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EC number: 236-675-5 | CAS number: 13463-67-7
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
![](https://echa.europa.eu./o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro DNA damage and/or repair study
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- significant methodological deficiencies
Data source
Reference
- Reference Type:
- publication
- Title:
- DNA damage and alterations in expression of DNA damage responsive genes induced by TiO2 nanoparticles in human hepatoma HepG2 cells.
- Author:
- Petković, J. et al.
- Year:
- 2 011
- Bibliographic source:
- Nanotoxicology 5(3): 341-353.
Materials and methods
Test guideline
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- In this study human hepatoma HepG2 cells were exposed to TiO2 NPs at the concentrations 0, 1, 10, 100, 250 µg/mL in a comet asssay.
- GLP compliance:
- not specified
- Remarks:
- publication
- Type of assay:
- comet assay
Test material
- Reference substance name:
- Titanium dioxide
- EC Number:
- 236-675-5
- EC Name:
- Titanium dioxide
- Cas Number:
- 13463-67-7
- Molecular formula:
- O2Ti
- IUPAC Name:
- dioxotitanium
- Test material form:
- solid: nanoform
- Details on test material:
- PARTICLE CHARACTERIZATION (by authors)
The size and morphology of TiO2 NPs were observed by field-emission-gun scanning electron microscopy. The crystal phase of the powders was identified by X-ray diffraction. UV/Vis (diffuse reflectance) spectroscopic characterization of the samples was recorded. The specific surface area was determined by gas adsorption using the BET method (Gemini 2370, Micromeritics).
- Name of test material (as cited in study): TiO2-Ru
- Source: purchased from Sigma-Aldrich
- Crystalline phase: rutile
- Size: ca. 10x40 nm
- Specific surface area (BET): 116.7 m²/g
- FEG-SEM (supplied material) Particle size within the agglomerates/aggregates: <100 nm
- Shape: elongated crystallites
- XRD: Rutile type: tetragonal crystallographic system; P42/mnm space group (according to JCPDS: 34–0180); characteristic reflections (110) at 27.3; (101) at 36.0; (211) at 54.1, 2θ.
- UV-Vis spectroscopy: rutile: absorption peaks at lmax = 305 nm and λmax = 205 nm
Constituent 1
Method
- Target gene:
- not applicable
Species / strain
- Species / strain / cell type:
- mammalian cell line, other: human hepatoma HepG2 cells
- Details on mammalian cell type (if applicable):
- HepG2 cells were obtained from European Collection of Cell Cultures (ECACC). Cells were grown in EMEM containing 10% fetal bovine serum, 1% non-essential amino acid solution, 2 mM L-glutamine and 100 U/mL penicillin plus 100 µg/mL streptomycin at 37°C in a humidified atmosphere with 5% CO2.
- Metabolic activation:
- not specified
- Test concentrations with justification for top dose:
- 1, 10, 100, and 250 µg/mL
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: phosphate buffered saline (PBS)
Powdered TiO2 NPs were suspended in PBS at a concentration 1 mg/mL and sonicated for 30 minutes in an ultrasonic bath (Sonorex, Bandelin electronic, Germany) at a frequency of 60 kHz, voltage of 220 V and an electric current of 0.5 A to ensure uniform suspension. This stock solution was subsequently diluted in the complete cell growth medium to yield concentrations. These samples were then sonicated for 30 minutes to produce a stable, less-agglomerated nanocrystalline suspension before exposure of cells in culture.
Controlsopen allclose all
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- cell growth medium containing 10% PBS
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: tert-butyl hydroperoxide
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- cell growth medium containing 10% PBS
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Details on test system and experimental conditions:
- PARTICLE SIZE DISTRIBUTION IN TREATMENT MEDIUM
Particle size distribution of TiO2 NPs in the treatment medium was measured by laser scattering, using a particle size distribution analyzer Horiba LA-920 (Japan). The particles were dispersed in Eagle Minimal Essential Medium (EMEM) medium at 30 µg/mL and sonicated using the same conditions as for preparation of particle suspensions for the cell treatments.
CYTOTOXICITY ASSAY
Cytotoxicity was determined with the MTT assay according to Mossman (1983)* with minor modifications (Zegura et al. 2003)*. The HepG2 cells were seeded onto 96-well microplates (Nunc, Naperville, IL, USA) at a density of 40,000 cells/mL and incubated for 20 hours at 37°C to attach. The medium was then replaced by fresh complete medium containing 0, 1, 10, 100 and 250 µg/mL of TiO2 NPs, and incubated for 4, 24 and 48 hours. In each experiment a vehicle control (cell growth medium containing 10% PBS) was included. MTT (final concentration 0.5 mg/mL) was then added, incubated for an additional 3 hours, the medium with MTT was then removed and the formed formazan crystals dissolved in DMSO. The optical density (OD) was measured at 570 nm (reference filter 690 nm) using a microplate reading spectrofluorometer (Tecan GENios, Austria). Viability was determined by comparing the OD of the wells containing the NPs treated cells with those of the vehicle (cell growth medium containing 10% PBS) treated cells. Five replicates per concentration point and three independent experiments were performed.
COMET ASSAY
HepG2 cells were seeded at a density of ~ 60,000 cells/mL into 12-well microtiter plates (Corning Costar Corporation, Corning, NY, USA). After incubating the cells at 37°C in 5% CO2 for 20 hours to attach to the plates, the growth medium was replaced with fresh medium containing 0, 1, 10, 100 and 250 µg/mL TiO2 NPs and incubated for 2, 4 and 24 hours. In each experiment positive controls (0.5 mM t-BOOH and 50 mM BaP) and a vehicle control (cell growth medium containing 10% PBS) were included.
At the end of the exposure the cells were harvested and the DNA damage determined by the protocol of Singh et al. (1988)* with minor modifications (Žegura and Filipic 2004)*. Images of 50 randomly selected nuclei per experimental point were analyzed with image analysis software Comet Assay IV (Perceptive Instruments, UK). Three independent experiments were performed for each of the treatment conditions. The percent of tail DNA was used to measure the level of DNA damage.
The level of oxidized purines/pyrimidines was determined with the modified comet assay as described by Collins et al. (1996)*. After the cell lysis the slides were washed three times for 5 minutes with endonuclease buffer (40 mM HEPES-KOH, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/mL bovine serum albumin, pH 8.0). Fifty microlitre aliquots of Fpg/Endo III solution or enzyme buffer without Fpg/Endo III were added, covered with a cover glass and incubated at 37°C for 30/45minutes. The slides were then processed as described above.
*References:
- Mossman T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63.
- Zegura B, Sedmak B, Filipic M. 2003. Microcystin-LR induces oxidative DNA damage in human hepatoma cell line HepG2. Toxicon 41:41–48.
- Singh NP, McCoy MT, Tice RR, Schneider EL. 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191.
- Zegura B, Lah TT, Filipic M. 2004. The role of reactive oxygen species in microcystin-LR-induced DNA damage. Toxicology
200:59–68.
- Collins AR, Dusinska M, Gedik CM, Stetina R. 1996. Oxidative damage to DNA: Do we have a reliable biomarker? Environ Health Perspect 104(3):465–469. - Evaluation criteria:
- no data
- Statistics:
- One-way analysis of variance (ANOVA, Kruskal-Wallis) was used to analyze the differences between treatments within each experiment. Dunnet’s test was used for comparing median values of percentage tail DNA; P < 0.05 was considered as statistically significant.
Results and discussion
Test results
- Species / strain:
- mammalian cell line, other: human hepatoma HepG2 cells
- Metabolic activation:
- not specified
- Genotoxicity:
- ambiguous
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Additional information on results:
- PARTICLE SIZE DISTRIBUTION IN TREATMENT MEDIUM
The particle size distribution determined by laser scattering particle size distribution analysis showed that in the medium both types of TiO2 NPs are highly aggregated and agglomerated with an average size of aggregates and agglomerate size at the micron level (TiO2-Ru: 1542 ± 760 nm). The portion of submicron-sized particles is much higher TiO2-Ru NPs compared to TiO2-An NPs. Micron-sized aggregates and agglomerates were confirmed also by FEG-SEM.
CYTOTOXICITY ASSAY
The viability of HepG2 cells exposed to 0, 1, 10, 100 and 250 µg/mL of TiO2-Ru NPs for 4, 24 and 48 h was not significantly affected.
COMET ASSAY
- Exposure to TiO2-Ru NPs induced significantly more strand breaks only in cells exposed for 4 hours to 100 µg/mL TiO2-Ru NPs.
- In cells exposed to TiO2-Ru NPs, a significant increase of Fpg-sensitive sites was detected only after 24 hours exposure to 10 and 100 µg/mL NPs.
- Induction of Endo III-sensitive sites by TiO2 NPs was much lower than that of Fpg-sensitive sites and was not dose-dependent. In cells exposed to TiO2-Ru, a significant increase of Endo III-sensitive sites was observed at the highest concentration (250 µg/mL) after 2 hours and after 24 hours exposure.
Applicant's summary and conclusion
- Conclusions:
- Petković, J. et al. (2011) examined the DNA damaging potential of TiO2-Ru NPs in Hep-G2 cells using the conventional as well as modified comet assays. The colorimetric MTT assay was exploited in order to investigate on NP’s cytotoxicity.
The authors conclude that TiO2-Ru NPs was practically ineffective to induce DNA damage.
According to the evaluator of the publication, TiO2-Ru did not show an increase of DNA damage.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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