Single Wall Carbon Nanotubes (SWCNT)

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:

2012

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

Test material information

Constituent 1

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

impurity 1

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

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 / concentrations

Doses / concentrations 1

Dose / conc.:

0.2 mg/kg bw (total dose)

Remarks:

single instillation

Doses / concentrations 2

Dose / conc.:

1 mg/kg bw (total dose)

Remarks:

single instillation

Doses / concentrations 3

Dose / conc.:

0.04 mg/kg bw (total dose)

Remarks:

repeated (intermittent) instillations

Doses / concentrations 4

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 results

Test results 1

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

Test results 2

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 0.04 mg/kg	SWCNT 0.2 mg/kg	SWCNT 1.0 mg/kg
Single instillation, 3 h after dosing
No. of rats examined	5	0	5	5
Hemorrhage in alveolus	0.2^a		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	0.2^a	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 (Mean ± SD)
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.