Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
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

Constituent 1
Chemical structure
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
Constituent 2
Reference substance name:
GRAPHISTRENGTH C100
IUPAC Name:
GRAPHISTRENGTH C100
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
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, 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.