Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 11 - November 5, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
According to OECD Guideline 471, with GLP.

Data source

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

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
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
A mixture of: propan-2-one-O,O'(methoxyvinylsilandiyl)dioxime; propan-2-one-O-(dimethoxyvinylsilyl)oxime; propan-2-one-O,O',O''-(vinylsilantriyl)trioxime
EC Number:
458-680-3
EC Name:
A mixture of: propan-2-one-O,O'(methoxyvinylsilandiyl)dioxime; propan-2-one-O-(dimethoxyvinylsilyl)oxime; propan-2-one-O,O',O''-(vinylsilantriyl)trioxime
Cas Number:
797751-44-1
Molecular formula:
not applicable, multiconstituent substance
IUPAC Name:
3-ethenyl-3-methoxy-6-methyl-2,4-dioxa-5-aza-3-silahept-5-ene; 5-ethenyl-2,8-dimethyl-5-{[(propan-2-ylidene)amino]oxy}-4,6-dioxa-3,7-diaza-5-silanona-2,7-diene; 5-ethenyl-5-methoxy-2,8-dimethyl-4,6-dioxa-3,7-diaza-5-silanona-2,7-diene
Test material form:
liquid

Method

Target gene:
histidine-requiring gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
other: S. typhimurium TA 97a
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction
Test concentrations with justification for top dose:
5000, 1667, 556, 185, and 62 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance hydrolyses rapidly in water
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
Positive controls:
yes
Remarks:
(without activation)
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
TA97a: 4-nitro-o-phenylene-diamine- 10 µg
Positive controls:
yes
Remarks:
(with activation)
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
TA97a:7,12-dimethylbenz(a)anthracene-10 µg
Positive controls:
yes
Remarks:
(without activation)
Positive control substance:
2-nitrofluorene
Remarks:
TA98: 2-nitrofluorene - 2 µg
Positive controls:
yes
Remarks:
(without activation)
Positive control substance:
sodium azide
Remarks:
TA100: sodium azide-2 µg and TA1535: sodium azide- 1 µg
Positive controls:
yes
Remarks:
(with activation)
Positive control substance:
other: 2- aminoanthracene
Remarks:
TA98:2- aminoanthracene- 1 µg , TA1535: aminoanthracene- 2 µg and TA100: aminoanthracene- 2 µg
Positive controls:
yes
Remarks:
(with activation)
Positive control substance:
other: 1,8-Dihydroxy-anthraquinone
Remarks:
TA102: 1,8-Dihydroxy-anthraquinone - 50 µg
Positive controls:
yes
Remarks:
(without activation)
Positive control substance:
other: t-Butyl-hydroperoxide
Remarks:
TA102: t-Butyl-hydroperoxide-50 µg
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation). Bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state. For each sample the following solutions were combined:
- 0.1 mL of the overnight culture of the bacteria,
- 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
- 0.1 mL of the appropriate test- or reference substance solution and
- 2 mL of top agar.

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates (an independent repetition of the experiment was performed)

DETERMINATION OF CYTOTOXICITY
Method: the citotoxicity was rated as follows:
A reduced bacterial background lawn (mottled instead of homogeneous).
Microcolonies of bacteria instead of a homogeneous background lawn.
No background lawn.
Clearly reduced numbers of revertant colonies.

Evaluation criteria:
The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
- For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2.5 fold of the amount of the spontaneous revertants.
- For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 1.67 fold of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of the Ames test.

Results and discussion

Test results
Key result
Species / strain:
other: TA97a, TA98, TA100, TA102, and TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test substance was seen in any on the concentrations groups

RANGE-FINDING/SCREENING STUDIES:
Preliminary experiment was performed: different concentrations of test substance solutions were mixed with phosphate buffer, bacteria (TA100) and top-agar, as described in 3.5 and spread over a plate with minimal agar. The plates were incubated at 37 °C for 2 days and the growth of the bacterial background and the density of revertant colonies was determined.

COMPARISON WITH HISTORICAL CONTROL DATA:
The numbers of spontaneous revertans were comparable with the historic control data for the negative controls.

Any other information on results incl. tables

Individual numbers of revertants per plates

 

StrainTA97a: experiment no.1

concentr.
µg/plate

revertants
without metabolisation

revertants
with metabolisation

5000

96

109

124

147

160

114

1667

114

103

103

117

121

135

556

111

100

91

129

128

122

185

90

98

85

134

122

133

62

98

90

75

131

145

120

solvent

41

94

66

98

82

78

solvent

70

64

70

133

102

110

positive

219

194

200

517

482

460

 

StrainTA97a: experiment no.2

concentr.
µg/plate

revertants
without metabolisation

revertants
with metabolisation

5000

137

130

120

125

134

142

1667

108

111

113

131

163

131

556

104

130

128

130

108

180

185

131

142

130

157

180

128

62

119

130

102

104

163

143

solvent

123

142

131

148

151

168

solvent

133

175

123

125

119

145

positive

465

675

569

645

526

601

 

solvent:     DMSO

positive:    without metabolisation: 4-Nitro-o-phenylene-diamine, 10 µg / plate

                  with metabolisation: 7,12-Dimethylbenz[a]anthracene, 10 µg / plate


StrainTA98: experiment no.1

concentr.
µg/plate

revertants
without metabolisation

revertants
with metabolisation

5000

7

8

6

4

13

5

1667

14

10

11

11

6

11

556

7

17

11

8

8

7

185

9

2

4

14

11

4

62

7

3

10

6

6

15

solvent

11

10

10

9

12

12

solvent

6

8

9

14

6

12

positive

155

131

110

216

227

209

 

StrainTA98: experiment no.2

concentr.
µg/plate

revertants
without metabolisation

revertants
with metabolisation

5000

10

0

11

17

13

13

1667

10

8

6

14

10

12

556

12

7

5

9

18

9

185

14

12

14

10

13

15

62

12

12

4

9

10

8

solvent

13

10

8

10

13

14

solvent

11

5

4

12

11

16

positive

343

450

306

338

273

329

 

solvent:     DMSO

positive:    without metabolisation: 2-Nitrofluorene, 2 µg / plate

                  with metabolisation: 2-Amino-anthracene, 1 µg / plate

 

StrainTA100:  experiment no.1

concentr.
µg/plate

revertants
without metabolisation

revertants
with metabolisation

5000

85

53

47

88

79

88

1667

91

76

108

85

85

79

556

81

96

78

107

73

73

185

81

81

70

91

96

96

62

90

102

73

99

96

102

solvent

73

59

88

102

101

122

solvent

66

82

102

113

108

73

positive

410

399

375

1096

1321

1081

 

strainTA100: experiment no.2

concentr.
µg/plate

revertants
without metabolisation

revertants
with metabolisation

5000

55

62

55

98

104

84

1667

101

93

101

102

104

127

556

78

91

69

91

130

105

185

69

44

34

88

96

96

62

84

90

67

90

108

99

solvent

87

59

69

84

91

81

solvent

87

84

67

94

84

76

positive

181

149

160

469

393

375

 

solvent:     DMSO

positive:    without metabolisation: Sodium azide, 2 µg / plate

                  with metabolisation: 2 -Amino-anthracene, 2 µg / plate

 

StrainTA102: experiment no.1

concentr.
µg/plate

revertants
without metabolisation

revertants
with metabolisation

5000

148

149

168

140

230

230

1667

166

194

170

233

250

215

556

157

151

140

267

224

239

185

166

146

152

239

213

248

62

172

200

195

247

274

247

solvent

169

162

157

251

250

265

solvent

169

206

187

248

239

241

positive

629

742

634

629

588

576

 

StrainTA102: experiment no.2

concentr.
µg/plate

revertants
without metabolisation

revertants
with metabolisation

5000

194

198

198

264

253

248

1667

215

213

181

251

232

250

556

198

183

169

270

273

227

185

194

209

187

268

253

265

62

181

218

206

248

245

224

solvent

183

206

183

242

267

224

solvent

157

183

148

238

247

248

positive

975

902

686

477

442

533

 

solvent:     DMSO

positive:    without metabolisation: t-Butyl-hydroperoxide, 50 µg / plate

                  with metabolisation: 1,8-Dihydroxy-anthraquinone, 50 µg / plate

 

StrainTA1535: experiment no.1

concentr.
µg/plate

revertants
without metabolisation

revertants
with metabolisation

5000

8

3

6

14

8

4

1667

4

7

8

14

3

6

556

7

7

5

7

6

5

185

8

5

8

8

10

7

62

7

16

8

10

7

4

solvent

6

10

6

12

5

8

solvent

8

4

8

10

15

10

positive

256

229

207

181

127

175

StrainTA1535: experiment no.2

concentr.
µg/plate

revertants
without metabolisation

revertants
with metabolisation

5000

6

12

13

11

9

10

1667

13

8

9

8

14

9

556

10

15

12

8

8

11

185

14

7

15

10

10

15

62

12

11

9

12

8

12

solvent

14

18

12

18

12

11

solvent

14

18

11

10

12

13

positive

253

244

274

154

154

184

 

solvent:     DMSO

positive:    without metabolisation: Sodium azide, 1 µg / plate

                  with metabolisation: 2-Amino-anthracene, 2 µg / plate

Applicant's summary and conclusion

Conclusions:
According to the results obtained in this study, the test substance is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolising system up to 5000 µg/plate.

Executive summary:

The Bacterial Reverse mutation Assay (Ames test) for the test substance was performed with five histidine dependents strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535. Plate Incorporation Method was carried out at the concentrations of 5000, 1667, 556, 185, and 62 µg/plate in the presence and absence of metabolic activation. No precipitation of the test substance and no signs of cytotoxicity were noted at any dose level. In none of the concentrations tested and with none of the strains used an significant increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA1535 and 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. Metabolic activation did not change these results.

According to the obtained results, the test substance is non-mutagenic in the Ames test with and without an external metabolizing system up to 5000 µg/plate.