Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Oct - 04 Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Remarks:
GLP-Landesleitstelle Bayern, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
His operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced male rats
Test concentrations with justification for top dose:
Pre-experiment: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Main experiment: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Controls
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 10 µg/plate for TA 98, 40 µg/plate for TA 1537, without metabolic activation; 2-aminoanthracene, 2.5 µg/plate for TA 98, TA 100, TA 1535, TA 1537, 10 µg/plate for TA 102, with metabolic activation
Remarks:
The profiency of S9-mix was characterised with benzo(a)pyrene (mutagen that requires metabolic activation by microsomal enzymes) before the experiment.
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1: plate incorporation; Experiemnt 2: pre-incubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 – 72 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: the diminution of the background lawn or a significant reduction in the number of revertants in solvent control.
Evaluation criteria:
Negative control data within historical range. Positive controls show a distinct enhancement of revertant rates over the control plate. At least five different concentrations of each tester strain are analyzable.
Positive response: a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as:
- if in tester strains TA 98 and TA 100 the number of reversions is at least and twice as high
- if in tester strains TA 102 the number of reversions is at least 1.5-fold higher
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
Negative response: neither a dose related increase in the number of revertants nor a reproducible biologically relevant increase.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: In the pre-experiment no toxic effects were observed at any concentration of the substance.

HISTORICAL CONTROL DATA
- Positive historical control data: Positive controls met the criteria of validity.
- Negative (solvent/vehicle) historical control data: Negative controls were within historical norms.

Any other information on results incl. tables

 Tester Strain: TA 98  Experiment: 1

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest.

 

16

 

 

19

 

 

 

 

14

19

7.6

22

32

20.6

0.8

1.7

28

 

 

56

 

 

 

 

DMSO

 

17

 

 

20

 

 

 

 

18

24

10.7

23

19

4.6

1.0

1.0

36

 

 

14

 

 

 

 

Test Item

31.6 µg

18

 

 

13

 

 

 

 

22

23

6.1

17

17

3.5

1.0

0.9

30

 

 

20

 

 

 

 

Test Item

100 µg

55

 

 

33

 

 

 

 

30

33

20.2

20

24

7.8

1.4

1.3

15

 

 

19

 

 

 

 

Test Item

316 µg

24

 

 

29

 

 

 

 

22

23

1.0

23

27

3.5

1.0

1.4

23

 

 

29

 

 

 

 

Test Item

1000 µg

20

 

 

15

 

 

 

 

19

17

4.4

22

18

3.5

0.7

1.0

12

 

 

18

 

 

 

 

Test Item

2500 µg

27

 

 

34

 

 

 

 

26

27

0.6

28

29

4.6

1.1

1.5

27

 

 

25

 

 

 

 

Test Item

5000 µg

12 P

 

 

42 P

 

 

 

 

14 P

14

1.5

20 P

28

12.2

0.6

1.5

15 P

 

 

22 P

 

 

 

 

4-NOPD

10 µg

461

 

 

/

 

 

 

 

543

515

46.8

/

/

/

21.8

/

541

 

 

/

 

 

 

 

2-AA

2.5 µg

/

 

 

1957

 

 

 

 

/

/

/

2402

2273

275.2

/

119.6

/

 

 

2460

 

 

 

 

SD: Standard-deviation P: Precipitation

B: Background lawn reduced C: Contamination

N: No background lawn

Mutationfactor = mean revertants (test item)/mean revertants (vehicle control)

 

Tester Strain: TA 100  Experiment: 1

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest.

 

92

 

 

86

 

 

 

 

87

92

5.0

80

85

4.2

1.2

1.1

97

 

 

88

 

 

 

 

DMSO

 

92

 

 

81

 

 

 

 

75

77

14.6

81

75

11.0

1.0

1.0

63

 

 

62

 

 

 

 

Test Item

31.6 µg

58

 

 

80

 

 

 

 

81

66

13.0

57

69

11.6

0.9

0.9

59

 

 

71

 

 

 

 

Test Item

100 µg

62

 

 

77

 

 

 

 

158

107

48.3

98

83

13.1

1.4

1.1

100

 

 

74

 

 

 

 

Test Item

316 µg

65

 

 

82

 

 

 

 

63

57

11.6

74

89

20.0

0.7

1.2

44

 

 

112

 

 

 

 

Test Item

1000 µg

70

 

 

66

 

 

 

 

52

61

9.0

88

79

11.5

0.8

1.1

61

 

 

83

 

 

 

 

Test Item

2500 µg

91

 

 

60

 

 

 

 

72

83

9.7

95

83

20.2

1.1

1.1

85

 

 

95

 

 

 

 

Test Item

5000 µg

69 P

 

 

82 P

 

 

 

 

63 P

74

14.2

66 P

77

9.9

1.0

1.0

90 P

 

 

84 P

 

 

 

 

NaN3

10 µg

796

 

 

/

 

 

 

 

845

837

37.2

/

/

/

10.9

/

869

 

 

/

 

 

 

 

2-AA

2.5 µg

/

 

 

2252

 

 

 

 

/

/

/

2181

2261

84.3

/

30.3

/

 

 

2349

 

 

 

 

 

Tester Strain: TA 1535  Experiment: 1

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest.

 

12

 

 

17

 

 

 

 

14

14

2.5

14

17

3.5

0.9

1.1

17

 

 

21

 

 

 

 

DMSO

 

20

 

 

14

 

 

 

 

12

16

4.0

15

16

3.2

1.0

1.0

17

 

 

20

 

 

 

 

Test Item

31.6 µg

14

 

 

11

 

 

 

 

15

13

2.1

10

13

3.8

0.8

0.8

11

 

 

17

 

 

 

 

Test Item

100 µg

13

 

 

14

 

 

 

 

14

13

1.5

17

14

2.5

0.8

0.9

11

 

 

12

 

 

 

 

Test Item

316 µg

15

 

 

15

 

 

 

 

17

16

1.0

20

16

4.0

1.0

1.0

16

 

 

12

 

 

 

 

Test Item

1000 µg

15

 

 

12

 

 

 

 

15

15

0.0

16

17

5.0

0.9

1.0

15

 

 

22

 

 

 

 

Test Item

2500 µg

12

 

 

16

 

 

 

 

12

14

2.9

16

14

3.5

0.8

0.9

17

 

 

10

 

 

 

 

Test Item

5000 µg

14 P

 

 

16 P

 

 

 

 

15 P

14

0.6

10 P

14

3.5

0.9

0.9

14 P

 

 

16 P

 

 

 

 

NaN3

10 µg

1038

 

 

/

 

 

 

 

1205

1147

94.7

/

/

/

70.2

/

1199

 

 

/

 

 

 

 

2-AA

2.5 µg

/

 

 

127

 

 

 

 

/

/

/

127

147

34.6

/

9.0

/

 

 

187

 

 

 

 

 

Tester Strain: TA 1537  Experiment: 1

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest.

 

8

 

 

9

 

 

 

 

10

9

1.2

9

10

2.3

0.9

1.1

8

 

 

13

 

 

 

 

DMSO

 

11

 

 

9

 

 

 

 

7

10

2.3

12

9

2.5

1.0

1.0

11

 

 

7

 

 

 

 

Test Item

31.6 µg

7

 

 

8

 

 

 

 

13

10

3.0

9

8

1.5

1.0

0.8

10

 

 

6

 

 

 

 

Test Item

100 µg

14

 

 

9

 

 

 

 

14

12

3.5

9

9

0.6

1.2

0.9

8

 

 

8

 

 

 

 

Test Item

316 µg

6

 

 

10

 

 

 

 

11

9

2.6

8

8

1.5

0.9

0.9

10

 

 

7

 

 

 

 

Test Item

1000 µg

10

 

 

8

 

 

 

 

5

8

2.5

9

9

0.6

0.8

0.9

8

 

 

9

 

 

 

 

Test Item

2500 µg

7

 

 

8

 

 

 

 

10

8

1.5

9

8

1.0

0.9

0.9

8

 

 

7

 

 

 

 

Test Item

5000 µg

6 P

 

 

7 P

 

 

 

 

9 P

8

1.5

6 P

7

1.0

0.8

0.8

8 P

 

 

8 P

 

 

 

 

4-NOPD

40 µg

160

 

 

/

 

 

 

 

124

141

18.1

/

/

/

14.6

/

139

 

 

/

 

 

 

 

2-AA

2.5 µg

/

 

 

181

 

 

 

 

/

/

/

184

179

6.8

/

19.1

/

 

 

171

 

 

 

 

 

Tester Strain: TA 102  Experiment: 1

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest.

 

267

 

 

368

 

 

 

 

285

280

11.7

398

362

39.3

1.2

1.1

289

 

 

320

 

 

 

 

DMSO

 

227

 

 

300

 

 

 

 

255

242

14.0

345

318

24.0

1.0

1.0

243

 

 

308

 

 

 

 

Test Item

31.6 µg

231

 

 

288

 

 

 

 

240

237

5.2

288

288

0.0

1.0

0.9

240

 

 

288

 

 

 

 

Test Item

100 µg

351

 

 

235

 

 

 

 

234

261

79.6

243

243

8.5

1.1

0.8

199

 

 

252

 

 

 

 

Test Item

316 µg

214

 

 

256

 

 

 

 

215

215

0.6

231

246

13.1

0.9

0.8

215

 

 

250

 

 

 

 

Test Item

1000 µg

248

 

 

250

 

 

 

 

249

247

3.2

321

266

49.0

1.0

0.8

243

 

 

227

 

 

 

 

Test Item

2500 µg

281

 

 

222

 

 

 

 

271

272

8.1

220

221

1.2

1.1

0.7

265

 

 

222

 

 

 

 

Test Item

5000 µg

165 P

 

 

213 P

 

 

 

 

190 P

210

58.2

267 P

245

28.5

0.9

0.8

276 P

 

 

256 P

 

 

 

 

MMS

1 µL

1212

 

 

/

 

 

 

 

1169

1298

186.9

/

/

/

5.4

/

1512

 

 

/

 

 

 

 

2-AA

10 µg

/

 

 

847

 

 

 

 

/

/

/

807

835

24.7

/

2.6

/

 

 

852

 

 

 

 

 

Tester Strain: TA 98  Experiment: 2

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest.

 

47

 

 

22

 

 

 

 

51

48

3.1

37

30

7.5

1.1

1.2

45

 

 

30

 

 

 

 

DMSO

 

47

 

 

26

 

 

 

 

38

43

4.7

30

25

5.0

1.0

1.0

45

 

 

20

 

 

 

 

Test Item

31.6 µg

57

 

 

30

 

 

 

 

57

57

0.6

20

26

5.3

1.3

1.0

58

 

 

28

 

 

 

 

Test Item

100 µg

51

 

 

18

 

 

 

 

52

50

2.6

30

25

6.4

1.2

1.0

47

 

 

28

 

 

 

 

Test Item

316 µg

46

 

 

23

 

 

 

 

33

37

7.5

24

22

2.1

0.9

0.9

33

 

 

20

 

 

 

 

Test Item

1000 µg

53

 

 

33

 

 

 

 

44

43

11.1

24

29

4.5

1.0

1.1

31

 

 

29

 

 

 

 

Test Item

2500 µg

52

 

 

38

 

 

 

 

53

52

1.5

24

31

7.0

1.2

1.2

50

 

 

32

 

 

 

 

Test Item

5000 µg

51 P

 

 

28 P

 

 

 

 

50 P

50

0.6

31 P

31

3.5

1.2

1.2

50 P

 

 

35 P

 

 

 

 

4-NOPD

10 µg

296

 

 

/

 

 

 

 

312

302

8.7

/

/

/

7.0

/

298

 

 

/

 

 

 

 

2-AA

2.5 µg

/

 

 

1529

 

 

 

 

/

/

/

1861

1971

506.0

/

77.8

/

 

 

2523

 

 

 

 

 

Tester Strain: TA 100  Experiment: 2

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest.

 

76

 

 

88

 

 

 

 

101

90

12.9

88

90

4.0

1.0

1.1

94

 

 

95

 

 

 

 

DMSO

 

99

 

 

74

 

 

 

 

95

94

5.0

93

81

10.2

1.0

1.0

89

 

 

77

 

 

 

 

Test Item

31.6 µg

92

 

 

86

 

 

 

 

90

92

2.5

76

80

5.5

1.0

1.0

95

 

 

77

 

 

 

 

Test Item

100 µg

86

 

 

76

 

 

 

 

90

85

5.0

92

83

8.1

0.9

1.0

80

 

 

82

 

 

 

 

Test Item

316 µg

102

 

 

88

 

 

 

 

79

87

13.3

89

88

1.5

0.9

1.1

79

 

 

86

 

 

 

 

Test Item

1000 µg

78

 

 

93

 

 

 

 

83

77

6.0

113

101

10.6

0.8

1.2

71

 

 

97

 

 

 

 

Test Item

2500 µg

76

 

 

119

 

 

 

 

85

85

9.0

99

109

10.0

0.9

1.3

94

 

 

110

 

 

 

 

Test Item

5000 µg

112 P

 

 

77 P

 

 

 

 

81 P

97

15.6

84 P

82

4.0

1.0

1.0

99 P

 

 

84 P

 

 

 

 

NaN3

10 µg

925

 

 

/

 

 

 

 

947

924

24.0

/

/

/

9.8

/

899

 

 

/

 

 

 

 

2-AA

2.5 µg

/

 

 

1508

 

 

 

 

/

/

/

1572

1485

101.0

/

18.3

/

 

 

1374

 

 

 

 

 

Tester Strain: TA 1535  Experiment: 2

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest.

 

24

 

 

18

 

 

 

 

22

22

1.5

15

18

3.0

1.0

0.9

21

 

 

21

 

 

 

 

DMSO

 

23

 

 

17

 

 

 

 

24

22

2.1

18

19

2.6

1.0

1.0

20

 

 

22

 

 

 

 

Test Item

31.6 µg

18

 

 

20

 

 

 

 

30

23

6.2

23

21

1.5

1.0

1.1

21

 

 

21

 

 

 

 

Test Item

100 µg

26

 

 

17

 

 

 

 

21

23

2.5

18

18

0.6

1.0

0.9

23

 

 

18

 

 

 

 

Test Item

316 µg

25

 

 

26

 

 

 

 

24

23

2.6

21

22

3.6

1.0

1.2

20

 

 

19

 

 

 

 

Test Item

1000 µg

26

 

 

20

 

 

 

 

27

26

0.6

18

19

1.2

1.2

1.0

26

 

 

18

 

 

 

 

Test Item

2500 µg

22

 

 

18

 

 

 

 

25

26

5.1

19

19

1.0

1.2

1.0

32

 

 

20

 

 

 

 

Test Item

5000 µg

31 P

 

 

17 P

 

 

 

 

25 P

25

5.5

18 P

17

0.6

1.1

0.9

20 P

 

 

17 P

 

 

 

 

NaN3

10 µg

1231

 

 

/

 

 

 

 

1272

1211

72.5

/

/

/

54.2

/

1131

 

 

/

 

 

 

 

2-AA

2.5 µg

/

 

 

254

 

 

 

 

/

/

/

200

226

27.1

/

11.9

/

 

 

223

 

 

 

 

 

Tester Strain: TA 1537  Experiment: 2

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest.

 

17

 

 

15

 

 

 

 

12

15

2.5

18

16

1.5

0.8

1.0

15

 

 

16

 

 

 

 

DMSO

 

17

 

 

17

 

 

 

 

19

18

1.0

15

16

1.0

1.0

1.0

18

 

 

16

 

 

 

 

Test Item

31.6 µg

18

 

 

15

 

 

 

 

16

18

1.5

14

15

1.5

1.0

1.0

19

 

 

17

 

 

 

 

Test Item

100 µg

17

 

 

17

 

 

 

 

15

16

1.0

18

17

1.5

0.9

1.0

16

 

 

15

 

 

 

 

Test Item

316 µg

17

 

 

16

 

 

 

 

20

18

1.7

17

16

1.0

1.0

1.0

17

 

 

15

 

 

 

 

Test Item

1000 µg

15

 

 

16

 

 

 

 

14

15

1.0

18

16

2.0

0.8

1.0

16

 

 

14

 

 

 

 

Test Item

2500 µg

17

 

 

17

 

 

 

 

15

17

1.5

18

18

1.0

0.9

1.1

18

 

 

19

 

 

 

 

Test Item

5000 µg

12 P

 

 

14 P

 

 

 

 

11 P

11

1.5

13 P

14

1.0

0.6

0.9

9 P

 

 

15 P

 

 

 

 

4-NOPD

40 µg

164

 

 

/

 

 

 

 

146

152

10.4

/

/

/

8.4

/

146

 

 

/

 

 

 

 

2-AA

2.5 µg

/

 

 

251

 

 

 

 

/

/

/

249

245

8.1

/

15.3

/

 

 

236

 

 

 

 

 

Tester Strain: TA 102  Experiment: 2

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest.

 

305

 

 

385

 

 

 

 

329

317

12.0

386

381

8.4

1.0

1.0

318

 

 

371

 

 

 

 

DMSO

 

295

 

 

387

 

 

 

 

313

313

18.0

357

385

26.6

1.0

1.0

331

 

 

410

 

 

 

 

Test Item

31.6 µg

292

 

 

375

 

 

 

 

299

295

3.6

379

378

2.6

0.9

1.0

294

 

 

380

 

 

 

 

Test Item

100 µg

302

 

 

380

 

 

 

 

313

312

9.5

354

365

13.3

1.0

0.9

321

 

 

362

 

 

 

 

Test Item

316 µg

327

 

 

375

 

 

 

 

307

316

10.1

365

367

7.6

1.0

1.0

314

 

 

360

 

 

 

 

Test Item

1000 µg

274

 

 

405

 

 

 

 

318

302

24.1

360

374

27.2

1.0

1.0

313

 

 

356

 

 

 

 

Test Item

2500 µg

250

 

 

343

 

 

 

 

276

276

26.0

366

363

18.2

0.9

0.9

302

 

 

379

 

 

 

 

Test Item

5000 µg

308 P

 

 

301 P

 

 

 

 

304 P

302

6.7

341 P

333

29.3

1.0

0.9

295 P

 

 

358 P

 

 

 

 

MMS

1 µL

1129

 

 

/

 

 

 

 

1315

1239

97.5

/

/

/

4.0

/

1273

 

 

/

 

 

 

 

2-AA

10 µg

/

 

 

830

 

 

 

 

/

/

/

911

899

64.3

/

2.3

/

 

 

957

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of the conducted test the substance was not mutagenic in any of the five bacterial strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) tested with and without metabolic activation.