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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:
01/09/2001 - 09/10/2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
3-trimethoxysilylpropane-1-thiol
EC Number:
224-588-5
EC Name:
3-trimethoxysilylpropane-1-thiol
Cas Number:
4420-74-0
IUPAC Name:
3-(trimethoxysilyl)propane-1-thiol
Test material form:
other: liquid

Method

Target gene:
Salmonella strains: histidine locus; E. coli: tryptophan locus
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1: (TA98, TA 100, TA1537, WP2 uvrA) 100, 333, 1000, 3333, 5000 μg/plate; (TA1535, TA1537) 105, 348, 1045, 3485, 5000 μg/plate (+/-S9)
(TA98, TA100, TA105, TA1535, TA1537, WP2 uvrA) 75, 200, 600, 1800, 5000 μg/plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2-aminoanthracene, 2-nitrofluorone, sodium azide, 9-aminoacridine, methyl methanesulfonate
Remarks:
historical negative and positive control values included.
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation, in agar

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48-72 hours

Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of animal top agar. Deionized water was used for the preparation of media and reagents. Bottom agar was 1.5% (w/v), nutrient bottom agar containing 1.5% agar and supplemented with 2.5% (w/v) nutrient broth. 0.5 ml S9 or sham mix, 100 μL of tester strain and 50 μL of vehicle or test article were added to 13x100 mm glass culture tubes pre-heated to 37°C. After vortexing, the mixtures were incubated for an hour in a shaker at 37°C. Following the preincubation, 2 mL of selective top agar was added to each tube and the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by 50 μL aliquot of relevant positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37°C. Plates that were not counted immediately following the incubation were stored at 2-8°C until colony counting could be conducted.

ACTIVATION:
Bulk S9 was assayed for ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to S. typhimurium TA 100. S9 mix contained 10% S9, 5mM glucose-6-phosphate, 4 mM NADP, 8mM MgCl2 and 33 mM KCl in 100 mM phosphate buffer pH 7.4. 0.5 ml S9 mix were added to a total volume of 2.65 ml giving a final concentration of approximately 2% S9 in the plates.


SELECTION AGENT (mutation assays): histidine/tryptophan deficient-agar


NUMBER OF REPLICATIONS: All dose levels of test article, vehicle controls and positive controls were plated in triplicate. The experiment was repeated.

DETERMINATION OF CYTOTOXICITY
- Method: condition of bacterial lawn/reduction in number of revertants

Evaluation criteria:
A result is positive if the number of revertants is significantly increased compared with the solvent control to at least 2-fold of the solvent control for TA 98, TA 100 and WP2 uvrA and 3-fold of the solvent control for TA 1535 and TA 1537. There must be a dose related increase in the mean
revertants per plate of at least one tester strain over a minimum of 2 increasing concentrations of test article.
Statistics:
Relevant statistics (standard deviation, mean) were calculated using software by BIOSYS.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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:
cytotoxicity
Remarks:
3485 and 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: No precipitate was observed.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxicity was evident at ≥3333 or 5000 μg/plate.
Remarks on result:
other: No mutagenic potential

Any other information on results incl. tables

Table 1. Experiment 1, preincubation. Number of revertants per plate (mean of 3 plates).

 

TA98

TA100

WP2 uvrA

Conc.
[μg/ plate]

- MA

+ MA

Cytotoxic
(yes/no)

-MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

DMSO

20

21

no

92

116

no

10

12

no

100

19

26

no

99

115

no

13

10

no

333

22

29

no

87

119

no

14

12

no

1000

25

26

no

93

95

no

10

11

no

3333

18

25

no

63

79

no

11

10

no

5000

1

7

yes

4

60

yes

10

10

no

 

 

 

Nitrofluorene

1μg

567

-

-

-

-

-

-

-

-

2-aminoanthacene 1μg

-

608

-

-

488

-

-

162

-

Sodium azide 1μg

-

-

-

408

-

-

-

-

-

Methyl methanesulfonate 1000μg

-

-

-

-

-

-

368

-

-

 

Table 2. Experiment 1, preincubation. Number of revertants per plate (mean of 3 plates).

 

TA1537

TA1535

Conc.
[
μg/ plate]

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

DMSO

5

7

no

12

9

no

100

-

7

no

-

13

no

105

6

-

no

10

-

no

333

-

5

no

-

10

no

348

6

-

no

12

-

no

1000

-

6

no

-

10

no

1045

4

-

no

10

-

no

3333

-

4

no

-

7

no

3485

3

-

yes

9

-

yes

5000

0

0

yes

0

1

yes

 

 

 

9-aminoacridine 75μg

580

-

-

-

-

-

2-aminoanthacene 1μg

40

-

-

35

-

Methyl methanesulfonate 1000μg

-

-

-

-

-

-

Sodium azide 1μg

 

 

 

 231

-

-

 

Table 3: Experiment 2, preincubation. Number of revertants per plate (mean of 3 plates).

 

TA98

TA100

TA1535

Conc.
[
μg/plate]

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

DMSO

13

13

no

85

97

no

6

8

no

75

13

15

no

86

108

no

9

7

no

200

15

19

no

89

123

no

9

7

no

600

12

16

no

95

85

no

10

7

no

1800

10

16

no

78

100

no

3

9

no

5000

0

0

yes

0

0

yes

0

8

yes

Nitrofluorene

1μg

308

 

 

 

2-aminoanthracene 1μg

258

385

39

Sodium azide 1μg

311

94

  

Table 4. Experiment 2, preincubation. Number of revertants per plate (mean of 3 plates).

 

[TA1537]

[WP2 uvrA]

Conc.
[
μg/plate]

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

DMSO

4

8

no

11

10

no

75

3

4

no

10

12

no

200

3

5

no

11

15

no

600

4

4

no

11

10

no

1800

3

4

no

9

11

no

5000

0

1

yes

7

10

yes

9- Aminoacridine 

75μg

218

 

2-aminoanthracene 1μg

45

 

57

 

Methyl methanesulfonate 1000μg

 

 

 

266

 

 

Applicant's summary and conclusion

Conclusions:
3-trimethoxysilylpropane-1-thiol has been tested for mutagenicity to bacteria, in a study which was conducted according to OECD TG 471, and in compliance with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in the initial or the repeat experiments up to limit concentrations in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and E coli WP2 uvrA. Cytotoxicity was observed at the highest concentration tested. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

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