Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: not mutagenic

Chromosome aberration test performed with human lymphocytes cells: not clastogenic

HPRT test performed with CHO cells: not mutagenic

Link to relevant study records

Referenceopen allclose all

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

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

 

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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-03-11 to 1997-04-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Adopted 4 April, 1984
Deviations:
yes
Remarks:
only 3 instead of 4 analysable concentrations were tested.
Qualifier:
according to
Guideline:
EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)
Version / remarks:
1983
Deviations:
yes
Remarks:
only 3 instead of 4 analysable concentrations were tested.
Qualifier:
according to
Guideline:
other: EEC Directive 87/302/EEC
Version / remarks:
1988
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
hemizygous hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: CHO-Kl-BH4 cells were obtained from the British Industrial Biological Research Association.
- Suitability of cells: These cells are functionally hemizygous at the HPRT locus.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
These cells are functionally hemizygous at the HPRT locus. Ham's F12 medium was supplemented with 2 mM L-glutamine and 50 µg/mL gentamicin. .
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9
Aroclor stimulated S9 liver mix from male Sprague-Dawley rats
- method of preparation of S9 mix
S-9 fraction was prepared from a group of c. 10 animals. Mixed function oxidase systems in the rat liver were stimulated by Aroclor 1254, administered as a single intraperitoneal injection in Arachis oil at a dosage of 500 mg/kg bodyweight. On the fifth day after injection, following an overnight starvation, the rats were killed and their livers aseptically removed. The following steps were carried out at 0 - 4°C under aseptic conditions. The livers were placed in 250 mM sucrose solution (3 mL of sucrose solution : 1 g of liver) before being transferred to an Ultra-Turrax homogeniser. Following preparation the homogenate was centrifuged at 9000 g for 10 minutes. The supernatant fraction (S-9 fraction) was dispensed into aliquots and stored at -80°C until required.
- concentration or volume of S9 mix and S9 in the final culture medium
S-9 mix contains: S-9 fraction (25% v/v), isocitric acid (43.5 mM), NADP (8.0 mM) in ice cold medium. The co-factors were prepared on the morning of the test, neutralised with 1N NaOH and filter sterilised before adding to S-9 fraction.
- quality controls of S9: All batches of S-9 fraction were tested using 20-methylcholanthrene before use.
Test concentrations with justification for top dose:
Mutation tests (with and without S9 mix; two independent experiments)
1250, 2500, 5000 µg/mL of a 24.9 % Sodium ethylenesulphonate solution

Vehicle / solvent:
Positive controls: DMSO
Test material: cell medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 20-methylcholanthrene
Remarks:
with S-9 Mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 Mix
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:
- Cell density at seeding: 7.5 x 10E5 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Test substance, solvent or positive control were added to the flasks and incubated for 4 hours.
- Harvest time after the end of treatment: At the end of the treatment period the cells were harvested, washed and seeded with 200 cells in medium. These plates were incubated at 37°C for 7 days after which time the colonies growing in the plates were fixed, stained and counted.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
The cultures were subcultured once during the expression period on Day 4 or 5 and, after a total of 7 days, were harvested by trypsinisation. For each treatment group, three 60 mm culture plates were each seeded with 200 cells in H5 and five 100 mm plates with 2 x 105 cells in selective medium.
- Selection time:
Further cells from each culture were seeded in flasks containing medium and incubated for 7 days to allow for expression of the mutant phenotype. After 7 days the cells were harvested and 200 cells per culture plate were seeded in medium or selection medium for 7 days. At the end of this incubation period colonies growing in the plates were fixed, strained and counted.
- Selective agent: The selective medium, in which only HPRT deficient cells grow, consisted of heat-inactivated fetal calf serum supplemented with 6-TG (Sigma) at a final concentration of 10 µg/mL.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: plating efficiency
Evaluation criteria:
The criteria for a positive response were:
- The demonstration of a statistically significant increase in mutant frequency following treatment with the test substance.
- Evidence of a dose relationship, over at least two dose levels, in any increase in mutant frequency.
- Demonstration of reproducibility in any increase in mutant frequency.
- In addition the mean mutant frequency must lie outside the upper limit of the historical control
Statistics:
The statistical significance of the data was analysed by weighted analysis of variance following the methods described by Arlett et al (1989), assuming the data are acceptable according to their criteria.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Some increases of pH were observed.
- Effects of osmolality: dose related increases in osmolarity were observed in all tests, both in the presence and the absence of S-9 mix.

TABLE 1

Test 1. Cytotoxicity in the absence of S-9 mix

Concentration of test item 24.9% (µg/mL)

Number of colonies on viability plates

 Cell survival (% control)

Mean
% survival

1

2

3

Total

0

112

79

96

287

100

100

87

98

85

270

(SDW control)

68

107

72

247

47

56

57

160

1250

129

114

104

347

144

122

81

77

84

242

100

2500

65

89

75

229

95

98

66

97

79

242

100

5000

48

67

79

194

80

105

108

94

111

313

130

Ems (250 µg/mL)

138

122

128

388

161

180

180

154

145

479

199

 


TABLE 2

Test 1. Mutant frequency in the absence of S-9 mix

Concentration of test item 24.9% (µg/mL)

No. of colonies on non-selective plates

Plating efficiency (%)

No. of colonies on selective plates

Mutant

frequency

per 106

survivors

Mean mutant frequency per 106survivors

1

2

3

Total

1

2

3

4

5

Total

0

(SDW control)

130

114

134

378

63

0

0

0

0

0

0

0

 

113

120

105

338

56

0

1

0

1

0

2

4

 

150

125

130

405

68

4

1

0

0

0

5

7

4

128

131

114

373

62

1

1

1

0

0

3

5

 

1250

96

104

91

291

49

0

0

0

0

0

0

0

2

113

109

107

329

55

0

0

1

1

0

2

4

 

2500

118

136

116

370

62

0

0

1

1

0

2

3

8

117

114

103

334

56

1

0

0

4

2

7

13

 

5000

117

140

114

371

62

0

0

0

0

1

1

2

2

112

114

104

330

55

0

0

1

0

0

1

2

 

Ems (250 µg/mL)

137

125

125

387

64

37

35

36

40

39

187

290

298

115

134

111

360

60

39

34

34

37

39

183

305

***

*** - Significantly different from control P<0.001

 

 

TABLE 3

Test 2. Cytotoxicity in the absence of S-9 mix

Concentration of test item 24.9% (µg/mL)

Number of colonies on viability plates

Cell survival (% control)

Mean
% survival

1

2

3

Total

0

81

77

70

228

100

100

73

81

80

234

(SDW control)

80

80

89

239

80

75

105

260

1250

81

79

C

(240)

100

99

78

C

C

(234)

98

2500

81

82

80

243

101

95

71

83

56

210

88

5000

108

91

94

293

122

124

87

107

105

299

125

Ems (250 µg/mL)

91

74

C

(248)

103

92

73

52

68

193

80

C Contaminated. Figure in brackets is adjustment made for contaminated plates and is used in all calculations


 

TABLE 4

Test 2. Mutant frequency in the absence of S-9 mix

Concentration of test item 24.9% (µg/mL)

No. of colonies on non-selective plates

Plating efficiency (%)

No. of colonies on selective plates

Mutant

frequency

per 106

survivors

Mean mutant frequency per 106survivors

1

2

3

Total

1

2

3

4

5

Total

0

(SDW control)

117

164

123

404

67

1

0

1

0

0

2

3

 

143

129

134

406

68

0

0

0

0

0

0

0

 

120

110

127

357

60

0

0

0

0

2

2

3

3

152

124

138

414

69

0

1

1

0

2

4

6

 

1250

116

143

102

361

60

0

3

1

2

0

6

10

9

128

143

127

398

66

1

1

2

1

0

5

8

 

2500

132

118

124

374

62

0

0

0

0

1

1

2

2

125

139

147

411

68

1

0

0

0

0

1

1

 

5000

101

122

115

338

56

0

0

0

0

0

0

0

1

128

115

110

353

59

0

0

0

0

1

1

2

 

Ems (250 µg/mL)

106

118

101

325

54

23

23

19

21

20

106

196

228

113

123

131

367

61

33

31

30

32

33

159

260

***

*** - Significantly different from control P<0.001

 

 

TABLE 5

Test 1. Cytotoxicity in the presence of S-9 mix

Concentration of test item 24.9% (µg/mL)

Number of colonies on viability plates

Cell survival (% control)

Mean
% survival

1

2

3

Total

0

109

91

70

270

100

100

96

87

107

290

(SDW control)

117

117

93

327

107

87

102

296

1250

245

224

248

717

242

168

101

82

91

274

93

2500

80

91

92

263

89

94

94

105

94

293

99

5000

85

90

99

274

93

92

88

89

88

265

90

MC (5 µg/mL)

81

81

105

267

90

82

80

50

89

219

74

 


 

TABLE 6

Test 1. Mutant frequency in the presence of S-9 mix

Concentration of test item 24.9% (µg/mL)

No. of colonies on non-selective plates

Plating efficiency (%)

No. of colonies on selective plates

Mutant

frequency

per 106

survivors

Mean mutant frequency per 106survivors

1

2

3

Total

1

2

3

4

5

Total

0

(SDW control)

115

145

161

421

70

1

0

2

0

1

4

6

 

139

142

122

403

67

3

0

0

0

1

4

6

 

118

121

159

398

66

1

1

0

1

1

4

6

7

159

137

124

420

70

1

3

1

1

0

6

9

 

1250

136

128

122

386

64

0

1

0

1

1

3

5

7

125

121

134

380

63

3

0

0

1

1

5

8

 

2500

133

127

127

387

64

0

0

1

0

0

1

2

3

122

131

121

374

62

0

0

0

0

2

2

3

 

5000

138

130

119

387

64

0

0

0

1

0

1

2

1

127

134

107

368

61

0

0

0

0

0

0

0

 

MC (5 µg/mL)

147

133

140

420

70

40

38

39

38

41

196

280

299

138

137

96

371

62

40

41

37

38

40

196

317

***

*** - Significantly different from control P<0.001

 

 

TABLE 7

Test 2. Cytotoxicity in the presence of S-9 mix

Concentration of test item 24.9% (µg/mL)

Number of colonies on viability plates

Cell survival (% control)

Mean
% survival

1

2

3

Total

0

90

102

92

284

100

100

89

107

99

295

(SDW control)

97

83

90

270

101

98

110

309

1250

128

106

116

350

121

117

113

104

112

329

113

2500

108

113

113

334

115

119

116

124

118

358

123

5000

135

135

120

390

134

125

116

100

117

333

115

MC (5 µg/mL)

129

143

125

397

137

137

131

136

129

396

137

 


 

TABLE 8

Test 2. Mutant frequency in the presence of S-9 mix

Concentration of test item 24.9% (µg/mL)

No. of colonies on non-selective plates

Plating efficiency (%)

No. of colonies on selective plates

Mutant

frequency

per 106

survivors

Mean mutant frequency per 106survivors

1

2

3

Total

1

2

3

4

5

Total

0

(SDW control)

138

103

113

354

59

2

2

0

1

1

6

10

 

124

184

111

419

70

1

0

1

1

2

5

7

 

106

143

160

409

68

2

3

0

2

0

7

10

8

160

145

C

(458)

76

1

1

0

0

2

4

5

 

1250

114

148

117

379

63

0

1

0

0

0

1

2

5

113

121

117

351

59

0

0

1

0

3

4

7

 

2500

127

123

133

383

64

1

0

2

2

1

6

9

8

135

115

135

385

64

0

0

0

3

1

4

6

 

5000

96

84

107

287

48

0

0

0

0

3

3

6

10

121

120

117

358

60

2

0

3

1

2

8

13

 

MC (5 µg/mL)

102

96

112

310

52

56

65

58

46

54

279

540

521

93

109

102

304

51

45

47

59

57

49

257

507

***

C - Contaminated. Figure in brackets is adjustment made for contaminated plates and is used in all calculations

*** - Significantly different from control P<0.001

 

 

TABLE 9

pH of the culture medium treated with test item - 0 hours

Concentration of test item 24.9% (µg/mL)

In the absence of S-8 mix

In the presence of S-9 mix

Test 1

Test 2

Test 1

Test 2

0

(SDW control)

8.2

7.5

7.2

7.5

1250

8.4

7.9

7.2

7.6

2500

8.3

8.1

7.3

7.6

5000

8.6

8.4

7.3

7.6

 

Conclusions:
The test solution, containing 24.9% Sodium ethylenesulphonate, did not demonstrate a mutagenic potential in mammalian cells. Under the conditions of the present mammalian cell gene mutation assay the maximum test concentration of Sodium ethylenesulphonate was 1250 µg/mL. This concentration is in the range of the maximum test concentration of 1300 µg/mL (0.01M) recommended according to the guideline.
Executive summary:

A test solution, containing 24.9 % Sodium ethylenesulphonate, was tested in a mammalian gene mutation test in CHO cells (HPRT assay) according to OECD guideline 476 and GLP (Huntigdon Life Science Ltd. (b), 1997). In two independent experiments concentrations of 1250, 2500 and 5000 µg/mL of the 25 % Sodium ethylenesulphonate solution were tested with and without metabolic activation (S9 -mix). Some increases of pH values and dose related increases in osmolarity were observed in all tests. No significant increases in mutant frequency were observed after treatment with 24.9 % Sodium ethylenesulphonate in either test in the absence and the presence of S-9 mix. Ethyl methanesulphonate and 20-Methylcholanthrene, the positive controls, induced highly significant increases in mutant frequency in both tests. It is concluded that the test solution, containing 24.9 % Sodium ethylenesulphonate, did not demonstrate mutagenic potential in this in vitro gene mutation assay. Under the conditions of the present mammalian gene mutation test the maximum test concentration of Sodium ethylenesulphonate was 1250 µg/mL (5000 µg/mL of a 25 % aqueous Sodium ethylenesulphonate solution). This concentration is in the range of the maximum test concentration of 1300 µg/mL (0.01M) recommended according to the guideline.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-02-26 to 1997-04-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Adopted: 26 May 1983
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
29.12.1992
Deviations:
no
Qualifier:
according to
Guideline:
other: UK Department of Health Repor on Health and Social Subjects No. 35. Guidelines for the testing of chemicals for mutagenicity (1989).
Version / remarks:
1989
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Sex, age and number of blood donors: male donors
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: pooled
- Mitogen used for lymphocytes: stimulated to divide by addition of phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature
37°C, 5% CO2, RPMI 1640 tissue culture medium (Imperial) containing 10% foetal calf serum (PAA)
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9
S9-mix (by Aroclor 1254 stimulated rat liver)

- method of preparation of S9 mix
Mixed function oxidase systems in the liver of a group of rats were stimulated by Aroclor 1254, administered as a single intraperitoneal injection in Arachis oil at a dosage of 500 mg/kg bodyweight. On the fifth day after injection, following an overnight starvation, the rats were killed and their livers asepticaily removed. The following steps were carried out at 0 - 4°C under aseptic conditions. The livers were placed in 0.15 M KCl (3 ml KCl : 1 g liver) before being transferred to an Ultra-Turrax homogeniser. Following preparation, the homogenates were centrifuged at 9000 g for 10 minutes. The supernatant fraction (S9 fraction) was dispensed into aliquots and stored at -80°C until required.

- concentration or volume of S9 mix and S9 in the final culture medium
S9 mix contained: S9 fraction (10% v/v), MgCI2 (8 mM), KCl (33 mM), sodium orthophosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filter-sterilised before use.

- quality controls of S9: All batches of S9 fraction were tested for sterility and efficiency.
Test concentrations with justification for top dose:
1250, 2500 and 5000 µg/mL of the 24.9 % Sodium ethylenesulphonate solution.

Positive control:
+ S9 mix: 20, 25 and 30 µg/mL (Cyclophoshamide)
- S9 mix: 0.2, 0.4 and 0.8 µg/mL (Mitomycin C)
Vehicle / solvent:
- Justification for choice of solvent/vehicle:
Sodium ethylenesulphonate 25 % was supplied as a 25 % aqueous solution and on dosing at 1 % v/v into aqueous tissue culture medium (RPMI 1640), giving a final concentration of 2500 µg/mL, no precipitate was observed. A concentration of 5000 µg/mL was achieved by the addition of undiluted Sodium etyhlenesulphonate (25 %) at 2 % v/v and again no precipitate was observed.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 Mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 Mix
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
First test:
with and without S9 Mix: 21 hours (with S9 Mix: 3 hours exposure and 18 hours postexposure)

Second test:
with and without S9 Mix: 21 hours (with S9 Mix: 3 hours exposure and 18 hours postexposure)
with and without S9 Mix: 45 hours (with S9 Mix: 3 hours exposure and 42 hours postexposure)

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor: Colcemid at a final concentration of 0.1 µg/mL after 2 hours incubation

- Methods of slide preparation and staining technique used including the stain used:
Each cell suspension was transferred to a conical centrifuge tube and centrifuged for 5 minutes at 500 g. The cell pellets were treated with a hypotonic solution (0.075M KCl prewarmed at 37°C). After a 10 minute period of hypotonic incubation at 37°C, the suspensions were centrifuged at 500 g for 5 minutes and the cell pellets fixed by addition of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid). The fixative was replaced several times. The pellets were resuspended, then centrifuged at 200 g for 10 minutes and finally resuspended in a small volume of fresh fixative. Two or three drops of the cell suspensions were dropped onto pre-cleaned microscope slides which were then allowed to air-dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and then mounted in DPX.

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
The slides were then coded. Metaphase figures were identified, using a magnification of xl60 and examined at a magnification of x1000 using an oil immersion objective. Approximately 100 metaphase figures were examined, where possible, from each culture. Only cells with 44 - 46 chromosomes were analysed. The Vernier readings of all aberrant metaphase figures were recorded.
- Determination of polyploidy: no data
- Determination of endoreplication: no data

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)
Statistics:
The number of aberrant metaphase figures in each treatment group was compared with the solvent control value using Fisher's test.
Key result
Species / strain:
lymphocytes: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
absence of S9 mix at 5000 µg/mL of the 24.9 % Sodium etyhlensulphonate solution, corresponding to 1250 µg/mL sodium ethylenesulphonate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No substantial increases in pH values were observed.
- Data on osmolality: Dose related increases in osmolality were observed both in the absence and presence of S9 mix.
- Precipitation and time of the determination: no data

STUDY RESULTS
- see attached results

- Results from cytotoxicity measurements:
FIRST TEST:
In the absence of S9 mix, the test item caused a reduction in the mitotic index to 55% of the solvent control value at 5000 µg/mL, corresponding to 1250 µg/mL Sodium ethylenesulphonate. All three concentrations were therefore acceptable for metaphase analysis.
In the presence of S9 mix, the test item caused no reduction in the mitotic index compared to the solvent control. All three dose levels were again subject to metaphase analysis.
SECOND TEST (21 hour harvest):
In the absence of S9 mix, the test item caused a reduction in the mitotic index to 66% of the solvent control level at 5000 µg/mL. All three concentrations were acceptable for metaphase analysis.
In the presence of S9 mix, the test item caused no reduction in mitotic index compared to the untreated cultures. All three dose levels were again subject to metaphase analysis.
SECOND TEST (45 hour harvest):
In both the absence and presence of S9 mix, the test item caused no reduction in the mitotic index compared to the solvent control value. 5000 µg/mL, was chosen for metaphase analysis as this was the highest concentration analysed at the 21 hour harvest time.
- Genotoxicity results:
The test item caused no statistically significant increase in the proportion of metaphase figures with chromosome aberrations, at any concentrations analysed, in either the absence or presence of S9 mix.
Both positive control compounds, mitomycin C and cyclophosphamide, caused large, statistically significant increases (P<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

HISTORICAL CONTROL DATA
- see attached results

TABLE 1

Mitotic index data - first test, 21 hour harvest

Without S9 mix

Concentration of test item 24.9% (µg/mL)

Mitotic index

Relative mitotic index (%)

Incidence

% Mean

0

80/1000

6.9

100

(De-ionised water)

57/1000

1250

51/1000

5.5

80

58/1000

2500

41/1000

4.3

62

45/1000

5000

40/1000

3.8

55

35/1000

 

Mitotic index data - first test, 21 hour harvest -continued

With S9 mix

Concentration of test item 24.9% (µg/mL)

Mitotic index

Relative mitotic index (%)

Incidence

% Mean

0

51/1000

5.5

100

(De-ionised water)

58/1000

1250

46/1000

3.8

69

30/1000

2500

40/1000

3.9

71

37/1000

5000

43/1000

5.9

107

75/1000

 


TABLE 2

Metaphase analysis data - first test, 21 hour harvest

Without S9 mix

Concentration of test item 24.9% (µg/mL)

No. cells examined

Aberrations

No of aberrant cells

Relative MI (%)

Chromatid type

Chromosome type

others

Gaps

Exc. gaps

Mean %

Inc. gap

Mean %

ctb

cte

csb

cse

ctg

csg

0

100

1

 

1

 

 

1

 

2

2.0

3

2.5

100

(De-ionised water)

100

2

 

 

 

 

 

 

2

 

2

 

 

1250

100

1

 

 

 

 

1

 

1

0.5

2

1.0

80

100

 

 

 

 

 

 

 

0

 

0

 

 

2500

100

1

 

 

 

 

2

 

1

1.0

1

2.0

62

100

1

 

 

 

 

 

 

1

 

3

 

 

5000

100

4

 

 

 

 

 

 

3

3.5

3

3.5

55

100

3

 

1

 

 

 

 

4

 

4

 

 

0.8

50

5

3

2

 

 

1

 

10

22.0

10

23.0

-

Mitomycin C

50

15

2

1

 

 

 

 

12

***

12

***

 

 

Metaphase analysis data - first test, 21 hour harvest - continued

With S9 mix

Concentration of test item 24.9% (µg/mL))

No. cells examined

Aberrations

No of aberrant cells

Relative MI (%)

Chromatid type

Chromosome type

others

Gaps

Exc. gaps

Mean %

Inc. gap

Mean %

ctb

cte

csb

cse

ctg

csg

0

100

 

 

 

 

 

 

 

0

1.0

0

1.0

100

(De-ionised water)

100

2

 

 

 

 

 

 

2

 

2

 

 

1250

100

1

 

 

 

 

 

 

1

1.0

1

1.0

69

100

1

 

 

 

 

 

 

1

 

1

 

 

2500

100

 

 

 

 

 

 

 

0

2.0

0

2.5

71

100

3

 

1

 

 

1

 

4

 

5

 

 

5000

100

 

 

 

 

 

 

 

0

0.5

0

0.5

107

100

1

 

 

 

 

 

 

1

 

1

 

 

25

50

16

1

3

 

 

 

 

13

23.0

13

23.0

-

Cyclophosphamide

50

15

 

1

 

 

 

 

10

***

10

***

 

ctb     - Chromatid break                              cte - Chromatid exchange

csb    - Chromosome break                          cse - Chromosome exchange

ctg     - Chromatid gap                                  csg - Chromosome gap
others
  - Cells with greater than 10 aberrations, pulverised cells and pulverised chromosomes

 

***      - P<0.001

Otherwise - P>0.01


TABLE 3

Mitotic index data - second test, 21 hour harvest

Without S9 mix

Concentration of test item 24.9% (µg/mL)

Mitotic index

Relative mitotic index (%)

Incidence

% Mean

0

78/1000

7.0

100

(De-ionised water)

61/1000

1250

60/1000

6.1

87

62/1000

2500

66/1000

6.1

87

56/1000

5000

44/1000

4.6

66

48/1000

 

 

Mitotic index data – second test, 21 hour harvest -continued

With S9 mix

Concentration of test item 24.9% (µg/mL)

Mitotic index

Relative mitotic index (%)

Incidence

% Mean

0

78/1000

7.8

100

(De-ionised water)

78/1000

1250

82/1000

7.6

97

70/1000

2500

90/1000

8.8

113

85/1000

5000

76/1000

7.8

100

80/1000

 


TABLE 4

Metaphase analysis data - second test, 21 hour harvest

Without S9 mix

Concentration of test item 24.9% (µg/mL)

No. cells examined

Aberrations

No of aberrant cells

Relative MI (%)

Chromatid type

Chromosome type

others

Gaps

Exc. gaps

Mean %

Inc. gap

Mean %

ctb

cte

csb

cse

ctg

csg

0

100

 

 

 

 

 

 

 

0

0.5

0

0.5

100

(De-ionised water)

100

 

1

 

 

 

 

 

1

 

1

 

 

1250

100

1

 

 

 

 

 

 

1

0.5

1

0.5

87

100

 

 

 

 

 

 

 

0

 

0

 

 

2500

100

1

 

 

 

 

 

 

1

0.5

1

1.0

87

100

 

 

 

 

 

1

 

0

 

1

 

 

5000

100

 

 

 

 

 

 

 

0

0.0

0

0.0

66

100

 

 

 

 

 

 

 

0

 

0

 

 

0.8

100

9

1

10

 

 

1

 

17

21.5

17

22.5

-

Mitomycin C

50

6

 

16

 

 

2

 

13

***

14

***

 

 

Metaphase analysis data - second test, 21 hour harvest - continued

With S9 mix

Concentration of test item 24.9% (µg/mL)

No. cells examined

Aberrations

No of aberrant cells

Relative MI (%)

Chromatid type

Chromosome type

others

Gaps

Exc. gaps

Mean %

Inc. gap

Mean %

ctb

cte

csb

cse

ctg

csg

0

100

2

 

 

 

 

 

 

2

1.0

2

1.0

100

(De-ionised water)

100

 

 

 

 

 

 

 

0

 

0

 

 

1250

100

 

 

 

 

 

 

 

0

0.0

0

0.0

97

100

 

 

 

 

 

 

 

0

 

0

 

 

2500

100

 

 

 

 

 

 

 

0

0.0

0

0.0

113

100

 

 

 

 

 

 

 

0

 

0

 

 

5000

100

 

 

 

 

 

 

 

0

0.5

0

0.5

100

100

1

 

 

 

 

 

 

1

 

1

 

 

25

100

13

 

1

 

 

 

 

11

10.0

11

10.0

-

Cyclophosphamide

100

10

1

 

 

 

 

 

9

***

9

***

 

ctb     - Chromatid break                              cte - Chromatid exchange

csb    - Chromosome break                          cse - Chromosome exchange

ctg     - Chromatid gap                                  csg - Chromosome gap
others
  - Cells with greater than 10 aberrations, pulverised cells and pulverised chromosomes

 

***      - P<0.001

Otherwise - P>0.01


TABLE 5

Mitotic index data - second test, 45 hour harvest

Without S9 mix

Concentration of test item 24.9% (µg/mL)

Mitotic index

Relative mitotic index (%)

Incidence

% Mean

0

66/1000

6.3

100

(De-ionised water)

60/1000

1250

73/1000

7.8

124

82/1000

2500

79/1000

7.4

117

68/1000

5000

64/1000

5.9

94

54/1000

 

 

Mitotic index data – second test, 45 hour harvest -continued

With S9 mix

Concentration of test item 24.9% (µg/mL)

Mitotic index

Relative mitotic index (%)

Incidence

% Mean

0

99/1000

9.3

100

(De-ionised water)

86/1000

1250

89/1000

8.6

92

83/1000

2500

93/1000

8.7

94

81/1000

5000

102/1000

9.7

104

91/1000

 


TABLE 6

Metaphase analysis data - second test, 45 hour harvest

Without S9 mix

Concentration of test item 24.9% (µg/mL)

No. cells examined

Aberrations

No of aberrant cells

Relative MI (%)

Chromatid type

Chromosome type

others

Gaps

Exc. gaps

Mean %

Inc. gap

Mean %

ctb

cte

csb

cse

ctg

csg

0

100

1

 

 

 

 

 

 

1

0.5

1

0.5

100

(De-ionised water)

100

 

 

 

 

 

 

 

0

 

0

 

 

5000

100

 

 

 

 

 

 

 

0

1.0

0

1.0

94

100

1

 

1

 

 

 

 

2

 

2

 

 

 

Metaphase analysis data - second test, 45 hour harvest - continued

With S9 mix

Concentration of test item 24.9% (µg/mL)

No. cells examined

Aberrations

No of aberrant cells

Relative MI (%)

Chromatid type

Chromosome type

others

Gaps

Exc. gaps

Mean %

Inc. gap

Mean %

ctb

cte

csb

cse

ctg

csg

0

100

 

 

 

 

 

 

 

0

0.5

0

0.5

100

(De-ionised water)

100

 

2

 

 

 

 

 

1

 

1

 

104

5000

100

 

 

 

 

 

 

 

0

0.0

0

0.0

 

100

 

 

 

 

 

 

 

0

 

0

 

 

ctb     - Chromatid break                              cte - Chromatid exchange

csb    - Chromosome break                          cse - Chromosome exchange

ctg     - Chromatid gap                                  csg - Chromosome gap
others
  - Cells with greater than 10 aberrations, pulverised cells and pulverised chromosomes

 

***      - P<0.001

Otherwise - P>0.01


 

Historical control data for human lymphocytes in whole blood culture,

treated with solvent controls from February 1995 until January 1997.

 

Aberrant cells

 

 

Total no. of cells

Without gaps

No. of aberrant cells

% mean

Range of means (%)

Upper 95% limit (%)

min

max

-S-9

55980

317

0.57

0

3.68

1.5

+S-9

57600

304

0.53

0

3.0

1.5

 

 

 

Total no. of cells

With gaps

No. of aberrant cells

% mean

Range of means (%)

Upper 95% limit (%)

min

max

-S-9

55980

403

0.72

0

4.47

2.0

+S-9

57600

391

0.68

0

3.0

2.0

 


 

Conclusions:
The test solution, containing 24.9% Sodium ethylenesulphonate, did not induce chomosomal aberrations over a background when tested up to cytotoxic concentrations. Under the conditions of the present mammalian chromosome aberraton test the maximum test concentration of Sodium ethylenesulphonate was 1250 µg/mL (5000 µg/mL of a 24.9 % aqueous Sodium ethylenesulphonate solution). This concentration is in the range of the maximum test concentration of 1300 µg/mL (0.01M) recommended according to the guideline.
Executive summary:

A test solution, containing 24.9 % Sodium ethylenesulphonate, was assessed to its ability to induce chromosomal aberrations in human lymphocytes according to OECD guideline 473 and GLP (Huntingdon Life Science Ltd. (a), 1997). In two independent experiments at least 100 metaphase figures were analysed at concentrations of 1250, 2500 and 5000 µg/mL of the 24.9 % Sodium ethylenesulphonate solution. In the current version of the OECD TG 473 (2014) it is recommended to analyse at least 300 well-spread metaphases per concentration and control to conclude a test item as clearly negative. However, in the OECD TG 473 from 1983 it was sufficient to score 100 metaphases per concentration to come to a conclusion on clastogenicity. 

A mitotic index (MI) of 55 -66% was observed after treatment with the highest concentration of test item in the absence of S-9 mix. No significant increases in mutant frequency were observed in cultures treated with the 24.9% Sodium ethylenesulphonate solution in any of the tests either in the absence or the presence of S-9 mix. The positive controls induced highly significant increases in mutant frequency in all of the tests in both the absence and presence of S-9 mix thus demonstrating adequate sensitivity of the test system. In conclusion, the test solution, containing 24.9% Sodium ethylenesulphonate, did not induce chromosomal aberrations over background when tested up to cytotoxic concentrations. Under the conditions of the present mammalian chromosome aberration test the maximum test concentration of Sodium ethylenesulphonate was 1250 µg/mL (5000 µg/mL of a 24.9% aqueous Sodium ethylenesulphonate solution). This concentration is in the range of the maximum test concentration of 1300 µg/mL (0.01M) recommended according to the guideline.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test

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 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.

In vitro mammalian chromosome aberration test

A test solution, containing 24.9 % Sodium ethylenesulphonate, was assessed to its ability to induce chromosomal aberrations in human lymphocytes according to OECD guideline 473 and GLP (Huntingdon Life Science Ltd. (a), 1997). In two independent experiments at least 100 metaphase figures were analysed at concentrations of 1250, 2500 and 5000 µg/mL of the 24.9 % Sodium ethylenesulphonate solution. In the current version of the OECD TG 473 (2014) it is recommended to analyse at least 300 well-spread metaphases per concentration and control to conclude a test item as clearly negative. However, in the OECD TG 473 from 1983 it was sufficient to score 100 metaphases per concentration to come to a conclusion on clastogenicity. 

A mitotic index (MI) of 55 -66% was observed after treatment with the highest concentration of test item in the absence of S-9 mix. No significant increases in mutant frequency were observed in cultures treated with the 24.9% Sodium ethylenesulphonate solution in any of the tests either in the absence or the presence of S-9 mix. The positive controls induced highly significant increases in mutant frequency in all of the tests in both the absence and presence of S-9 mix thus demonstrating adequate sensitivity of the test system. In conclusion, the test solution, containing 24.9% Sodium ethylenesulphonate, did not induce chromosomal aberrations over background when tested up to cytotoxic concentrations. Under the conditions of the present mammalian chromosome aberration test the maximum test concentration of Sodium ethylenesulphonate was 1250 µg/mL (5000 µg/mL of a 24.9% aqueous Sodium ethylenesulphonate solution). This concentration is in the range of the maximum test concentration of 1300 µg/mL (0.01M) recommended according to the guideline.

 

In vitro mammalian cell gene mutation test

A test solution, containing 24.9 % Sodium ethylenesulphonate, was tested in a mammalian gene mutation test in CHO cells (HPRT assay) according to OECD guideline 476 and GLP (Huntigdon Life Science Ltd. (b), 1997). In two independent experiments concentrations of 1250, 2500 and 5000 µg/mL of the 25 % Sodium ethylenesulphonate solution were tested with and without metabolic activation (S9 -mix). Some increases of pH values and dose related increases in osmolarity were observed in all tests. No significant increases in mutant frequency were observed after treatment with 24.9 % Sodium ethylenesulphonate in either test in the absence and the presence of S-9 mix. Ethyl methanesulphonate and 20-Methylcholanthrene, the positive controls, induced highly significant increases in mutant frequency in both tests. It is concluded that the test solution, containing 24.9 % Sodium ethylenesulphonate, did not demonstrate mutagenic potential in this in vitro gene mutation assay. Under the conditions of the present mammalian gene mutation test the maximum test concentration of Sodium ethylenesulphonate was 1250 µg/mL (5000 µg/mL of a 25 % aqueous Sodium ethylenesulphonate solution). This concentration is in the range of the maximum test concentration of 1300 µg/mL (0.01M) recommended according to the guideline.

In the present three in vitro genotoxicity studies (Ames test, HPRT assay and chromosome aberration test) no mutagenic potential was indicated. Under the conditions of the mammalian chromosome aberraton and gene mutation test the maximum test concentration of Sodium ethylenesulphonate was in the range of the maximum test concentration recommended according to the guideline. In the Ames test the maximum test concentration of Sodium ethylenesulphonate was minimally lower (3000 µg/plate instead of 5000 µg/plate) than specified in the guideline. However, since the result of the Ames test was in line with the mammalian chromosome aberration and gene mutation test and overall no signs of genotoxicity were indicated, it is concluded that Sodium ethylenesulphonate will not demonstrate mutagenicity in the Ames test in concentrations up to 5000 µg/plate. Of note, in a QSAR analysis by means of the software toxtree v. 2.5.0 (Benigni/Bossa rulebase for mutagenicity and carcinogenicity) Sodium ethylenesulphonate was no potenital S.typhimurium TA 100 mutagen based on available structure attributes.


Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available results Sodium ethylenesulphonate was not classified and labelled for genotoxicity according to Regulation (EC) No 1272/2008 (CLP).