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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:
25 July - 11 Aug 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from livers treated with Aroclor 1254 (500 mg/kg bw)
Test concentrations with justification for top dose:
Experiment 1a, plate incorporation method
S. typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 1535: 5000, 1500, 500, 150 and 50 μg/plate with and without metabolic activation

Experiment 1b, plate incorporation method
S. typhimurium TA102: 5000 μg/plate, 1500, 500, 150, 50, 15 and 5 μg/plate with and without metabolic activation

Experiment 2, pre-incubation method
S. typhimurium TA 97a, TA 98, TA 100 and TA 1535): 5000, 2500, 1250, 625, 313, 156 and 78 μg/plate with and without metabolic activation
S. typhimurium TA 102: 500, 250, 125, 62.5, 31.3, 15.6 and 7.8 μg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test item was not sufficiently soluble in demineralized water, dimethyl sulfoxide (DMSO), ethanol and tetrahydrofurane. Based on these results, ethanol was used as solvent in all experiments, because the test item showed in ethanol the best result for handling in the experiments. The test suspension was homogeneous and pipettable. In addition, ethanol does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-Amino-anthracene (2-AA): +S9: 1 µg/plate (in DMSO) for TA 97a, TA 100, TA 102, TA 1535; 4-Nitro-1,2-phenylenediamine (NPD): -S9: 20 µg/plate (in DMSO) for TA 97a, TA 98, TA 102
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, experiments 1a and 1b) and pre-incubation (experiment 2)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments (experiments 1a/1b and 2)

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn and of the number of revertants
Evaluation criteria:
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.

A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
Statistics:
The colonies were counted visually and the numbers were recorded. A validated spreadsheet was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA 97a, TA 98, TA 100 and 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 applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
-/+S9: 5000 and 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed in any experiment at any concentration

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) : data is summarised in "Any other information on results incl. tables"

Any other information on results incl. tables

Table 1: Mean Revertants Experiment 1a (all strains tested)

 

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin. water

Mean

85

80

29

34

73

93

307

377

27

26

sd

6.9

12.7

6.4

9.8

5.9

13.6

48.4

61.1

1.5

7.0

DMSO

Mean

98

97

35

38

78

78

320

388

22

24

sd

19.6

4.0

5.9

6.7

5.5

3.5

31.2

28.8

3.2

4.0

Ethanol

Mean

76

83

24

26

72

72

295

313

22

22

sd

11.4

7.0

6.4

6.0

10.6

8.6

96.4

37.2

3.8

5.9

Positive Controls*

Mean

637

781

383

86

451

1528

1411

1472

323

183

sd

99.4

64.2

27.2

8.1

22.7

424.2

100.0

36.7

56.6

28.4

f(I)

6.50

8.05

10.94

2.26

6.18

19.59

4.41

3.79

11.96

7.63

5000

µg/plate

Mean

99

93

22

26

76

82

79

48

20

19

sd

18.9

15.1

3.1

5.9

4.0

18.9

12.6

14.5

2.1

1.5

f(I)

1.30

1.12

0.92

1.00

1.06

1.14

0.27

0.15

0.91

0.86

1500

µg/plate

Mean

79

98

24

21

75

74

66

95

17

19

sd

16.2

9.0

4.0

6.4

11.5

8.0

19.9

23.1

1.5

2.5

f(I)

1.04

1.18

1.00

0.81

1.04

1.03

0.22

0.30

0.77

0.86

500

µg/plate

Mean

90

94

26

24

82

77

285

331

23

19

sd

22.0

15.9

5.5

2.9

6.0

13.6

60.2

10.1

4.5

3.2

f(I)

1.18

1.13

1.08

0.92

1.14

1.07

0.97

1.06

1.05

0.86

150

µg/plate

Mean

79

84

26

20

66

76

281

384

18

23

sd

15.0

20.3

6.9

2.6

5.5

10.7

89.3

10.6

3.6

0.6

f(I)

1.04

1.01

1.08

0.77

0.92

1.06

0.95

1.23

0.82

1.05

 

50 µg/plate

Mean

88

89

25

32

83

64

291

355

22

24

sd

5.2

6.4

4.0

1.2

14.5

6.2

63.8

64.7

4.6

4.2

f(I)

1.16

1.07

1.04

1.23

1.15

0.89

0.99

1.13

1.00

1.09

f(I) = increase factor

* Different positive controls were used

Table 2: Mean Revertants Experiment 1b (strain TA 102)

 

Strain

TA102

Induction

-S9

+S9

Demin. water

Mean

356

345

sd

62.9

72.6

DMSO

Mean

257

324

sd

26.6

20.8

Ethanol

Mean

292

258

sd

58.9

24.0

Positive Controls*

Mean

689

1083

sd

60.2

400.0

f(I)

2.68

3.34

5000

µg/plate

Mean

12

4

sd

1.0

1.5

f(I)

0.04

0.02

1500

µg/plate

Mean

80

56

sd

10.0

9.0

f(I)

0.27

0.22

500

µg/plate

Mean

315

268

sd

46.4

58.9

f(I)

1.08

1.04

150

µg/plate

Mean

284

284

sd

52.3

52.0

f(I)

0.97

1.10

 

50 µg/plate

Mean

355

292

sd

50.0

34.6

f(I)

1.22

1.13

 

15 µg/plate

Mean

339

349

sd

47.7

120.2

f(I)

1.16

1.35

 

5 µg/plate

Mean

304

287

sd

65.8

33.5

f(I)

1.04

1.11

f(I) = increase factor

* Different positive controls were used

Table 3: Mean Revertants Experiment 2 (strains TA 97a, TA 98, TA 100 and TA 1535)

 

Strain

TA97a

TA98

TA100

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin. water

Mean

64

81

24

28

51

64

22

23

sd

4.0

17.9

4.7

2.1

4.0

6.7

4.2

6.1

DMSO

Mean

82

67

28

34

44

62

24

24

sd

13.7

11.0

8.2

5.6

3.6

7.5

4.4

4.2

Ethanol

Mean

61

87

32

35

40

48

21

23

sd

8.5

14.2

4.0

4.2

1.0

1.5

5.0

4.9

Positive Controls*

Mean

631

237

459

165

429

491

280

141

sd

83.2

59.8

88.1

8.1

113.5

132.8

59.7

9.2

f(I)

7.70

3.54

16.39

4.85

8.41

7.92

12.73

5.88

5000

µg/plate

Mean

71

80

22

29

54

64

15

17

sd

5.7

1.2

7.2

3.0

6.1

8.5

1.7

2.6

f(I)

1.16

0.92

0.69

0.83

1.35

1.33

0.71

0.74

2500

µg/plate

Mean

69

76

25

29

46

73

18

26

sd

2.5

5.0

4.9

1.5

9.1

6.1

2.0

2.5

f(I)

1.13

0.87

0.78

0.83

1.15

1.52

0.86

1.13

1250

µg/plate

Mean

86

103

21

31

52

63

18

23

sd

14.7

19.3

2.1

10.1

7.0

14.5

2.0

5.5

f(I)

1.41

1.18

0.66

0.89

1.30

1.31

0.86

1.00

625

µg/plate

Mean

71

85

26

28

57

68

18

28

sd

12.7

17.2

9.6

4.7

4.6

9.0

1.5

3.5

f(I)

1.16

0.98

0.81

0.80

1.43

1.42

0.86

1.22

313

µg/plate

Mean

66

72

28

25

42

56

17

23

sd

0.0

4.2

5.2

4.5

7.0

5.6

2.3

2.1

f(I)

1.08

0.83

0.88

0.71

1.05

1.17

0.81

1.00

156

µg/plate

Mean

74

95

30

35

55

69

21

26

sd

2.9

4.0

7.2

7.9

8.7

8.1

6.6

4.0

f(I)

1.21

1.09

0.94

1.00

1.38

1.44

1.00

1.13

 

78 µg/plate

Mean

72

74

27

28

37

45

16

23

sd

4.6

6.4

8.0

9.0

7.9

11.4

3.1

2.9

f(I)

1.18

0.85

0.84

0.80

0.93

0.94

0.76

1.00

f(I) = increase factor

* Different positive controls were used

Table 4: Mean Revertants Experiment 2 (strain TA 102)

 

Strain

TA102

Induction

-S9

+S9

Demin. water

Mean

287

283

sd

19.7

28.1

DMSO

Mean

287

268

sd

42.0

38.2

Ethanol

Mean

253

335

sd

42.4

43.9

Positive Controls*

Mean

952

1325

sd

298.2

51.4

f(I)

3.32

4.94

500

µg/plate

Mean

320

332

sd

39.4

35.6

f(I)

1.26

0.99

250

µg/plate

Mean

292

292

sd

13.9

10.6

f(I)

1.15

0.87

125

µg/plate

Mean

301

287

sd

58.0

26.6

f(I)

1.19

0.86

62.5

µg/plate

Mean

271

269

sd

24.1

64.8

f(I)

1.07

0.80

31.3

µg/plate

Mean

276

283

sd

52.9

42.0

f(I)

1.09

0.84

15.6

µg/plate

Mean

289

315

sd

42.8

77.7

f(I)

1.14

0.94

7.8

µg/plate

Mean

280

275

sd

17.4

15.1

f(I)

1.11

0.82

f(I) = increase factor

* Different positive controls were used

Table 5: Historical Data of Spontaneous Revertants and Comparison with Experiments 1a, 1b and 2

 

Strain

 

TA97a

TA98

TA100

TA102

TA1535

Induction

 

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

 

 

H2O de-min.

Mean

92

97

15

18

93

97

279

296

17

17

Min

60

63

6

8

62

66

85

67

6

7

Max

144

138

37

41

141

141

425

511

31

33

SD

19

16

6

6

15

15

65

75

6

6

Exp 1a

85

80

29

34

73

93

307

377

27

26

Exp 1b

n.r.

n.r.

n.r.

n.r.

n.r.

n.r.

356

345

n.r.

n.r.

Exp 2

64

81

24

28

51

64

287

283

22

23

 

 

DMSO

Mean

91

100

15

16

90

93

279

289

17

16

Min

58

70

7

8

60

63

79

80

8

6

Max

135

144

46

36

136

199

393

459

33

31

SD

18

16

7

6

16

19

61

65

6

6

Exp 1a

98

97

35

38

78

78

320

388

22

24

Exp 1b

n.r.

n.r.

n.r.

n.r.

n.r.

n.r.

257

324

n.r.

n.r.

Exp 2

82

67

28

34

44

62

287

268

24

24

 

 

Ethanol

Mean

89

99

16

17

86

90

284

275

16

16

Min

57

65

8

9

61

69

111

141

9

9

Max

181

205

35

40

116

129

368

339

28

29

SD

24

29

7

8

13

16

52

47

6

6

Exp 1a

76

83

24

26

72

72

295

313

22

22

Exp 1b

n.r.

n.r.

n.r.

n.r.

n.r.

n.r.

292

258

n.r.

n.r.

Exp 2

61

87

32

35

40

48

253

335

21

23

 

 

Positive Controls*

Mean

552

501

394

83

513

716

1160

1237

254

115

Min

264

241

100

39

223

273

491

408

55

45

Max

1152

1181

793

333

984

1912

2331

6083

484

712

SD

169

144

143

49

153

290

452

659

89

79

Exp 1a

637

781

383

86

451

1528

1411

1472

323

183

Exp 1b

n.r.

n.r.

n.r.

n.r.

n.r.

n.r.

689

1083

n.r.

n.r.

Exp 2

631

237

459

165

429

491

952

1325

280

141

* Different positive controls were used

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative