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Toxicological information

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:
1988-03-15 to 1988-03-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
used dose level too low according to current requirements

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

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

Method

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:
Type and composition of metabolic activation system:
- source of S9 :
microsomal enzyme system (S-9 mix) from Aroclor 1254 induced Sprague-Dawley rat liver
- method of preparation of S9 mix:
Male Sprague Dawley rats (200 - 300 g) received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bodyweight) 5 days before sacrifice. Preparation was performed at 0 to 4°C using cold sterile solution and glassware. The livers from at least 5-6 animals are removed and pooled, washed in 150 mM KCL (approximately 1 mL/g wet livers). The washed livers are cut into small pieces and homogenized in three volumes of KCL. The homogenate is centrifuged at 9000 g for 10 minutes. The supernatant is the S-9 fraction. It is divided into small portions, rapidly frozen and storage at -80°C for not longer than three months.
- concentration or volume of S9 mix and S9 in the final culture medium :10% S9-Mix
Test concentrations with justification for top dose:
Toxicity test (first and second experiment) and mutagenicity test (first experiment): 0, 4, 20, 100, 500, 2500, 10000 µg/plate of the 30 % Sodium ethylenesulphonate solution.
Mutagenicity test (second experiment): 0, 4, 20, 100, 500, 2500, 10000 µg/plate of the 30 % Sodium ethylenesulphonate solution.
Vehicle / solvent:
aqua bidest
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 Mix (TA 100, TA 1535)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 Mix (TA 1537)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9 Mix (TA 98, TA 1538)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 Mix (TA 98, TA 100, TA 1535, TA 1537, TA 1538, WPuvrA)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
no
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with S9 Mix (TA 98, TA 100, TA 1535, TA 1537, TA 1538, WPuvrA)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-Methyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 Mix (WP2uvraA)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 to 72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: thinning of the bacterial lawn
Evaluation criteria:
no data
Statistics:
no data

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

First experiment

Table 1: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 100

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 100

Dose

µg/plate

Metabolic activation

Mean value

Colonies per plate

0

-

169

180

178

150

4

-

178

184

188

163

20

-

175

176

172

178

100

-

190

186

204

180

500

-

202

209

202

194

2500

-

185

199

172

185

10000

-

182

185

180

180

 

0

+

180

185

172

182

4

+

175

185

171

171

20

+

175

166

175

183

100

+

196

202

176

210

500

+

191

200

183

189

2500

+

177

179

182

169

10000

+

179

183

188

167

 

 

Table 2: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 1535

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1535

Dose

µg/plate

Metabolic activation

Mean value

Colonies per plate

0

-

12

10

12

14

4

-

13

15

10

15

20

-

13

14

15

11

100

-

13

11

12

17

500

-

12

15

12

10

2500

-

14

11

15

16

10000

-

11

11

11

10

 

0

+

13

15

11

13

4

+

13

14

13

12

20

+

12

13

12

11

100

+

14

17

13

11

500

+

15

11

16

17

2500

+

14

13

14

15

10000

+

13

11

15

12

Compound dissolved in 100 microliter Aqua bidest.

- '       : absence

+        : presence


 

Table 3: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 1537

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1537

Dose

µg/plate

Metabolic activation

Mean value

Colonies per plate

0

-

10

11

8

11

4

-

10

11

9

11

20

-

8

9

7

8

100

-

8

7

9

7

500

-

9

8

9

9

2500

-

8

7

10

7

10000

-

8

9

7

8

 

0

+

10

10

9

11

4

+

10

10

10

11

20

+

11

11

9

12

100

+

11

10

12

12

500

+

12

11

13

11

2500

+

10

9

13

9

10000

+

10

10

10

11

 

 

Table 4: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 1538

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1538

Dose

µg/plate

Metabolic activation

Mean value

Colonies per plate

0

-

12

13

11

13

4

-

13

14

14

12

20

-

13

11

16

12

100

-

14

12

14

15

500

-

15

15

19

11

2500

-

13

15

14

9

10000

-

12

15

11

10

 

0

+

18

18

16

19

4

+

18

23

16

15

20

+

15

16

14

14

100

+

17

18

16

18

500

+

19

17

20

19

2500

+

18

14

16

21

10000

+

17

19

14

18

Compound dissolved in 100 microliter Aqua bidest.

- '       : absence

+        : presence


 

Table 5: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 98

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 98

Dose

µg/plate

Metabolic activation

Mean value

Colonies per plate

0

-

25

23

26

25

4

-

26

21

28

28

20

-

25

22

24

28

100

-

23

25

25

20

500

-

23

23

27

20

2500

-

23

20

22

27

10000

-

25

25

28

23

 

0

+

30

29

32

29

4

+

34

34

35

33

20

+

31

33

32

29

100

+

33

30

38

32

500

+

35

34

32

38

2500

+

35

32

35

37

10000

+

32

31

34

30

 

 

Table 6: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

WP2uvrA

Number of revertant colonies per plate and mean values using Escherichia coli strain WP2uvrA

Dose

µg/plate

Metabolic activation

Mean value

Colonies per plate

0

-

40

45

39

35

4

-

38

39

40

35

20

-

40

40

40

39

100

-

37

37

36

37

500

-

42

42

43

40

2500

-

41

43

37

42

10000

-

40

42

40

37

 

0

+

44

48

36

48

4

+

42

42

45

39

20

+

45

42

51

42

100

+

45

41

48

45

500

+

39

41

39

38

2500

+

42

43

43

39

10000

+

43

36

46

46

Compound dissolved in 100 microliter Aqua bidest.

- '       : absence

+        : presence


 

Second experiment

Table 7: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 100

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 100

Dose

µg/plate

Metabolic activation

Mean value

Colonies per plate

Surviving fraction

0

-

143

152

143

133

1.0

4

-

147

150

136

154

1.0

20

-

142

143

119

164

1.0

100

-

156

161

150

157

1.0

500

-

148

158

119

168

1.0

2500

-

141

129

127

167

0.9

5000

-

142

140

126

160

1.0

 

0

+

183

183

195

172

1.0

4

+

165

155

158

182

1.0

20

+

174

181

158

182

1.0

100

+

178

197

163

173

0.9

500

+

155

187

155

154

1.0

2500

+

168

191

159

153

0.9

5000

+

172

200

158

159

0.9

 

 

Table 8: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 1535

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1535

Dose

µg/plate

Metabolic activation

Mean value

Colonies per plate

0

-

13

13

12

14

4

-

13

12

14

14

20

-

12

12

10

14

100

-

12

14

10

11

500

-

14

13

11

17

2500

-

11

10

12

10

5000

-

11

9

14

11

 

0

+

12

12

12

13

4

+

12

10

13

13

20

+

13

11

11

16

100

+

13

11

15

14

500

+

12

10

13

12

2500

+

12

13

15

9

5000

+

11

12

11

10

Compound dissolved in 100 microliter Aqua bidest.

- '       : absence

+        : presence


 

Table 9: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 1537

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1537

Dose

µg/plate

Metabolic activation

Mean value

Colonies per plate

0

-

7

9

7

6

4

-

8

7

8

8

20

-

7

5

9

6

100

-

7

6

8

8

500

-

7

5

7

8

2500

-

7

6

9

6

5000

-

7

9

6

7

 

0

+

9

10

8

8

4

+

9

9

10

7

20

+

10

10

10

11

100

+

10

8

12

11

500

+

8

7

9

9

2500

+

9

9

9

8

5000

+

10

10

10

9

 

 

Table 10: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 1538

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1538

Dose

µg/plate

Metabolic activation

Mean value

Colonies per plate

0

-

12

12

11

13

4

-

12

13

12

10

20

-

14

15

15

13

100

-

12

13

13

12

500

-

11

9

11

13

2500

-

14

14

14

15

5000

-

12

10

11

14

 

0

+

19

16

21

20

4

+

17

15

17

19

20

+

19

17

20

20

100

+

17

18

19

15

500

+

17

17

18

15

2500

+

19

17

22

17

5000

+

17

15

18

18

Compound dissolved in 100 microliter Aqua bidest.

- '       : absence

+        : presence


 

Table 11: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 98

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 98

Dose

µg/plate

Metabolic activation

Mean value22

Colonies per plate

0

-

22

23

22

22

4

-

22

20

21

25

20

-

27

26

27

27

100

-

23

24

24

20

500

-

21

24

19

19

2500

-

24

30

20

21

5000

-

22

21

22

24

 

0

+

25

23

26

27

4

+

29

28

28

32

20

+

28

30

27

27

100

+

26

30

20

27

500

+

28

31

25

29

2500

+

27

23

31

27

5000

+

25

23

28

25

 

 

Table 12: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

WP2uvrA

Number of revertant colonies per plate and mean values using Escherichia coli strain WP2uvrA

Dose

µg/plate

Metabolic activation

Mean value

Colonies per plate

0

-

51

52

56

45

4

-

50

43

46

60

20

-

54

54

56

53

100

-

51

52

49

51

500

-

52

51

55

50

2500

-

53

51

57

50

5000

-

57

54

61

55

 

0

+

56

54

56

59

4

+

58

52

61

62

20

+

54

53

54

54

100

+

56

54

53

60

500

+

59

59

60

58

2500

+

57

56

60

56

5000

+

53

52

49

58

Compound dissolved in 100 microliter Aqua bidest.

- '       : absence

+        : presence


 

First experiment

Table 13: mutability (positive controls) and sterility test of the experiment with Vinylsulfonsaures Natrium 30%ig

 

Number of revertant colonies per plate and mean values using Salmonella typhimurium strains and Escherichia coli

Strain

Compound

Dose µg/plate

Metab. Activ.

Mean Value

Colonies per plate

TA100

Sodium-azide

1

-

526

456

549

574

TA1535

Sodium-azide

1

-

409

410

398

419

TA1537

9-Aminoacridine

50

-

95

81

102

103

TA1538

2-Nitrofluorene

2.5

-

462

432

487

467

TA98

2-Nitrofluorene

2.5

-

327

294

355

333

WP2uvrA

MNNG

1.5

-

261

285

256

243

-

Vinylsulfon- saures Natrium 30%ig

10000

-

0

0

0

0

- : absence

 

 

Table 13a : mutability (positive controls) and sterility test of the experiment with Vinylsulfonsaures Natrium 30%ig

 

Number of revertant colonies per plate and mean values using Salmonella typhimurium strains and Escherichia coli

Strain

Compound

Dose ug/plate

Metab. Activ.

Mean Value

Colonies per plate

TA100

2-Aminoanthracen

0.5

+

818

874

806

775

TA1535

2-Aminoanthracen

1

+

140

151

126

142

TA1537

2-Aminoanthracen

1

+

126

122

130

125

TA1538

2-Aminoanthracen

0.5

+

458

438

463

473

TA98

2-Aminoanthracen

0.5

+

426

407

444

426

WP2uvrA

2-Aminoanthracen

10

+

440

428

442

451

 

TA100

Benzo[a]pyrene

10

+

1311

1352

1298

1283

TA1535

Benzo[a]pyrene

10

+

18

15

18

21

TA1537

Benzo[a]pyrene

10

+

96

85

100

102

TA1538

Benzo[a]pyrene

10

+

176

206

173

149

TA98

Benzo[a]pyrene

10

+

550

535

585

531

WP2uvrA

Benzo[a]pyrene

10

+

87

97

75

88

-

S-9 mix

500 µl

+

0

0

0

0

-

Vinylsulfon- saures Natrium 30%ig

1000 µg

+

0

0

0

0

+ :presence

 


 

Second experiment

Table 14: mutability (positive controls) and sterility test of the experiment with Vinylsulfonsaures Natrium 30%ig

 

Number of revertant colonies per plate and mean values using Salmonella typhimurium strains and Escherichia coli

Strain

Compound

Dose µg/plate

Metab. Activ.

Mean Value

Colonies per plate

TA100

Sodium-azide

1

-

293

29+2

306

280

TA1535

Sodium-azide

1

-

272

277

287

252

TA1537

9-Aminoacridine

50

-

157

160

163

178

TA1538

2-Nitrofluorene

2.5

-

459

469

430

478

TA98

2-Nitrofluorene

2.5

-

340

314

379

328

WP2uvrA

MNNG

2.5

-

223

206

223

241

-

Vinylsulfon- saures Natrium 30%ig

5000

-

0

0

0

0

- : absence

 

 

Table 14a : mutability (positive controls) and sterility test of the experiment with Vinylsulfonsaures Natrium 30%ig

 

Number of revertant colonies per plate and mean values using Salmonella typhimurium strains and Escherichia coli

Strain

Compound

Dose µg/plate

Metab. Activ.

Mean Value

Colonies per plate

TA100

2-Aminoanthracen

0.5

+

465

399

497

502

TA1535

2-Aminoanthracen

1

+

88

77

84

103

TA1537

2-Aminoanthracen

1

+

90

79

108

84

TA1538

2-Aminoanthracen

0.5

+

494

474

549

458

TA98

2-Aminoanthracen

0.5

+

577

589

563

579

WP2uvrA

2-Aminoanthracen

10

+

481

463

496

483

 

TA100

Benzo[a]pyrene

10

+

685

746

670

639

TA1535

Benzo[a]pyrene

10

+

18

16

15

24

TA1537

Benzo[a]pyrene

10

+

86

81

96

80

TA1538

Benzo[a]pyrene

10

+

183

176

184

188

TA98

Benzo[a]pyrene

10

+

551

552

547

583

WP2uvrA

Benzo[a]pyrene

10

+

74

66

79

76

-

S-9 mix

500 ul

+

0

0

0

0

-

Vinylsulfon- saures Natrium 30%ig

5000 ug

+

0

0

0

0

+ :presence

 

Applicant's summary and conclusion

Conclusions:
The test solution, containing 30% Sodium ethylenesulphonate, did not demonstrate mutagenic potential. Under the conditions of the present bacterial reverse mutation assay the maximum test concentration of Sodium ethylenesulphonate was 3000 µg/plate.
Executive summary:

Sodium ethylenesulphonate was tested as a 30 % aqueous solution in the Salmonella typhimurium reverse mutation assay (Hoechst AG (b), 1988) with the strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA) similar to OECD guideline 471 and GLP. Two independent mutagenicity studies were conducted (plate incorporation method), each in the absence and in the presence of a metabolising system derived from a rat liver homogenate. For both studies each bacterial strain was exposed to 6 dose levels. In the first experiment concentrations ranged from 4 to 10000 µg/plate and in the second experiment from 4 to 5000 µg/plate.Control plates without mutagen showed that the number of spontaneous revertant colonies was similiar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies. The test solution proved to be not toxic to the bacterial strains. In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with the 30 % Sodium ethylenesulphonate solution did not result in relevant increases in the number of revertant colonies.In conclusion thetest solution, containing 30% Sodium ethylenesulphonate, did not demonstrate mutagenic potential. Under the conditions of the present bacterial reverse mutation assay the maximum test concentration of Sodium ethylenesulphonate was 3000 µg/plate.