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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:
2010-09-28 to 2011-01-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Test according to EU-Method B.13/14 adopted 31. May, 2008 "Mutagenicity - Reverse mutation test using bacteria", Test according to OECD Guideline for Testing of Chemicals Part 471, adopted 21. Jul. 1997 "Bacterial Reverse Mutation Test", Test according to "Revised OECD Principles of Good Laboratory Practice" (Paris, 1997).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

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)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
CITROWANIL B, colourless to pale yellow liquid
IUPAC Name:
CITROWANIL B, colourless to pale yellow liquid
Constituent 2
Reference substance name:
2-benzyl-2-methyl-3-butennitril
IUPAC Name:
2-benzyl-2-methyl-3-butennitril
Constituent 3
Chemical structure
Reference substance name:
2-benzyl-2-methylbut-3-enenitrile
EC Number:
407-870-4
Cas Number:
97384-48-0
Molecular formula:
C12H13N
IUPAC Name:
2-benzyl-2-methylbut-3-enenitrile
Details on test material:
Citrowanil B; C12H13N

Method

Target gene:
TA97a: hisD6610, uvrB, pKM 101, rfa
TA98: hisD3052, uvrB, pKM 101, rfa
TA100: hisG46, uvrB, pKM 101, rfa
TA102: hisG428,pKM 101, rfa
TA1535: hisG46, uvrB, rfa
Species / strainopen allclose all
Species / strain / cell type:
other: S. typhimurium TA97a
Additional strain / cell type characteristics:
other: hisD6610, uvrB, pKM 101, rfa
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: hisG428,pKM 101, rfa
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: hisG46, uvrB, pKM 101, rfa
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: hisD3052, uvrB, pKM 101, rfa
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: hisG46, uvrB, rfa
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix with 4% S9, produced from the livers of male Spraque-Dawley rats wich were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally
Test concentrations with justification for top dose:
First Experiment: 4998 / 1499 / 500 / 150 / 50 µg/plate
Second Experiment: 501 / 150 / 50 / 15 / 5 µg/plate
Third Experiment: 504 / 252 / 126 / 63 / 32 / 16 / 8 µg/plate
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: 20 µg/plate in DMSO for TA98 with metabolic activation
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: 1 µg/plate in water for TA100, TA1535 without metabolic activation
Positive controls:
yes
Positive control substance:
other: 1 µg/plate 2-Amino-anthracene in DMSO for TA97a, TA100, TA102, TA1535 with metabolic activation
Positive controls:
yes
Positive control substance:
other: 20 µg/plate 4-Nitro-1,2-phenylene diaminein DMSO for TA97a, TA98, TA102 without metabolic activation
Details on test system and experimental conditions:
Per strain and dose, four plates with and four plates without S9 were used.
The first and the second experiment were carried out according to the plate incorporation method, the third experiment was carried out according to the Pre-incubation method.
Each test item solution was membrane filtrated before use to accomplish sterility.

Plate incorporation method:
0.1 mL of the appropriate solution of the test item was given into a sterile tube. After mixing with 0.1 mL overnight culture of the respective strain and 0.5 mL phosphate buffer (treatement without S9) or 0.5 mL S9 mix (treatement with S9), 2 mL Top-Agar, containing 10 mL 0.5 mMol histidine-biotin-solution per 100 mL, were added.
Pre-incubation method:
0.1 mL of the appropriate solution of the test item was given into a sterile tube. After mixing with 0.1 mL overnight culture of the respective strain and 0.5 mL phosphate buffer (treatement without S9) or 0.5 mL S9 mix (treatement with S9) were added. The mixture was incubated in an incubating chamber at 37°C for 20 minutes. During this time the vessles were aerated through careful shaking. Then 2 mL Top-Agar, containing 10 mL 0.5 mMol histidine-biotin-solution per 100 mL, was added.
In each case the mixture was vortexed gently, then poured on a minimal glucose plate and distributed evenly, rotating the plate. The plates were closed and left to harden for a few minutes, then inverted and placed in the dark incubator at 37°C.
Evaluation criteria:
The colonies were counted visually after 48 hours incubation, the numbers were recorded.

A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor f(I) ≥ 2) in at least one strain can be observed. A concentration-releated increase over the range tested can also be taken as a sign of mutagenic activity.
Statistics:
A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The increase factor f(I) of revertant induction (mean revetants divided by mean spontaneous revertants [vehicle control]) and the absolute number of revertants (mean revetants less mean spontaneous revertants [vehicle control]) were calculated, too.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: yes at 4998 / 1499 µg/plate, no at 500 / 150 / 50 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: yes at 4998 / 1499 µg/plate, no at 500 / 150 / 50 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: yes at 4998 / 1499 µg/plate, no at 500 / 150 / 50 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: yes at 4998 / 1499 µg/plate, no at 500 / 150 / 50 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: Plate incorporation and Pre-incubation method
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Survey of the Findings:

The mean revertant values of the four replicates are presented in the following table.

 

Exp.

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

1

H2O

Mean

124

115

11

17

128

113

218

233

14

12

DMSO

Mean

112

105

11

14

112

117

191

294

12

13

Pos. Contr.

Mean

f(I)

768

6.9

789

7.5

752

68.4

142

10.1

633

5.0

496

4.2

817

4.3

657

2.2

242

17.3

144

11.1

4998 µg/pl.

Mean

f(I)

110

0.98

67

0.64

1

0.09

0

0.00

0

0.00

0

0.00

20

0.10

0

0.00

0

0.00

0

0.00

1499 µg/pl.

Mean

f(I)

57

0.51

115

1.10

0

0.00

0

0.00

0

0.00

27

0.23

23

0.12

15

0.05

0

0.00

0

0.00

500 µg/pl.

Mean

f(I)

101

0.90

111

1.06

9

0.82

10

0.71

123

1.10

129

1.10

229

1.20

217

0.74

10

0.83

15

1.15

150 µg/pl.

Mean

f(I)

113

1.01

132

1.26

10

0.91

11

0.79

130

1.16

130

1.11

206

1.08

241

0.82

11

0.92

12

0.92

50 µg/pl.

Mean

f(I)

116

1.04

113

1.08

9

0.82

12

0.86

125

1.12

111

0.95

282

1.48

234

0.80

13

1.08

13

1.00

 

2

H2O

Mean

112

101

11

15

99

115

208

200

14

14

DMSO

Mean

105

97

11

16

90

111

253

176

10

12

Pos. Contr.

Mean

f(I)

310

3.0

414

4.3

305

27.7

260

16.3

674

6.8

1084

9.8

639

2.5

796

4.5

226

16.1

122

10.2

501 µg/pl.

Mean

f(I)

114

1.09

107

1.10

10

0.91

12

0.75

108

1.20

99

0.89

151

0.60

139

0.79

10

1.00

13

1.08

150 µg/pl.

Mean

f(I)

117

1.11

125

1.29

11

1.00

15

0.94

100

1.11

148

1.33

190

0.75

176

1.00

13

1.30

9

0.75

50 µg/pl.

Mean

f(I)

109

1.04

99

1.02

11

1.00

12

0.75

126

1.40

83

0.75

158

0.62

168

0.95

13

1.30

14

1.17

15 µg/pl.

Mean

f(I)

110

1.05

91

0.94

11

1.00

13

0.81

88

0.98

113

1.02

179

0.71

182

1.03

11

1.10

18

1.50

5

µg/pl.

Mean

f(I)

94

0.90

105

1.08

12

1.09

12

0.75

111

1.23

84

0.76

213

0.84

205

1.16

13

1.30

16

1.33

 

3

H2O

Mean

110

108

11

16

78

127

199

174

16

14

DMSO

Mean

105

105

14

11

98

106

196

186

17

17

Pos. Contr.

Mean

f(I)

320

3.1

303

2.9

231

16.5

117

10.6

254

3.3

221

2.1

886

4.5

514

2.8

237

14.8

208

12.2

504 µg/pl.

Mean

f(I)

115

1.10

135

1.29

6

0.43

9

0.82

29

0.30

91

0.86

222

1.13

224

1.2

9

0.53

10

0.59

252 µg/pl.

Mean

f(I)

106

1.01

104

0.99

5

0.36

12

1.09

29

0.30

84

0.79

230

1.17

188

1.01

13

0.76

14

0.82

126 µg/pl.

Mean

f(I)

121

1.15

111

1.06

8

0.57

13

1.18

79

0.81

104

0.98

268

1.37

192

1.03

10

0.59

19

1.12

63 µg/pl.

Mean

f(I)

111

1.06

112

1.07

10

0.71

14

1.27

99

1.01

126

1.19

159

0.81

174

0.94

11

0.65

16

0.94

32 µg/pl.

Mean

f(I)

108

1.03

107

1.02

10

0.71

13

1.18

108

1.10

88

0.83

211

1.08

258

1.39

11

0.65

14

0.82

18 µg/pl

Mean

f(I)

112

1.07

108

1.03

9

0.64

18

1.64

88

0.90

82

0.77

219

1.12

228

1.23

12

0.71

14

0.82

8

µg/pl

Mean

f(I)

112

1.07

113

1.08

8

0.57

16

1.45

107

1.09

110

1.04

218

1.11

156

0.84

11

0.65

12

0.71

 f(I) = increase factor

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

CITROWANIL B is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535.
Executive summary:

Three valid experiments were performed to evaluate the mutagenic potential of CITROWANIL B in the Bacterial Reverse Mutation Test.

 

First Experiment:

Five concentrations of the test item, dissolved in dimethyl sulfoxid (DMSO) (ranging from 4998 to 50 µg/plate) were used. Five genetically manipulated strains ofSalmonella typhimurium(TA97a, TA98, TA100, TA102 and TA1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9) for 48 hours using the plate incorporation method.

Signs of toxicity towards the bacteria could be observed in the two highest concentrations (4998 and 1499 µg/plate).

None of the three last concentrations (500, 150 and 50 µg/plate) caused a significant increase in the numbers of revertant colonies in the tested strains. The test item didn’t show any mutagenic effect in the first experiment.

The sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were within the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

 

Second Experiment:

Because of the results of the first experiment, a second experiment was performed, using five concentrations of the test item (ranging from 501 to 5 µg/plate), using the plate incorporation method, too.

The test item didn’t show mutagenic effects in the second experiment, either.

No signs of toxicity towards the bacteria could be observed.

The sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were within the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

 

Third Experiment:

To verify the first and the second experiment, a third experiment was performed, using seven concentrations of the test item (ranging from 504 to 8 µg/plate) and a modification in study performance(pre-incubation method).

The test item didn’t show mutagenic effects in the third experiment, either.

No signs of toxicity towards the bacteria could be observed.

The sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were within the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

 

Under the conditions of the test, the test item didn’t show mutagenic effects in Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535.

Therefore, no concentration-effect relationship could be determined.

 

The test item CITROWANIL B is considered as “not mutagenic under the conditions of the test”.