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Genetic toxicity: in vitro

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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:
16 Jun - 05 Jul 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

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)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-Propanediol, 2-methyl-, reaction products with ethenyltrimethoxysilane
EC Number:
700-957-0
Molecular formula:
UVCB
IUPAC Name:
1,3-Propanediol, 2-methyl-, reaction products with ethenyltrimethoxysilane
Constituent 2
Reference substance name:
Reaction products of trimethoxy(vinyl)silane and 2-methylpropane-1,3-diol (2:5-6)
IUPAC Name:
Reaction products of trimethoxy(vinyl)silane and 2-methylpropane-1,3-diol (2:5-6)
Details on test material:
- Name of test material (as cited in study report): Y-15866
- Analytical purity: 72%
- Lot/batch No.: 3710-10
- Expiration date of the lot/batch: 2013-07-16
- Storage condition of test material: at room temperature

Method

Target gene:
his operon (S. thyphimurium) and trp operon (E. coli)
Species / strainopen allclose all
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:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Wistar rats induced with 80 mg/kg bw phenobarbital i.p. and β-naphthoflavone p.o. each on three consecutive days
Test concentrations with justification for top dose:
Pre-experiment (Experiment I): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle/solvent used: tetrahydrofuran
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4NOPD (-S9; 10 µg/plate (TA 98) + 50 µg/plate (TA 1537)); NaN3 (-S9; 10 µg/plate; TA 1535 + TA 100); MMS (-S9; 3 μL/plate; WP2 uvrA); 2AA (+S9; 2.5 µg/plate (TA 1535, TA 1537, TA 98, TA 100) + 10 μg/plate (WP2 uvrA))
Remarks:
4NOPD: 4-nitro-o-phenylene diamine; NaN3 = sodium azide; MMS = methyl methane sulfonate; 2AA: 2-amino-anthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION 1: in agar (plate incorporation) for pre-experiment (Experiment I)

DURATION
- Exposure duration: 48 h

METHOD OF APPLICATION 2: preincubation followed by plate incorporation for Experiment II

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicate each in both experiments

DETERMINATION OF CYTOTOXICITY
- Method: determination of the number of revertants per plate and inspection of the bacterial background lawn
Evaluation criteria:
The test substance was considered mutagenic if:
- a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control was observed,
- the threshold is exceeded at more than one concentration (dose-dependency),
- an increase exceeding the threshold at only one concentration was reproduced in an independent second experiment,
- a dose dependent increase in the number of revertant colonies below the threshold was reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

The assay was considered acceptable if the following criteria were met:
- regular background growth in the negative and solvent control,
- the spontaneous reversion rates in the negative and solvent control were in the range of the historical data,
- the positive control substances produced a significant increase in mutant colony frequencies.
Statistics:
Mean values and standard deviations were calculated for revertant colonies.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test item precipitated in the overlay agar in the test tubes from 2500 μg/plate up to 5000 μg/plate in Experiment I and II. Precipitation on the incubated agar plate was observed from 2500 μg/plate up to 5000 μg/plate in both experiments with and without S9 mix. The undissolved particles of the test item had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: in a pre-experiment (Experiment I) using the plate incorporation method, each strain was incubated with concentrations ranging from 3 to 5000 μg/plate of the test substance. Three replicates for each concentration were performed both in the presence and absence of metabolic activation. Since no toxic effects were observed at any concentration, 5000 μg/plate was chosen as maximal concentration.

COMPARISON WITH HISTORICAL CONTROL DATA: in Experiment I, the number of revertant colonies of the 5 tester strains remained in the range of the historical control values for these strains. In Experiment II, without metabolic activation, the number of colonies did not quite reach the lower limit of the historical control data in strain TA 100 (untreated control). Since this deviation was rather small, this effect was judged to be based upon statistical fluctuations and had no detrimental impact on the outcome of the study.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test results of pre-experiment/Experiment I (plate incorporation)

Y-15866: Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD)

EXPERIMENT I (plate incorporation)

S9-Mix

Without

 

Concentration (per plate)

TA 1535

TA1537

TA98

TA100

E.coli WP2 uvrA

SC (THF)

11 ± 3

12 ± 3

23 ± 6

116 ± 4

47 ± 3

NC

10 ± 3

13 ± 1

25 ± 5

133 ± 4

46 ± 2

Y-15866

 

 

 

 

 

5000 µg

11 ± 4P

15 ± 2P

22 ± 6P

132 ± 5P

61 ± 4P

2500 µg

12 ± 2P

12 ± 3P

30 ± 1P

119 ± 5P

57 ± 1P

1000 µg

10 ± 1

14 ± 2

25 ± 3

117 ± 12

52 ± 2

333 µg

10 ± 1

13 ± 1

23 ± 6

126 ± 11

58 ± 5

100 µg

10 ± 3

12 ± 2

26 ± 4

117 ± 20

51 ± 1

33 µg

11 ± 5

11 ± 2

21 ± 5

116 ± 6

56 ± 11

10 µg

12 ± 3

12 ± 5

26 ± 8

124 ± 6

55 ± 5

3 µg

11 ± 4

12 ± 3

20 ± 8

123 ± 12

47 ± 3

PC

 

 

 

 

 

NaN3 (10 µg)

1666 ± 29

-

-

1875 ± 97

-

4NOPD (10 µg)

-

-

325 ± 15

-

-

4NOPD (50 µg)

-

72 ± 14

-

-

-

MMS (3 µL)

-

-

-

-

942 ± 29

S9-Mix

 

With

Concentration (per plate)

TA 1535

TA1537

TA98

TA100

E.coli WP2 uvrA

SC (THF)

19 ± 4

18 ± 3

38 ± 8

138 ± 3

71 ± 4

NC

23 ± 7

20 ± 3

40 ± 4

136 ± 9

70 ± 9

Y-15866

 

 

 

 

 

5000 µg

18 ± 4P

20 ± 8P

39 ± 4P

149 ± 11P

92 ± 8

2500 µg

20 ± 4P

16 ± 3P

43 ± 3P

143 ± 16P

60 ± 9

1000 µg

21 ± 1

19 ± 7

33 ± 8

146 ± 12

70 ± 4

333 µg

22 ± 4

20 ± 6

38 ± 6

128 ± 5

59 ± 7

100 µg

16 ± 1

16 ± 6

33 ± 4

135 ± 13

62 ± 8

33 µg

18 ± 2

17 ± 5

29 ± 3

152 ± 3

74 ± 15

10 µg

17 ± 4

19 ± 5

40 ± 2

145 ± 6

66 ± 9

3 µg

20 ± 3

19 ± 3

38 ± 4

133 ± 10

79 ± 6

PC

 

 

 

 

 

2AA (2.5 µg)

196 ± 10

457 ± 42

2442 ± 291

3058 ± 180

-

2AA (10 µg)

-

-

-

-

316 ± 25

NC = Negative Control; SC = Solvent control;

PC = Positive control substances; SD = standard deviation;

NaN3 = sodium azide; 4NOPD = 4-nitro-o-phenylene-diamine; MMS = methyl methane sulfonate; 2AA = 2-aminoan-thracene

P = Precipitate

Table 2. Test results of Experiment II (pre-incubation)

Y-15866: Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD)

EXPERIMENT II (pre-incubation)

S9-Mix

Without

 

Concentration (per plate)

TA 1535

TA1537

TA98

TA100

E.coli WP2 uvrA

SC

16 ± 2

10 ± 2

30 ± 8

100 ± 8

40 ± 2

NC

17 ± 5

16 ± 4

27 ± 7

76 ± 13

39 ± 7

Y-15866

 

 

 

 

 

5000 µg

18 ± 5P

10 ± 2P

22 ± 3P

101 ± 11P

27 ± 3P

2500 µg

17 ± 1P

11 ± 1P

29 ± 7P

93 ± 13P

24 ± 3P

1000 µg

15 ± 2

12 ± 3

23 ± 2

103 ± 10

32 ± 2

333 µg

13 ± 1

12 ± 4

26 ± 5

86 ± 14

39 ± 5

100 µg

18 ± 4

13 ± 3

24 ± 12

84 ± 8

39 ± 4

33 µg

14 ± 1

9 ± 3

26 ± 9

103 ± 8

34 ± 9

PC

 

 

 

 

 

NaN3 (10 µg)

1864 ± 62

-

-

702 ± 148

-

4NOPD (10 µg)

-

-

435 ± 56

-

-

4NOPD (50 µg)

-

107 ± 7

-

-

-

MMS (3 µL)

-

-

-

-

2539 ± 40

S9-Mix

 

With

Concentration (per plate)

TA 1535

TA1537

TA98

TA100

E.coli WP2 uvrA

SC

17 ± 2

17 ± 4

44 ± 2

110 ± 5

48 ± 5

NC

22 ± 7

22 ± 9

39 ± 6

112 ± 7

58 ± 16

Y-15866

 

 

 

 

 

5000 µg

22 ± 3P

17 ± 3P

38 ± 9P

111 ± 37P

50 ± 6P

2500 µg

15 ± 2P

19 ± 8P

27 ± 1P

129 ± 13P

54 ± 6P

1000 µg

16 ± 5

16 ± 4

40 ± 10

136 ± 12

54 ± 11

333 µg

19 ± 6

15 ± 1

41 ± 7

113 ± 18

54 ± 4

100 µg

18 ± 3

16 ± 3

40 ± 4

121 ± 13

51 ± 3

33 µg

20 ± 1

18 ± 2

55 ± 13

116 ± 18

56 ± 1

PC

 

 

 

 

 

2AA (2.5 µg)

302 ± 24

271 ± 15

2094 ± 279

1687 ± 280

-

2AA (10 µg)

-

-

-

-

349 ± 17

NC = Negative Control; SC = Solvent control; PC = Positive control substances; SD = standard deviation;

NaN3 = sodium azide; 4NOPD = 4-nitro-o-phenylene-diamine; MMS = methyl methane sulfonate; 2AA = 2-aminoan-thracene

P = Precipitate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions reported, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of metabolic activation in the selected strains of S. typhimurium (TA 98, TA 100, TA 1535 and TA 1537) and E. coli WP2 uvrA.
Executive summary:

The bacterial gene mutation assay (Ames test) was conducted in compliance with OECD guideline 471 and under GLP conditions. The assay was performed in two independent experiments both performed in the absence and in the presence of liver microsomal activation system (S9 mix). In the first experiment (pre-experiment), the direct plate incorporation procedure was conducted with the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and the Escherichia coli strain WP2 uvrA at concentrations ranging from 3 to 5000 µg/plate for a period of 48 h. In the second experiment, concentrations ranging from 33 to 5000 µg/plate were analysed using the same strains, but with modification of the study design (with pre-incubation period of 60 min). No signs of cytotoxicity, evident as a reduction in the number of revertants, were observed in both experiments. Precipitation of the test substance occurred at doses ≥ 2500 µg/plate, but did not influence data evaluation. The number of revertants was not increased at any concentrations tested. The positive and negative controls included showed the expected results in each experiment. Under the experimental conditions reported, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of metabolic activation in the selected strains of S. typhimurium (TA 98, TA 100, TA 1535 and TA 1537) and E. coli WP2 uvrA.