Miura, N. et al. (2017) (Mouse): Male C57BL/6J mice were injected intravenously with TiNPs ( Aeroxide-P25, at doses of 0.1, 1, 2, and 10 mg/kg bw) once per week for 4 consecutive weeks. Body weights, liver weights as well as Ti liver content and hepatic function parameters were analyzed.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: nanomaterial
Details on test material:
STUDY 1 (Rat; Meena & Paulraj (2012)):
- Name of test material (as cited in study report): Titanium dioxide anatase nanoparticle (purchased from Sigma Aldrich, U.S.A.)

STUDY 2 (Mouse; Han, W. et al. (2009)):
TiO2 nanomaterials with different dimensions and crystal structures were used as test items.
- Name of test material (as cited in study report): Titanium dioxide nanoparticle material
1) P25 TiO2
2) rutile TiO2, 20 nm
3) rutile TiO2, 50 nm
4) rutile TiO2, 100 nm
5) anatase TiO2, 10 nm
6) anatase TiO2, 20 nm
7) anatase TiO2, 100 nm
8) TiO2 nanotube

STUDY 3 (Mouse; Moon, E-Y. et al. (2011)):
- Name of test material (as cited in study report): TiO2 nanoparticles (purchased from Sigma (St. Louis, MO)).
- Particle size: <25 nm or <100 nm


Test animals

Species:
other: Meena, R. et al. (2012): rat; Han, W. et al. (2009): mouse; Moon, E.Y. et al. (2011): mouse; Miura, N. et al. (2017): mouse
Strain:
other: Meena, R. et al. (2012): Wistar; Han, W. et al. (2009): Balb-c; Moon, E.Y. et al. (2011): C57BL/6J; Miura, N. et al. (2017): C57BL/6J
Details on test animals and environmental conditions:
Meena, R. et al. (2012) (Rat):
TEST ANIMALS - Wistar rat
- Source: Jawaharlal Nehru University, New Delhi, India
- Age at study initiation: eight weeks old
- Weight at study initiation: 200 g
- Diet (ad libitum)
- Water (ad libitum)

Han, W. et al. (2009) (Mouse):
TEST ANIMALS - Balb-c mice
- Source: Beijing Experimental animal Center
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: 18 - 25 g
- Housing: housed in stainless steel cages containing sterile paddy husk as bedding in ventilated animal rooms
- Diet (ad libitum): commercial laboratory complete food
- Water (ad libitum)
- Acclimation period: yes, but stated for how long

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 1°C
- Humidity: 60 ± 10%
- Photoperiod (hrs dark / hrs light): 12/12

Moon, E.Y. et al. (2011) (Mouse):
TEST ANIMALS - C57BL/6J mice
- Source: Dae-Han Biolink (Chungju, South Korea)
- Housing: pathogen-free authorized facility in Sejong University (Seoul, South Korea)
- Diet (ad libitum): mouse (rodent) chow (Purina Co., Seoul, South Korea)
- Water (ad libitum)

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 22°C
- Relative humidity: 50 - 60%
- Photoperiod (hrs dark / hrs light): 12/12

Miura, N. et al. (2017) (Mouse):
TEST ANIMALS - C57BL/6J mice
- Source: Japan SLC (Shizioka, Japan)
- Age at study initiation: 8 weeks
- Housing: in cages under standard conditions
- Diet (ad libitum): sterilized commercial pellet diet (CE-2, Clea Japan, Inc., Tokyo, Japan)
- Water (ad libitum): sterilized filtered tap water

ENVIRONMENTAL CONDITIONS
- Temperature: 24 ± 1 °C
- Relative humidity: 55 ± 5 %
- Photoperiod (hrs dark / hrs light): 12/12 (lights on at 08:00 hr)

Administration / exposure

Route of administration:
other: Meena, R. et al. (2012) (Rat): intravenous; Han, W. et al. (2009) (mouse): intraperitoneal; Moon, E. Y. et al. (2011) (Mouse): intraperitoneal; Miura, N. et al. (2017): intravenous injection
Vehicle:
other: Meena, R. et al. (2012) (Rat): distilled water; Han, W. et al. (2009) (mouse): natural salt; Moon, E.Y. et al. (2011) (Mouse): normal saline; Miura, N. et al. (2017): disodium phosphate (DSP)
Details on exposure:
Meena, R. et al. (2012) (Rat):
CHARCTERIZATION OF NANO-TiO2
Nano-TiO2 was characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Nano-TiO2 (50 µg/mL) was dissolved in distilled water and ultrasonicated for 30 minutes to make the homogenous suspension. The diameters of ranadomly selected particles were measured at 8000 x magnification, and elemental analysis was conducted using an energy-dispersive X-ray (EDX) analyzer. The hydrodynamic diameters of nano-TiO2 were evaluated by DLS.
Results:
Transmission electron microscopic images showed that the nano-TiO2 in clustered suspension had an average diameter of 10-20 nm. EDX spectral analysis of the particle surface chemical composition revealed that the nano-TiO2 contained 56% titanium (Ti), 26% oxygen (O), and 18% copper (Cu) elements, respectively. The DLS results demonstrate the well-disturbed and steady state nano-TiO2 in the prepared solution with an average size of 72 nm, with the size range of 41 - 102 nm.

TREATMENT:
Animals were injected with 200 µL of 5, 25, or 50 mg/kg of nano-TiO2, respectively. Control animals were treated with equal volume of PBS. Each animal was injected intravenously (through caudal vein) at weekly interval for 30 days.

Han, W. et al. (2009):
The different TiO2 nanomaterials were distributed into natural salt to become 2 mg/mL suspension, then were high temperature & pressure sterilized and ultrasonic oscillated 20 minutes before usage (Han, W. et al., 2008)*.
Every suspension was administered at a dose of 20 mg/kg bw intraperitoneally with same cubage. Injection dose was equal normal salts in control group.

Moon, E.Y. et al. (2011) (Mouse):
TEST SUBSTANCE PREPARATION
TiO2 nanoparticles were suspended in normal saline to yield a stock 1.0 mg particle/mL solutions. Prior to use for injection, the suspensions underwent sonication to assure no significant agglomeration in the aqueous solution took place.

TREATMENT
Mice were intraperitoneally injected with ~200 µL of the suspension of <25- or <100-nm TiO2 nanoparticles such that a dose of 10 mg particle/kg was achieved. The amount of saline needed to dilute the stock solution (so as to prepare the 200 µL injection volume) was adjusted according to the body weight of the mice, which was recorded daily.

*Reference:
W. Han, Y.D. Wang and Y. F. Zheng: Adv. Mater.Res. Vol.47 - 50 (2008), p. 1438.

Miura, N. et al. (2017) (Mouse):
TEST SUBSTANCE PREPARATION
After sterilization of TiNPs by dry heat sterilizer (180 °C for 1 hr), TiNPs were suspended in 2 mg/mL disodium phosphate (DSP) in a glass vial to generate the 10 mg/mL TiNP suspension. The TiNp suspension was sonicated in an ultrasonic water bath (Brasonic 2510; Branson, Danbury, CT, USA) for 30 min. The zeta potential of TiNPS after sonication was determined using the particle and molecular size analyzer, Zetasizer Nano-ZS (Malvern, Worcestershire, UK).

TREATMENT
Male C57BL/6J mice were injected intravenously with TiNPs (Aeroxide-P25, at doses of 0.1, 1, 2, and 10 mg/kg bw) once per week for 4 consecutive weeks. Control animals recieved only sonicated DSP.
Duration of treatment / exposure:
Meena, R. et al. (2012) (Rat): 30 days
Han, W. et al. (2009) & Moon, E.Y. et al. (2011) (Mouse): 7 days
Miura, N. et al. (2017) (Mouse): 4 weeks
Frequency of treatment:
Meena, R. et al. (2012) (Rat): weekly interval
Han, W. et al. (2009) & Moon, E.Y. et al. (2011) (Mouse): daily
Miura, N. et al. (2017) (Mouse): once per week
Doses / concentrations
Remarks:
Doses / Concentrations:
Meena, R. et al. (2012) (Rat): 5, 25, or 50 mg/kg
Han, W. et al. (2009) (Mouse): not stated
Moon, E.Y. et al. (2011) (Mouse): 10 mg particle/kg
Miura, N. et al. (2017) (Mouse): 0.1, 1, 2 or 10 mg/kg bw
No. of animals per sex per dose:
Meena, R. et al. (2012) (Rat): 6 animals
Han, W. et al. (2009) (Mouse): 6 animals
Moon, E.Y. et al. (2011) (Mouse): 5 animals
Miura, N. et al. (2017) (Mouse): not specified
Control animals:
yes

Examinations

Observations and examinations performed and frequency:
Meena, R. et al. (2012) (Rat):
At the end of exposure time, after overnight fasting, all animals were anaesthetized to collect blood. The serum was harvested for biochemical assay.
The liver function was evaluated with serum levels of total bilirubin, alkaline phosphate, alanine aminotransferase, and asparate aminotransferase. Nephrotoxicity was determined by uric acid, blood urea nitrogen, and creatinine. Total protein, albumin, and globulin were assayed.
Furthermore, ROS assay, SOD assay, CAT assay, GPx assay, lipid peroxidation assay were carried out with liver and kidney tissue.
Food and water consumption of rats was observed.

Han, W. et al. (2009) (Mouse):
Clinical signs and mortality were noted.

Moon, E.Y. et al. (2011) (Mouse):
Body weight was recorded daily.

Miura, N. et al. (2017) (Mouse):
Hepatotoxicity (ALT and AST), body weight, liver weight, Ti liver content
Sacrifice and pathology:
Meena, R. et al. (2012) (Rat):
The organs such as liver, kidney, spleen, and brain were excised for various other examinations. The organ weights of the excised organs was obtained the organ to body weight ratio was calculated.
Liver and kidney tissue were dissected and histopathologically examined. Furthermore, for ultrastructure analysis these tissues were examined by TEM. In addition, liver and kidney of all animals were analysed for titanium concentration with energy dispersive X-ray fluorescence (EDXRF) spectroscopy and cell apoptosis was investigated in these organs.

Han, W. et al. (2009) (Mouse):
Necropsy was performed on all animals at the end of the study. Animals were fasted overnight prior to necropsy. All mice were killed by exsanguinations following pelltobarbitalum natricum anaesthesia. The blood was conserved by adding heparin. All animals were examined grossly. Tissues including liver, kidney, heart and lung were collected and histopathologically examined.
Oxygen stress level was evaluated with total anti-oxidation capability and catalase activity in both blood and tissue.

Moon, E.Y. et al. (2011) (Mouse):
After euthanization, spleens and thymuses were dissected from the control or TiO2 nanoparticle-injected mice and placed in RPMI 1640 supplemented with 2% FBS. Splenocyte and thymocytes were obtained. Aliquots of 1.0 x 10^6 thymocytes or splenocytes (as separate populations) from control or TiO2-injected mice were suspended in 2% FCS containing RPMI 1640. Thymocytes were then treated with FITC-conjugated anti-CD4 and PE-conjugated anti-CD8 antibodies; splenocytes received FITC-conjugated anti-CD43, -CD21, -IgD antibodies, and/or PE-conjugated anti-IgM antibodies. Splenocytes were also treated with PE-conjugated anti-NK1.1 antibodies and FITC-conjugated anti-CD11b antibodies for analysis of NK cells. Each suspension was incubated with antibodies for 20 minutes on ice. Thereafter, the cells were washed with medium and analysed by CELLQuest TM software in an FACScalibur TM system (Becton Dickison, Franklin lakes, NJ). A minimum of 10,000 events were analysed/cell population of interest.
In vitro experiments and ex vivo experiments were also conducted, but were here not further discussed due to lack of relevance.

Miura, N. et al. (2017) (Mouse):
At 3 days after the last injection, mice were sacrificed under carbon dioxide anesthesia. Body and liver weight were recorded. Blood samples were also collected from the inferior vena cava using Supercath (Medikit Co. Ltd., Tokyo, Japan), in which the needle was pre-saturated with heparin, and centrifuged at 1,000 x g (4 °C, 10 min) to seperate plasma samples. Plasma samples were stored at -80 °C until analyses of levels of ALT and AST.
Statistics:
Meena, R. et al. (2012) (Rat):
All data were expressed as mean ± standard deviation (SD). The statistical software SPSS for windows 13.0 was used to perform a post-hoc multiple comparison test such as LSD and t-test following a one-way analysis of variances (ANOVA). p<0.05 and p<0.01 were considered as statistically significance.

Han, W. et al. (2009) (Mouse): not stated

Moon, E.Y. et al. (2011) (Mouse):
Experimental differences were tested for statistical significance using analysis of variance (ANOVA) and Student's t-test. A P value of <0.05 or <0.01 was considered to be significant.

Miura, N. et al. (2017) (Mouse):
Data were analyzed by a one-way ANOVA. Statistical significance of differences between control and TiNPs-treated groups was determined with Dunnett´s test. In all cases, p< 0.05 was considered statistically significant.

Results and discussion

Results of examinations

Details on results:
Meena, R. et al. (2012) (Rat):
- no significant differences in food and water intake in the nao-TiO2-treated animals compared to control groups.
- organ to body weight rations of the liver, kidney, spleen, and brain of animals treated with lower and medium dose nano-Tio2 did not show any significant differences form control group.
- animals treated with 50 mg/kg nano-TiO2, ratio of the liver, kidney, and spleen to bodyweight increased significantly.
- no marked changes in brain to body weight ratio.
- accumulation pattern of titanium in different organs was liver>spleen>kidney>brain.
- in animals treated with 50 mg/kg of TiO2-nano, the concentration of titanium in liver and kidney was 31.8 ± 1.5 and 17.4 ± 0.4 µg/g, respectively. In the control group, it was 2.7 ± 0.4 and 1.5 ± 0.1 µg/g in liver and kidney, respectively.
- aspartate aminotransferase, alanine aminotransferase, and alkaline phosphate activities increased significantly.
- aspartate aminotransferase and alanine aminotransferase showed 99% significant rise in the 50 mg/kg group, but no significant changes were observed in the 5 and 25 mg/kg groups.
- alkaline phosphate rose significantly in 50 mg/kg of nao-TiO2 group.
- the levels of total protein, albumin, and globulin significantly changed in the 50 mg/kg groups.
- levels of the bilirubin increased in the blood.
- as nao-TiO2 dose increased, the contents of creatinine, calcium and phosphate of the kidney function parameters rose gradually (lower doses no significant differences).
- the urea and blood urea nitrogen were decreased gradually, with 50 mg/kg of nano-TiO2.
- ROS assay: 50 mg/kg of nano-TiO2 significantly increased ROS levels in liver and kidney. Liver was more sensitive to nano-TiO2-induced oxidative stress compared to kidney.
-CAT activity: activity of liver and kidney was significantly decreased in 50 mg/kg of TiO2-nano-treated groups. The liver CAT level fell by 44%, whereas the kidney CAT activity was reduced by 33%.
There was a negative correlation between the ROS level and CAT levels in both liver and kidney cells.
- SOD levels: compared to control, the liver SOD activity was reduced by 11% and 26% after exposure to 25 and 50 mg/kg nano-TiO2, respectively. In kidney cells a significant decrease (36%) was observed in the 50 mg/kg test group. There was a significant negative correlation between the ROS levels and SOD levels in both liver and kidney.
- GSH level: liver GSH levels were reduced significantly by 25% compared to control, after exposure to 50 mg/kg nano-TiO2. The cellular GSH level of kidney cells were reduced significantly by 44% compared to control in 50 mg/kg group. There was a significant negative correlation between ROS levels and GSH levels in both liver and kidney cells.
- MDA levels: liver MDA levels elevated significantly by 45% and 73% at the 5 and 50 mg/kg groups, respectively. The cellular MDA level was not significantly changed in kidney cells. There was a significant correlation between the level of ROS and MDA in liver and kidney cells.
- Cell apoptosis: a significant increase in apoptosis was observed in liver cells of nano-TiO2-treated groups (10.8%, 17.60% and 31.1%) compared to control (5.98%). Kidney cells treated with nano-TiO2 show an increase in apoptosis (6.79%, 9.66%, and 16.67%) compared to control (2.11%).
- Microscopic analysis: pathological changes were observed in the liver of 50 mg/kg group animals. Spotty necrosis around the central vein was induced. Mild pigment accumulation, the lymphocyte clusters, and particle laden macrophages concentrated at the portal area. In renal proximal tubules, the deposition of proteinic fluid and swelling inrenal glomerulus was observed in animals which received 50 mg/kg. Ultrastructure of hepatocytes cells in higher doses of nano-TiO2 showed tumescent mitochondria, particle-laden lysosomes, vacuolization in cytoplasm, and distorted nuclear membrane. Ultrastructure of kidney cells in animals treated with higher dose of nano-TiO2 shows characteristics of apoptosis, cell membrane shrinkage, cytoplasm budding, condensation and fragmentation of nuclear chromatin.

Han, W. et al. (2009) (Mouse):
- the animals in all groups were in good conditions. No overt toxic effects were observed.
- no mortality occurred during the study.
- there were no obvious changes observed in heart, lung, liver and kidney, and these tissue presented no histopathological changes.
- there were no notable structural change among TiO2 nanomaterials treated mice and the control.
- total anti-oxidation capability and catalase activity levels of all groups were elevated compared to the control group. The effect of TiO2 nanoparticles with same crystal structure was increased by the decrease of diameter. With the same diameter, the effect was ranked in the following sequence: anatase > rutile> nanotube>P25. There were no statistically significant differences among all experimental groups.
- TiO2 nanomaterials in lives can generate reactive oxygen spices and induce lipid-peroxided reaction.
- according to the authors, the results showed that nano TiO2 materials can enter circulation system from multiple approaches, and then distribute to tissues and organs.

Moon, E.Y. et al. (2011) (Mouse):
- TiO2 nanoparticles reduced B-lymphocytes development and the percentage of NK cells in the spleen.

Miura, N. et al. (2017) (Mouse):
- The general appearance and amount of spontaneous behaviour if TiNP-treated animals seemed to be normal throughout experiments. The body weight gain was also normal and no differences between control and TiO2-treated animals could be observed.
- liver weights were also not influenced by TiO2-treatment
- the ALT values were not significantly changed in the treatment group, although the values tended to be slightly higher, except for the 1 mg/kg group. The AST values were also not changed overall.
- Ti concentrations within the liver seemed to be elevated dose-dependently, although only the value of 10 mg/kg bw group presented a significantly increase.

Effect levels

open allclose all
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
Basis for effect level:
other: Moon, E.Y. et al. (2011) (Mouse): according to the authors, TiO2 nanoparticles reduced B-lymphocytes development and the percentage of NK cells in the spleen.
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
Basis for effect level:
other: see remarks
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Two references on the effect of TiO2 nanoparticles on kidney, liver, lung and/or heart in rats and mice were identified.
The test item was administered either intravenously (rats/mice) or intraperitoneally (mice). The administration duration was either weekly for 30 days (rats) or daily for 7 days (mice). The study conducted with the rats showed that TiO2 nanoparticles have an effect on the liver and the kidney, whereas the reference describing the study conducted with the mice showed that TiO2 nanoparticles had no effect on heart, lung, liver and kidney.
A third reference on the effect of TiO2 nanoparticles on immune cells in mice was identified. The mice were exposed to the test item by intraperitoneally injection daily for 7 days. According to the authors, TiO2 nanoparticles reduced B-lymphocytes development and the percentage of NK cells in the spleen.


The references contained in this summary entry represent mechanistic investigations on the effect of titanium dioxide nanoparticles administered via unphysiological routes on on various organs with very limited value for risk assessment purposes. All references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The information contained therein were included for information purposes only.

Miura, N. et al. (2017): The study conducted by Miura, N. et al. (2017) is not in accordance to any repeated dose toxicity guideline. Since the test item is poorly described and also the influence of the other simultaneously administered process chemicals is not addressed, this raises doubts as to whether (i) nano-sized TiO2 particles at all were used in this experiment, and (ii) whether the effects can really be attributed to titanium dioxide. Additionally, no justification for the dosing regime is included (once per week for 4 weeks) and investigators suspended the acidic P25 in an assumed alkaline DSP solution. No investigations regarding pH effects were performed. No food and water consumption were recorded and the number of animals per group is too low for statistical analysis.