Single Wall Carbon Nanotubes (SWCNT)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method).

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). Small, statistically significant increases in TA1535 revertant colony frequency were observed in the first mutation test at 50, 150 and 1500 μg/plate in the absence of S9-mix only. These increases were considered to be of no biological relevance because there was no clear evidence of a dose-response relationship or reproducibility and the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain. The response was more likely due to a slightly low vehicle control count.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 June 2016 - 14 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 471 (1997) "Bacterial Reverse
Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine or Tryptophan locus.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

10% liver S9 in standard co-factors

Test concentrations with justification for top dose:

The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item
(1.5, 5, 15, 50, 150, 500, 1500 and 5000 g/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

Distilled water.
In solubility checks performed in–house, the test item was noted to be insoluble in sterile
distilled water at 12.5, 25 and 50 mg/mL, dimethyl sulphoxide at 25 and 50 mg/mL and
dimethyl formamide and polyethylene glycol 400 at 50 mg/mL. The test item formed the
best doseable suspension in sterile distilled water at a maximum concentration of
12.5 mg/mL, therefore, this solvent was selected as the vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

0.4 mL of the appropriate concentration of test item or solvent vehicle or 0.1 mL of
appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented
media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate
buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative
(untreated) controls were also performed on the same day as the mutation test. Each
concentration of the test item, appropriate positive, vehicle and negative controls, and each
bacterial strain, was assayed using triplicate plates. For metabolic activation, 0.5 mL of S9-mix was added to the
molten, trace amino-acid supplemented media instead of phosphate buffer.
All of the plates were incubated at 37 ± 3 C for approximately 48 hours and scored for the
presence of revertant colonies using an automated colony counting system. The plates were
viewed microscopically for evidence of thinning (toxicity). Manual counts were performed
at and above 1500 μg/plate because of test item precipitation. Several further manual counts
were also performed due to bubble interference and artefacts on the plates, thus distorting the
actual plate count.

Evaluation criteria:

A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
A reproducible increase at one or more concentrations.
Biological relevance against in-house historical control ranges.
Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996).

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). A black test item precipitate was noted by eye at and above 5 g/plate, this observation did not prevent the scoring of revertant colonies.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). Small, statistically significant increases in TA1535 revertant colony frequency were observed in the first mutation test at 50, 150 and 1500 μg/plate in the absence of S9-mix only. These increases were considered to be of no biological relevance because there was no clear evidence of a dose-response relationship or reproducibility and the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain. The response was more likely due to a slightly low vehicle control count.

Conclusions:

The test item was considered to be non-mutagenic under the conditions of this test.

Executive summary:

A bacterial reverse mutation test was performed according to OECD TG 471.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 g/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate. Six test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in both mutation tests.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix), in both experiments.

Small, statistically significant increases in TA1535 revertant colony frequency were observed in the first mutation test at 50, 150 and 1500 μg/plate in the absence of S9-mix only. These increases were considered to be of no biological relevance because there was no clear evidence of a dose-response relationship or reproducibility and the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain. The response was more likely due to a slightly low vehicle control count.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies generally within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Test item was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

To get more clarity on mutagenicity of singe wall carbon nanotubes (Tuball™) an in vitro mutagenicity study was ordered (In Vitro Mammalian Chromosome Aberration Test with Tuball™ (1RW02)). However, following several attempts to achieve a homogenous, stable solution suitable for dosing the material, the attempt to perform such study had to be terminated, as no stable solution could be formed. The attached statement describes the attempts made in more detail and their outcome.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

To get more clarity on mutagenicity of singe wall carbon nanotubes (Tuball™) an in vitro mutagenicity study was ordered (In Vitro Mammalian Cell Gene Mutation Test: HPRT Assay with Tuball™ (1RW02)). However, following several attempts to achieve a homogenous, stable solution suitable for dosing the material, the attempt to perform such study had to be terminated, as no stable solution could be formed. The attached statement describes the attempts made in more detail and their outcome.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

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.

Link to relevant study records

Reference

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

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

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.

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.

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.

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).

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.

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).

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Test item was considered to be non-mutagenic under the conditions of an Ames study. Other in-vitro studies were technically not feasible. A published in vivo Comet assay performed with SWCNT resulted negative for mutagenicity, although broncheolar tissue inflammation was achieved. Thus, the substance is not considered to be mutagenic according to CLP (Regulation EC No. 1272/2008).