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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 Nov 2011 - 22 Mar 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep the substance in cool and dark place; tightly closed, in refrigerator (1 - 10 °C); fill nitrogen after opening of package.
- Stability under test conditions: The test substance is stable. The stability is confirmed at the end of exposure using IR spectroscopy.
- Solubility and stability of the test substance in the solvent/vehicle: soluble in DMSO at 50 mg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No reactivity


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: Test material was measured and diluted in DMSO at 50 mg/mL. This solution was diluted sequencly in the appropriate concentration.

Method

Target gene:
trp operon and his operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, prepared from the livers of rats treated with phenobarbital and 5-6-benzoflavone
Test concentrations with justification for top dose:
Experiment 1 and 2: 39.1, 78.1, 156, 313, 652, and 1250 μg/mL were tested for TA1535 (up to the cytotoxic concentration)
Experiment 1 and 2: 156, 313, 652, 1250, 2500 and 5000 μg/mL were tested for TA100 , WP2uvrA, TA98 and TA1537
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: due to the limited solubility of the test substance in water, DMSO was selected as the vehicle
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
furylfuramide
other: 6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine dihydrochloride for TA1537 without S9 mix, 2-amonoanthracene for TA1535 and WP2uvrA with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 49.5 h for the first experiment and 48 h for the second experiment

NUMBER OF REPLICATIONS:
3 replication each in 2 independent experiments

NUMBER OF CELLS EVALUATED: all cells

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
A test substance was considered positive in the Ames test if:
a) a more than 2-fold increase in the number of revertants was observed
b) the positive result was reproducible inspite of dose-related increase.
Statistics:
Mean values and standard deviations were calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: TA 98, TA 100, TA 1535, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Preliminary test was performed with 19.5, 78.1, 313, 1250 and 5000 μg/mL with/without S9.
There was no reverse mutation observed in all species at all concentration. Furthermore, cytotoxicity was observed at 1250 μg/mL and more (TA1535) and at 5000 μg/mL (WP2urvA, TA98, TA100 and TA1537) with and without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: number of revertant colonies

Any other information on results incl. tables

Table 1. Test results of the experiment 1 with or without S9 mix for triethoxy(methyl)silane

With or without S9 mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(Mean of 3 replicate ± SD)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2urvA

TA98

TA1537

-

0

84 ± 17.6

6 2.0±

20± 3.5

12± 3.8

5± 2.0

39.1

-

7± 1.5

-

-

-

78.1

-

6± 1.0

-

-

-

156

72± 12.1

8± 0.6

16± 4.9

13± 7.6

5± 0.6

313

87± 26.2

10± 3.5

17± 2.1

11± 4.6

5± 1.0

625

71± 10.2

7± 3.5

13± 2.0

9± 3.2

4± 1.2

1250

67± 7.4

7± 2.6

14± 5.1

14± 7.0

5± 1.5

2500

0± 0.0*

NT

10± 0.6*

0± 0.0*

0± 0.0*

5000

0± 0.0*

NT

5± 2.3*

0± 0.0*

0± 0.0*

Positive controls,

– S9 mix

Name

AF2

NaN3

AF2

AF2

ICR-191

Concentration (µg/plate)

0.01

0.5

0.01

0.1

1.0

Mean No. of colonies/plate

± SD

764± 8.1

350± 11.0

54± 21.5

365± 60.1

908± 69.8

+

0

80± 15.5

6± 1.2

15± 6.1

18± 4.9

10± 1.0

39.1

NT

8± 1.5

NT

NT

NT

78.1

NT

8± 2.5

NT

NT

NT

156

83± 12.1

8± 3.1

17± 3.1

15± 6.5

5± 1.5

313

93± 10.0

8± 1.5

13± 2.3

22± 7.0

6± 1.0

625

85± 9.0

8± 0.6

18± 5.0

14± 6.4

4± 2.3

1250

84± 8.2

6± 2.6*

11± 1.5

15± 5.7

6± 1.5

2500

51± 8.1*

NT

12± 3.5*

13± 4.7*

0 ± 0*

5000

0 ± 0*

NT

5 ± 2.0*

0 ± 0*

0 ± 0*

Positive controls,

+ S9 mix

Positive controls

B[a]P

2AA

2AA

B[a]P

B[a]P

Concentration (µg/plate)

5.0

2.0

10.0

5.0

5.0

Mean No. of colonies/plate

± SD

957 ± 93.6

254± 16.5

812± 66.4

250± 44.3

67± 3.1


Table 2. Test results of the experiment 2 with or without S9 mix for triethoxy(methyl)silane

With or without S9 mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate 

(Mean of 3 replicate ± SD)

-

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2urvA

TA98

TA1537

128 ± 11.5

8 ± 1.5

13 ± 1.2

14 ± 2.5

7 ± 1.2

39.1

-

4 ± 2.5

-

-

-

78.1

-

6 ± 0.6

-

-

-

156

116 ± 7.8

8 ± 4.6

13 ± 2.0

17 ± 3.8

10 ± 4.4

313

98 ± 6.8

8 ± 6.7

12 ± 2.9

11 ± 4.2

6 ± 1.0

625

126 ± 14.8

6 ± 3.2

19 ± 3.5

12 ± 1.0

4 ± 0.0

1250

107 ± 12.5

4 ± 1.5*

12 ± 3.5

12 ± 6.1

6 ± 1.5

2500

0 ± 0.0*

-

12 ± 2.9*

5 ± 2.3*

0 ± 0.0*

5000

0 ± 0.0*

-

0 ± 0.0*

0 ± 0.0*

0 ± 0.0*

Positive controls,

– S9 mix

Name

AF2

NaN3

AF2

AF2

ICR-191

Concentration (µg/plate)

0.01

0.5

0.01

0.1

1.0

Mean No. of colonies/plate

± SD

638 ± 47.9

161 ± 3.2

70 ± 7.6

491 ± 31.2

1232 ± 27.8

+

0

118 ± 9.6

8 ± 1.5

22 ± 5.6

24 ± 9.9

7 ± 1.0

39.1

NT

7 ± 2.5

NT

NT

NT

78.1

NT

8 ± 2.5

NT

NT -

NT

156

127 ± 21.4

8 ± 1.5

14 ± 3.6

19 ± 2.5

7 ± 2.3

313

119 ± 13.7

8 ± 1.5

18 ± 9.9

23 ± 3.1

6 ± 3.2

625

123 ± 4.2

8 ± 2.6

15 ± 5.8

25 ± 4.2

8 ± 3.1

1250

114 ± 8.1

8 ± 2.9*

14 ± 2.1

17 ± 3.5

6 ± 1.0

2500

52 ± 17.3*

NT

7 ± 2.1

8 ± 1.7*

0 ± 0*

5000

0 ± 0*

NT

9 ± 2.6*

0 ± 0*

0 ± 0*

Positive controls,

+ S9 mix

Name

B[a]P

2AA

2AA

B[a]P

B[a]P

Concentration (µg/plate)

5.0

2.0

10.0

5.0

5.0

Mean No. of colonies/plate

± SD

937 ± 45.0

191 ± 13.0

776 ± 14.2

311 ± 24.0

97 ± 2.4

 

AF2: Furylfulamide

NaN3: sodium azide

ICR-191: 6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine dihydrochloride

B[a]P: Benzo(a)pyrene

2AA: 2-aminoanthracene

SD:  standard deviation

*: Growth inhibition

NT: This concentration was not tested.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the Ames test the substance was not mutagenic in any of the five strains (S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA) tested with and without metabolic activation up to 1250 µg/plate for TA1535 and 5000 µg/plate for other strains.