Tangled Multi-Walled Carbon Nanotubes

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

march 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD 471)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

GRAPHISTRENGTH C100

IUPAC Name:

GRAPHISTRENGTH C100

Constituent 2

Reference substance name:

Tangled Multi-Walled Carbon Nanotubes

EC Number:

701-160-0

Cas Number:

7782-42-5

Molecular formula:

Hollow tubular carbon, 1-dimensional nano structures with hexagonal arrangement of carbon atoms

IUPAC Name:

Tangled Multi-Walled Carbon Nanotubes

Test material form:

solid: nanoform

Method

Target gene:

Histidine operon

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Arochlor1254-induced liver rats

Test concentrations with justification for top dose:

1, 10, 50, 100, 250 and 500 µg/plate for the preliminary toxicity test.
15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for both mutagenicity experiments

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:ethanol
- Justification for choice of solvent/vehicle:
The test item was found not soluble in the vehicles usually used for this type of assay.
Consequently, a suspension was selected for the treatment. An homogenous suspension (to the naked eye) was obtained in ethanol at the concentration of 10 mg/mL.

For positive control : DMSO was used as solvent (except for Mitomycin C which was dissolved in distilled water)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: azide de sodium, 9-Aminoacridine, 2-Nitrofluorène, Mitomycin C, 2-Anthramine, Benzo(a)pyrène

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).

DURATION
- Preincubation period: During the preincubation method, test item solution, S9 mix and the bacterial suspension were incubated for 60 minutes at 37°C, under shaking.
- Exposure duration: 48 to 72 hours of incubation at 37°C

NUMBER OF REPLICATIONS: Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level).

DETERMINATION OF CYTOTOXICITY
- Method: The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.
The test item did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the five strains.

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as
a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

no

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the dose-level of 500 µg/plate in the five strains used without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the dose-level of 500 µg/plate in the five strains used without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the dose-level of 500 µg/plate in the five strains used without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the dose-level of 500 µg/plate in the five strains used without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the dose-level of 500 µg/plate in the five strains used without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

See tables 1, 2.

Any other information on results incl. tables

Table 1: First experiment

              Direct plate incorporation method

Strain	Compound	Dose level per plate (µg)	S9 mix	Mean revertant colony counts	SD	Ration treated / solvent
TA 1535	ETHANOL		-	16	5
	TEST ITEM	15.6	-	12	5	0.8
		31.3	-	21	7	1.3
		62.5	-	21	8	1.4
		125	-	11	3	0.7
		250	-	15	6	1.0
		500	-	0	1	0.0
	NAN3	1	-	610	39	38.9
	ETHANOL		+	19	3
	TEST ITEM	15.6	+	12	2	0.6
		31.3	+	16	3	0.8
		62.5	+	16	2	0.8
		125	+	20	7	1.1
		250	+	16	8	0.8
		500	+	16	6	0.8
	2AM	2	+	286	16	14.8
TA 1537	ETHANOL		-	6	1
	TEST ITEM	15.6	-	9	8	1.6
		31.3	-	9	3	1.4
		62.5	-	12	3	2.1
		125	-	9	3	1.4
		250	-	10	6	1.6
		500	-	0	0	0.0
	9AA	50	-	1168	324	194.7
	ETHANOL		+	8	2
	TEST ITEM	15.6	+	10	4	1.2
		31.3	+	9	5	1.1
		62.5	+	9	4	1.0
		125	+	10	2	1.2
		250	+	8	5	1.0
		500	+	5	5	0.6
	2AM	2	+	144	15	17.3
TA 98	ETHANOL		-	30	4
	TEST ITEM	15.6	-	36	6	1.2
		31.3	-	28	5	0.9
		62.5	-	27	8	0.9
		125	-	31	7	1.0
		250	-	30	1	1.0
		500	-	1	2	0.0
	2NF	0.5	-	248	16	8.3
	ETHANOL		+	35	6
	TEST ITEM	15.6	+	38	4	1.1
		31.3	+	40	11	1.1
		62.5	+	36	2	1.0
		125	+	43	5	1.2
		250	+	40	4	1.1
		500	+	35	7	1.0
	2AM	2	+	1462	112	41.4
TA 100	ETHANOL		-	95	19
	TEST ITEM	15.6	-	125	22	1.3
		31.3	-	120	10	1.3
		62.5	-	112	30	1.2
		125	-	132	14	1.4
		250	-	138	1	1.5
		500	-	36	52	0.4
	NAN3	1	-	744	82	7.8
	ETHANOL		+	106	16
	TEST ITEM	15.6	+	117	8	1.1
		31.3	+	118	22	1.1
		62.5	+	119	8	1.1
		125	+	117	10	1.1
		250	+	122	6	1.2
		500	+	129	15	1.2
	BAP	5	+	957	28	9.0
TA 102	ETHANOL		-	290	52
	TEST ITEM	15.6	-	349	51	1.2
		31.3	-	241	34	0.8
		62.5	-	344	53	1.2
		125	-	345	16	1.2
		250	-	403	53	1.4
		500	-	12	5	0.0
	MMC	0.5	-	2764	159	9.5
	ETHANOL		+	382	41
	TEST ITEM	15.6	+	433	10	1.1
		31.3	+	433	22	1.1
		62.5	+	407	54	1.1
		125	+	327	27	0.9
		250	+	469	41	1.2
		500	+	505	22	1.3
	2AM	10	+	2778	162	7.3

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

GRAPHISTRENGTH C100 MICRONISED did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

The potential of the test item GRAPHISTRENGTH C100 MICRONISED to induce reverse mutation in Salmonella typhimurium was evaluated. The study was performed according to the international guidelines OECD 471 and Good Laboratory Practices.

A preliminary toxicity test was performed to define the dose-levels of GRAPHISTRENGTH C100 MICRONISED to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item GRAPHISTRENGTH C100 MICRONISED was suspended in ethanol.

The test item was found not soluble in the vehicles usually used for this type of assay. Consequently, a suspension was selected for the treatments. An homogenous suspension (to the naked eye) was obtained in ethanol at the concentration of 10 mg/mL. Since the test item was not soluble and non-toxic in the preliminary test, the highest dose-level was selected on the basis of the precipitate observed in the Petri plates, according to the criteria specified in the international guidelines. The selected treatment-levels were 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate, for both mutagenicity experiments with and without S9 mix.

A moderate to marked precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. In the first experiment without S9 mix, a marked toxicity was noted at the dose-level of 500 µg/plate in the five strains used. The test item did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the five strains. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

GRAPHISTRENGTH C100 MICRONISED did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.