Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Method

Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal fraction of Phenobarbital and β-Naphthoflavone induced rat livers
Test concentrations with justification for top dose:
Experiment I, with and without metabolic activation:
- TA 98, TA 100: 31.6, 100, 316, 1000, 2500, 5000 µg/plate
- TA 1535, TA 1537, TA 102: 10, 31.6, 100, 316, 1000, 2500 µg/plate
Experiment II, with and without metabolic activation: 62.5, 125, 250, 500, 1000, 3000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: the solvent was compatible with the survival of the bacteria and S9 activity
Controlsopen allclose all
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535, TA 100, without metabolic activation
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
TA 1537, TA 98, without metabolic activation
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 102, without metabolic activation
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535, TA1537, TA 98, TA 100, TA 102, with metabolic activation
Details on test system and conditions:
ACTIVATION: The protein concentration in the S9 preparation was 36 mg/ml. The S9 mix contained the following co-factors: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 5 mM NADP in 100 mM sodium-orthophosphate-buffer, pH 7.4, and 8.5 parts of cofactor solution were added to 1.5 parts of S9. 0.5 ml S9 mix was added to 0.1 ml test solution and 2 ml of overlay agar, giving a final concentration of approximately 3% S9 in the plates.

METHOD OF APPLICATION: in agar: plate incorporation

DURATION
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours

SELECTION AGENT (mutation assays): histidine deficient agar

NUMBER OF REPLICATIONS: triplicate plates were used, and an independent repeat experiment was conducted.

DETERMINATION OF CYTOTOXICITY
- Method: condition of bacterial lawn/reduction in number of revertants
Evaluation criteria:
A test item is considered mutagenic when: a dose related increase in the number of revertants occurs; a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
Statistics:
Relative statistics (standard deviation, mean) were calculates.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
from 316 µg/plate, TA 102 without metabolic activation
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment I:
- TA 1535, TA 1537, TA 100, TA 98: 2500 µg/plate and higher, with and without metabolic activation
- TA 102: at 316 µg/plate and higher, without metabolic activation; at 2500 µg/plate, with metabolic activation
Experiment II:
- TA 1535, TA 1537, TA 100, TA 98: at 3000 µg/plate, with and without metabolic activation
- TA 102: at 1000 and higher µg/plate, without metabolic activation; at 3000 µg/plate, with metabolic activation

PRECIPITATION
No precipitation of test item was observed.

Any other information on results incl. tables

Table 1. Experiment I, plate incorporation, revertant colonies per plate (mean of three plates), with and without metabolic activation

Concentration

μg/plate

TA 1535

TA 1537

TA 100

TA 98

TA 102

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

A dest

15

18

24

16

139

100

25

27

237

293

Ethanol

16

20

24

15

120

122

37

30

237

237

31.6

19

19

28

18

139

97

31

41

219

212

100

22

14

21

19

140

111

22

37

175

251

316

27

16

21

17

116

105

27

41

165

249

1000

25

19

16

16

81

106

28

33

260

225

2500

16

15

21

13

59

72

14

25

235

234

5000

6

7

3

3

0

0

0

0

118

219

4-NOPD 10

 

 

 

 

 

 

 

798

 

 

4-NOPD 40

 

 

 

199

 

 

 

 

 

 

2-AA 2.5

190

 

147

 

1296

 

1269

 

 

 

2-AA 10

 

 

 

 

 

 

 

 

558

 

Sodium azide 10

 

1163

 

 

 

1121

 

 

 

 

MMS 1μL

 

 

 

 

 

 

 

 

 

2136

Table 2. Experiment II, plate incorporation, revertant colonies per plate (mean of three plates), with and without metabolic activation

Concentration

µg/plate

TA 1535

TA 1537

TA 100

TA 98

TA 102

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

A dest

14

14

18

14

118

109

37

19

214

311

Ethanol

20

18

15

11

117

119

45

28

216

273

62.5

18

14

22

12

119

95

41

29

212

254

125

15

12

17

14

123

109

41

33

207

284

250

17

13

23

14

114

118

34

25

214

291

500

17

19

17

13

122

116

33

29

187

268

1000

19

16

18

10

107

97

40

27

202

189

3000

2

9

7

0

71

40

22

16

98

190

4-NOPD 10

 

 

 

 

 

 

 

1339

 

 

4-NOPD 40

 

 

 

228

 

 

 

 

 

 

2-AA 2.5

233

 

239

 

2227

 

1756

 

 

 

2-AA 10

 

 

 

 

 

 

 

 

521

 

Sodium azide 10

 

1324

 

 

 

1348

 

 

 

 

MMS 1μL

 

 

 

 

 

 

 

 

 

1819

 

4 -NOPD: 4-nitro-o-phenylene-diamine

2 -AA: 2-aminoanthracene

MMS: methylmethanesulphonate

 

 

 

Applicant's summary and conclusion

Conclusions:
1,1-Dimethyl-N,N'-bis(1-methylpropyl)silanediamine has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD TG 471, 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 Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or TA 102 in the initial or the repeat experiments using the plate incorporation method up to limit concentrations. Appropriate positive, solvent and negative 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.