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EC number: 943-098-9 | CAS number: -
- 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
Genetic toxicity: in vivo
Administrative data
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Data source
Reference
- Reference Type:
- publication
- Title:
- Unnamed
- Year:
- 2 012
- Report date:
- 2012
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
- GLP compliance:
- yes
- Remarks:
- Compliant to Guidelines for Animal Experimentation, Biosafety Research Center, Foods, Drugs & Pesticides, in line with ethics criteria contained in the bylaws of the Committee of National Institute of Advanced Industrial Science and Technology (AIST).
- Type of assay:
- mammalian comet assay
Test material
- Reference substance name:
- carbon
- EC Number:
- 943-098-9
- Cas Number:
- 7440-44-0
- Molecular formula:
- Carbon. Morphological features: - Tube cylindrical surface is formed by 6-membered rings consisting of carbon atoms linked by a double bond. - Carbon nanotube chirality is random. - Tube length: 1 - 10 μm. - Agglomeration: CNTs tend for form bundles
- IUPAC Name:
- carbon
- Reference substance name:
- Iron
- EC Number:
- 231-096-4
- EC Name:
- Iron
- Cas Number:
- 7439-89-6
- Molecular formula:
- Fe
- IUPAC Name:
- iron
- Test material form:
- solid: nanoform, no surface treatment
- Details on test material:
- Diameter was ~1.8 nm, length was 4.4 µm on average with a range from 1.3 to 18 µm; the BET surface area was measured as 878 m²/g
Constituent 1
impurity 1
Test animals
- Species:
- rat
- Strain:
- other: Crl: CD (SD)
- Details on species / strain selection:
- 61 male Crl: CD (SD) rats (7 weeks old) were obtained from Charles River Laboratories, Japan, Inc. (Yokohama, Japan) and used for the study
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- The rats were kept individually in a positive-pressure air-conditioned unit (20–26 °C, 35 – 75% relative humidity) for animal housing on a 12:12-h light/dark cycle. After a 6-day acclimation, 55 rats were assigned to the study. A standard rodent pellet diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) and drinking water were provided ad libitum.
Administration / exposure
- Route of administration:
- intratracheal
- Vehicle:
- Suspensions of SWCNTs were dispersed in 1% Tween 80 and instilled in a volume of 1.0 mL/kg body weight.
- Details on exposure:
- Based on the results of the dose-finding test, 1.0 mg/kg SWCNT was used for the high dosage group, expected to induce lung inflammation, and 0.2 mg/kg was used for the low dosage group, expected not to induce inflammation, in a single instillation study. In the repeated (intermittent) instillation study, a dosage of 0.2 or 0.04 mg/kg body weight once a week for 5 weeks was selected because it was expected to induce sub-acute lung inflammation or not.
- Duration of treatment / exposure:
- Single exposure with sacrifice 3hours and 24 hours after instillation. For repeated (intermittent) instillations exposure was once per week for 5 weeks.
- Frequency of treatment:
- Once for single treatment groups and once per week for repeated treatment group over 5 weeks
- Post exposure period:
- In the single instillation group, rats were anesthetized and sacrificed 3 or 24 h after the treatment, while in the repeated instillation group, rats were anesthetized and sacrificed 3 h after the last treatment.
Doses / concentrationsopen allclose all
- Dose / conc.:
- 0.2 mg/kg bw (total dose)
- Remarks:
- single instillation
- Dose / conc.:
- 1 mg/kg bw (total dose)
- Remarks:
- single instillation
- Dose / conc.:
- 0.04 mg/kg bw (total dose)
- Remarks:
- repeated (intermittent) instillations
- Dose / conc.:
- 0.2 mg/kg bw (total dose)
- Remarks:
- repeated (intermittent) instillations
- No. of animals per sex per dose:
- Five rats per group were used for each time point throughout the study.
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Ethyl methanesulfonate (EMS, sourced from Sigma–Aldrich Corporation, USA); 500 mg/kg of EMS was administered orally once 3 h before sacrifice in both the single and repeated study.
Examinations
- Tissues and cell types examined:
- The lungs were excised immediately after sacrifice. The left lobe was used for the histopathological examination, and the right lobe, for the comet assay.
- Details of tissue and slide preparation:
- The left lobes of the lungs were inflated and fixed in 10% neutral buffered formalin. All fixed tissues were routinely processed, embedded in paraffin, sectioned at 3 µm, and stained with hematoxylin and eosin (H&E) for light microscopic examination. The slides were scored double blind.
The comet assay was conducted in accordance with the standard protocol ‘‘International Validation of the In Vivo Rodent Alkaline Comet Assay for the Detection of Genotoxic Carcinogens’’ issued by the JaCVAM. Briefly, the right lobes of the lungs were washed out with homogenizing buffer (Hanks’s balanced salt solution containing 25 mmol/L EDTA-2Na and 10% v/v DMSO) and then homogenized in 5 mL of the homogenizing buffer using a Dounce-type tissue grinder (Wheaton Science Products, New Jersey, USA). Cell suspensions were chilled on ice for 5 min and centrifuged at 800 rpm for 5 min. After the supernatant was removed, the cells were re-suspended in homogenizing buffer. Ten microliters of the single cell suspension were mixed with 90 µL of 0.5% low-melting agarose gel, and 90 µL of the mixture was placed on a slide pre-coated with 1.0% agarose gel. Another 90 µL of low melting agarose was added. Two slides were prepared from each rat. The slides were transferred to lysing solution (2.5 mol/L NaCl, 100 mmol/L EDTA-2Na, 10 mmol/L, pH 10 Tris buffer, 10 vol.% DMSO and 1 vol.% Triton X-100) for at least one night at 4 °C in the dark. The slides were next covered with chilled electrophoresis buffer (pH > 13) for 20 min to allow DNA to unwind. Electrophoresis was then conducted at a constant voltage of 0.7 V/cm (25 V) (current at the start: 300 mA) for 20 min. The slides were transferred into neutralization buffer and left to stand for about 10 min. Subsequently, they were dehydrated with ethanol, and air-dried. The slides were stained with SYBR® Gold nucleic acid gel stain which was diluted 5000-fold with TE buffer solution. Images of DNA migration were examined using a fluorescence microscope (Olympus Corporation, Tokyo, Japan).
The final magnification was 200x. The images were analyzed using a Comet assay analyzer (Comet Assay IV system, Perceptive Instruments Ltd., Suffolk, UK). The comet parameter to measure DNA damage in the cells was the percentage of DNA in the tail (% Tail DNA), because% Tail DNA could be considered meaningful and easy to conceptualize (Kumaravel and Jha, 2006). Images of 100 (50 x 2) cells per rat were analyzed. The mean of the % Tail DNA value (mean value for 100 cells) of each group was calculated. - Statistics:
- Data for the SWCNTs treated groups and negative and positive control group were analyzed using the Dunnett’s multiple comparison test (two-sided, 0.05). Data for the positive control was compared to that for the negative control with Aspin-Welch’s t test (one-sided, 0.025).
Results and discussion
Test resultsopen allclose all
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- inflammation
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: single instillation
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- inflammation
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: repeated instillation
- Additional information on results:
- Single instillation
A single intratracheal instillation of the SWCNTs was performed. Rats were instilled with 0.2 or 1.0 mg/kg bw of the SWCNTs, and euthanized and necropsied 3 or 24 h later. Clinical signs and mean body weights of all SWCNT-treated groups were comparable to the negative control. Histopathological examination of the lungs revealed hemorrhage in the alveolus, and the infiltration of alveolar macrophages and neutrophiles in the 0.2 and 1.0 mg/kg bw groups at 3 h after treatment. In addition, thickening of the alveolar wall or edema was observed in the 0.2 and 1.0 mg/kg bw groups at 24 h after treatment. In the negative control groups, hemorrhage in the alveolus was observed in one of five rats at 3 h after treatment. In the comet assay, % tail DNA in lung epithelial cells exposed to the SWCNTs was comparable to that of the negative control at both 3 and 24 h. EMS, the positive control, induced significant DNA damage after 3 h of exposure as compared to the negative control.
Repeated instillation
Repeated (intermittent) intratracheal instillation of the SWCNTs was performed. Rats were instilled at a dosage of 0.04 or 0.2 mg/kg bw once a week for 5 weeks. They were euthanized and necropsied 3 h after the last treatment. Clinical signs, mean body weights and mean body weight changes for all SWCNT-treated groups were comparable to the negative control. Histopathological examination of the lungs revealed hemorrhage, infiltration of alveolar macrophages and neutrophiles in the alveolus, and thickening of the alveolus wall in the 0.04 and 0.2 mg/kg groups. In the negative control groups, hemorrhage in the alveolus was observed in one of five rats. In the comet assay, there was no significant difference in % tail DNA between the SWCNT groups and negative control.
Any other information on results incl. tables
Histopathology severity scores of lung on SWCNTs.
|
Tween 80 |
SWCNT |
SWCNT |
SWCNT |
Single instillation, 3 h after dosing |
||||
No. of rats examined |
5 |
0 |
5 |
5 |
Hemorrhage in alveolus |
0.2a |
|
0.4 |
0.8 |
Infiltration of macrophages 0 |
|
0.4 |
0.6 |
|
Infiltration of neutrophils in alveolus |
0 |
|
1.0 |
1.0 |
Single instillation, 24 h after dosing |
||||
No. of rats examined |
5 |
0 |
5 |
5 |
Edema in alveolus |
0 |
|
0 |
0.6 |
Hemorrhage in alveolus |
0 |
|
0.6 |
0.2 |
Infiltration of macrophages |
0 |
|
1.4 |
1.6 |
Infiltration of neutrophils in alveolus |
0 |
|
1.4 |
1.6 |
Thickening of alveolar wall |
0 |
|
1.2 |
1.2 |
Repeated (intermittent) instillation, 3 h after final dosing |
||||
No. of rats examined |
5 |
5 |
5 |
0 |
Hemorrhage in alveolus |
0.2a |
0.4 |
0.2 |
|
Infiltration of macrophages |
0 |
1.0 |
1.2 |
|
Infiltration of neutrophils in alveolus |
0 |
1.0 |
1.0 |
|
Thickening of alveolar wall |
0 |
0.2 |
0.8 |
|
Severity scores given to individual animals from a complete pathological examination are 0, not remarkable; 1, minimal; 2, moderate; and 3, marked; based upon relative evaluation of lesions. Severity scores for each animal within a group (5 rats) were added, and an average score per animal was calculated, which is shown in the table.
(a) One of five rats in the vehicle control groups showed minimal hemorrhage in the alveolus.
Results of comet assay on SWNCTs.
Compound |
Dose (mg/kg) |
No. of rats |
No. of cells analyzed |
% Tail DNA |
Single instillation, 3 h after dosing |
||||
Tween 80 |
0 |
5 |
500 |
3.90 ± 1.97 |
SWCNT |
0.2 |
5 |
500 |
3.40 ± 0.72 |
|
1.0 |
5 |
500 |
3.26 ± 0.74 |
EMS |
500 |
5 |
500 |
19.51 ± 3.92* |
Single instillation, 24 h after dosing |
||||
Tween 80 |
0 |
5 |
500 |
4.35 ± 1.53 |
SWCNT |
0.2 |
5 |
500 |
5.03 ± 1.57 |
|
1.0 |
5 |
500 |
5.50 ± 1.55 |
Repeated (intermittent) instillation, 3 h after final dosing |
||||
Tween 80 |
0 |
5 |
500 |
2.49 ± 0.68 |
SWCNT |
0.04 |
5 |
500 |
1.82 ± 0.54 |
|
0.2 |
5 |
500 |
2.20 ± 0.25 |
EMS |
500 |
5 |
500 |
11.52 ± 1.81* |
EMS: Ethyl methansulfonate (positive control).
* Significantly different from negative control at p < 0.025 (Aspin-Welch’s t-test).
Applicant's summary and conclusion
- Conclusions:
- In the present Comet Assay study, the bundled SWCNTs with length from 1.3 µm to 18 µm (median = 4.8 µm) and a diameter of 1.8 nm (very similar to Tuball SWCNT) were administered to rats with a single or repeated (intermittent) instillation for inducing acute or subacute inflammatory responses. The study design was suitable for observation of inflammatory response and/or DNA damage. In conclusion, the present findings showed that SWCNTs did not induce DNA damage in the lung cells of rats intratracheally instilled, even at doses that elicited both acute and subacute inflammatory responses. These findings suggest that SWCNTs have no potential for genotoxicity in vivo.
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