Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was considered to be non-mutagenic with and without metabolic activation in bacteria.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 February 2018 - 23 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Batch No.: 000071307
Purity: 69.9%
Species / strain / cell type:
other: S. typhimurium TA 97a, TA 98, TA 100, TA102 and TA 1535,
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9 mix
Test concentrations with justification for top dose:
Concentrations:
- Experiment 1a: 50, 150, 500, 1500 and 5000 µg/plate
- Experiment 1b: TA97a and TA102: 15, 50, 150, 500, 1500 and 5000 µg/plate; TA98 and TA100: 0.5, 1.5, 5, 15, 50, and 150 µg/plate; TA1535: 5, 15, 50, 150, 500 and 1500 µg/plate
- Experiment 2: TA97a and TA102: 39, 78, 156, 313, 625, 1250, 2500 and 5000 µg/plate; TA98 and TA100: 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75 and 150 µg/plate; TA1535: 12, 23, 47, 94, 188, 375, 750 and 1500 µg/plate;
Justification for top dose: tested up to recommended maximum test concentration according to OECD 471 in experiment 1a; due to observed toxicity lower concentrations were chosen for the experiment 1b and 2
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: In a non-GLP pre-test, the solubility of the test item was determined. Based on these results, DMSO was chosen as solvent.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO and demineralised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: Nitrophenylendiamine and 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: experiment 1a and 1b: in agar (plate incorporation); experiment 2: preincubation

DURATION
- Preincubation period: 20-30 minutes, 37 °C
- Exposure duration: 48-72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: determination revertant colony number and
Evaluation criteria:
The mean values and standard deviations of each threefold determination are calculated as well as the increase factor 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 (mean revertants minus mean spontaneous revertants) is given. A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant 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:
Not performed as not mandatory for this test system.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 97
Remarks:
TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated 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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed

Table 1: Mean Revertants Experiment 1a

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin.

water

Mean

69

66

32

27

78

72

225

263

21

19

sd

16.2

6.0

2.6

3.5

15.4

10.0

19.6

56.0

1.5

3.2

DMSO

Mean

69

95

27

28

73

78

225

287

19

21

sd

10.4

14.0

6.0

1.2

12.0

9.5

16.7

18.9

3.8

3.6

Positive
Controls*

Mean

329

657

s.g.

101

323

s.g.

692

727

257

143

sd

99.6

79.4

0.0

24.6

54.5

0.0

36.0

47.4

14.0

23.0

f(I)

4.77

6.92

37.07

3.61

4.14

12.83

3.08

2.53

12.24

6.81

5000 µg/plate

Mean

0

0

0

0

0

0

0

0

0

0

sd

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

f(I)

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

Background lawn

-

-

-

-

-

-

-

-

-

-

1500 µg/plate

Mean

73

79

0

0

27

46

201

256

12

15

sd

10.6

12.6

0.0

0.0

5.9

4.4

32.3

4.0

1.0

4.4

f(I)

1.06

0.83

0.00

0.00

0.37

0.59

0.89

0.89

0.63

0.71

Background lawn

++

++

++

++

++

++

++

++

++

++

500 µg/plate

Mean

89

73

1

2

25

35

256

228

19

19

sd

20.4

6.7

0.0

1.2

3.0

4.4

13.9

44.0

3.6

4.2

f(I)

1.29

0.77

0.04

0.07

0.34

0.45

1.14

0.79

1.00

0.90

Background lawn

++

++

++

++

++

++

++

++

++

++

150 µg/plate

Mean

74

82

3

4

33

44

247

252

22

16

sd

10.6

19.2

3.5

1.7

7.5

1.5

38.9

32.7

1.2

1.2

f(I)

1.07

0.86

0.11

0.14

0.45

0.56

1.10

0.88

1.16

0.76

Background lawn

++

++

++

++

++

++

++

++

++

++

50 µg/plate

Mean

78

110

28

31

82

78

213

243

21

18

sd

10.4

21.4

3.8

2.9

5.9

10.4

36.3

28.1

5.5

2.5

f(I)

1.13

1.16

1.04

1.11

1.12

1.00

0.95

0.85

1.11

0.86

Background lawn

++

++

++

++

++

++

++

++

++

++

++= background lawn, + = background lawn reduced, - = no background lawn, sd = standard deviation, f(I) = increase factor,s.g. = strong growth, too strong for counting of revertants

 

Table 2: Mean Revertants Experiment 1b

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin.

water

Mean

73

84

36

33

92

108

249

259

21

22

sd

7.6

5.1

6.2

5.5

8.0

15.9

26.6

53.3

1.2

1.5

DMSO

Mean

74

110

38

36

89

91

239

249

26

18

sd

5.2

21.1

4.4

11.9

9.2

1.2

28.9

46.0

1.7

0.6

Positive
Controls*

Mean

479

463

268

176

379

s.g.

657

1323

220

175

sd

67.2

132.3

28.0

24.3

62.3

0.0

9.2

60.0

10.6

31.1

f(I)

6.47

4.21

7.05

4.89

4.12

11.00

2.75

5.31

10.48

9.72

5000 µg/plate

Mean

6

55

n.d.

n.d.

n.d.

n.d.

0

0

n.d.

n.d.

sd

5.8

11.9

n.d.

n.d.

n.d.

n.d.

0.0

0.0

n.d.

n.d.

f(I)

0.08

0.50

n.d.

n.d.

n.d.

n.d.

0.00

0.00

n.d.

n.d.

Background lawn

++

++

 

 

 

 

-

-

n.d.

n.d.

1500 µg/plate

Mean

76

91

n.d.

n.d.

n.d.

n.d.

221

213

3

3

sd

5.5

22.0

n.d.

n.d.

n.d.

n.d.

4.6

24.1

1.7

1.2

f(I)

1.03

0.83

n.d.

n.d.

n.d.

n.d.

0.92

0.86

0.12

0.17

Background lawn

++

++

 

 

 

 

++

++

++

++

500 µg/plate

Mean

74

87

n.d.

n.d.

n.d.

n.d.

227

251

20

17

sd

11.1

17.1

n.d.

n.d.

n.d.

n.d.

12.9

48.1

2.1

0.6

f(I)

1.00

0.79

n.d.

n.d.

n.d.

n.d.

0.95

1.01

0.77

0.94

Background lawn

++

++

 

 

 

 

++

++

++

++

150 µg/plate

Mean

77

93

6

7

0

0

223

199

19

17

sd

11.5

9.5

2.9

1.7

0.0

0.0

14.0

9.2

3.5

0.0

f(I)

1.04

0.85

0.16

0.19

0.00

0.00

0.93

0.80

0.73

0.94

Background lawn

++

++

++

++

-

-

++

++

++

++

50 µg/plate

Mean

96

116

32

35

102

102

220

196

20

16

sd

13.4

7.2

4.4

5.8

18.5

12.2

20.8

21.2

3.0

4.0

f(I)

1.30

1.05

0.84

0.97

1.15

1.12

0.92

0.79

0.77

0.89

Background lawn

++

++

++

++

++

++

++

++

++

++

15 µg/plate

Mean

77

101

37

37

75

92

203

229

17

18

sd

14.0

10.3

2.5

5.2

9.2

8.7

20.5

30.0

1.0

2.0

f(I)

1.04

0.92

0.97

1.03

0.84

1.01

0.85

0.92

0.65

1.00

Background lawn

++

++

++

++

++

++

++

++

++

++

5 µg/plate

Mean

n.d.

n.d.

30

33

95

93

n.d.

n.d.

19

17

sd

n.d.

n.d.

3.5

1.7

18.9

9.5

n.d.

n.d.

3.6

2.1

f(I)

n.d.

n.d.

0.79

0.92

1.07

1.02

n.d.

n.d.

0.73

0.94

Background lawn

 

 

++

++

++

++

 

 

++

++

1.5 µg/plate

Mean

n.d.

n.d.

32

34

101

106

n.d.

n.d.

n.d.

n.d.

sd

n.d.

n.d.

8.1

3.5

5.0

11.9

n.d.

n.d.

n.d.

n.d.

f(I)

n.d.

n.d.

0.84

0.94

1.13

1.16

n.d.

n.d.

n.d.

n.d.

Background lawn

 

 

++

++

++

++

 

 

 

 

0.5 µg/plate

Mean

n.d.

n.d.

34

36

99

101

n.d.

n.d.

n.d.

n.d.

sd

n.d.

n.d.

2.5

2.9

9.9

4.2

n.d.

n.d.

n.d.

n.d.

f(I)

n.d.

n.d.

0.89

1.00

1.11

1.11

n.d.

n.d.

n.d.

n.d.

Background lawn

 

 

++

++

++

++

 

 

 

 

++= background lawn, + = background lawn reduced, - = no background lawn, sd = standard deviation, n.d. = not determined, due to the toxicity results of the experiment 1a, f(I) = increase factor,s.g. = strong growth, too strong for counting of revertants. * Different positive controls were used

 

Table 3: Mean Revertants Experiment 2 (TA97a, TA102)

Strain

TA97a

TA102

Induction

-S9

+S9

-S9

+S9

Demin.

water

Mean

79

76

308

284

sd

3.2

11.3

35.6

30.2

DMSO

Mean

79

95

308

261

sd

15.9

5.8

55.6

64.0

Positive
Controls*

Mean

577

433

1339

1181

sd

182.5

32.3

72.6

257.2

f(I)

7.30

4.56

4.35

4.52

5000 µg/plate

Mean

4

14

33

29

sd

1.0

2.1

12.4

21.4

f(I)

0.05

0.15

0.11

0.11

Background lawn

++

++

++

++

2500 µg/plate

Mean

18

27

229

295

sd

7.4

7.6

47.7

26.6

f(I)

0.23

0.28

0.74

1.13

Background lawn

++

++

++

++

1250 µg/plate

Mean

82

80

233

331

sd

0.6

7.5

32.3

61.2

f(I)

1.04

0.84

0.76

1.27

Background lawn

++

++

++

++

625 µg/plate

Mean

80

84

259

347

sd

13.9

7.1

18.9

57.7

f(I)

1.01

0.88

0.84

1.33

Background lawn

++

++

++

++

313 µg/plate

Mean

79

85

321

273

sd

3.5

18.6

2.3

45.5

f(I)

1.00

0.89

1.04

1.05

Background lawn

++

++

++

++

156 µg/plate

Mean

77

76

301

240

sd

6.7

9.6

35.9

4.0

f(I)

0.97

0.80

0.98

0.92

Background lawn

++

++

++

++

78 µg/plate

Mean

71

67

264

263

sd

11.3

2.5

0.0

39.5

f(I)

0.90

0.71

0.86

1.01

Background lawn

++

++

++

++

39 µg/plate

Mean

71

84

261

241

sd

10.6

25.2

24.1

24.1

f(I)

0.90

0.88

0.85

0.92

Background lawn

++

++

++

++

++= background lawn, + = background lawn reduced, - = no background lawn, sd = standard deviation, f(I) = increase factor,* Different positive controls were used

Table 4: Mean Revertants Experiment 2 (TA98, TA100)

Strain

TA98

TA100

Induction

-S9

+S9

-S9

+S9

Demin.

water

Mean

37

37

85

93

sd

5.9

8.7

9.5

12.5

DMSO

Mean

37

42

75

84

sd

3.6

3.1

18.9

7.6

Positive
Controls*

Mean

621

264

699

717

sd

56.8

18.3

25.7

37.0

f(I)

16.78

6.29

8.22

8.54

150 µg/plate

Mean

3

5

15

16

sd

0.6

1.7

3.1

2.6

f(I)

0.08

0.12

0.20

0.19

Background lawn

++

++

++

++

75 µg/plate

Mean

34

37

88

78

sd

3.5

9.3

6.4

3.1

f(I)

0.92

0.88

1.17

0.93

Background lawn

++

++

++

++

37.5 µg/plate

Mean

44

44

76

86

sd

4.7

4.5

7.6

5.8

f(I)

1.19

1.05

1.01

1.02

Background lawn

++

++

++

++

18.8 µg/plate

Mean

40

30

84

72

sd

5.6

7.8

4.0

1.7

f(I)

1.08

0.71

1.12

0.86

Background lawn

++

++

++

++

9.4 µg/plate

Mean

42

46

82

77

sd

1.0

12.9

1.7

4.6

f(I)

1.14

1.10

1.09

0.92

Background lawn

++

++

++

++

4.7 µg/plate

Mean

38

37

76

77

sd

4.6

9.3

7.2

3.8

f(I)

1.03

0.88

1.01

0.92

Background lawn

++

++

++

++

2.3 µg/plate

Mean

36

35

82

87

sd

5.0

8.1

6.9

8.7

f(I)

0.97

0.83

1.09

1.04

Background lawn

++

++

++

++

1.2 µg/plate

Mean

38

33

92

87

sd

10.3

3.8

13.7

3.1

f(I)

1.03

0.79

1.23

1.04

Background lawn

++

++

++

++

++= background lawn, + = background lawn reduced, - = no background lawn, sd = standard deviation, f(I) = increase factor,* Different positive controls were used

 

Table 5: Mean Revertants Experiment 2 (TA1535)

Strain

TA1535

Induction

-S9

+S9

Demin.

water

Mean

10

12

sd

2.1

3.5

DMSO

Mean

11

12

sd

2.5

4.0

Positive
Controls*

Mean

249

113

sd

72.0

29.5

f(I)

24.90

9.42

1500 µg/plate

Mean

2

3

sd

0.6

1.2

f(I)

0.18

0.25

Background lawn

++

++

750 µg/plate

Mean

12

13

sd

0.6

1.2

f(I)

1.09

1.08

Background lawn

++

++

375 µg/plate

Mean

12

12

sd

1.7

1.2

f(I)

1.09

1.00

Background lawn

++

++

188 µg/plate

Mean

12

15

sd

2.1

2.1

f(I)

1.09

1.25

Background lawn

++

++

94 µg/plate

Mean

12

13

sd

1.2

3.2

f(I)

1.09

1.08

Background lawn

++

++

47 µg/plate

Mean

9

10

sd

1.5

1.5

f(I)

0.82

0.83

Background lawn

++

++

23 µg/plate

Mean

11

9

sd

3.5

1.5

f(I)

1.00

0.75

Background lawn

++

++

12 µg/plate

Mean

11

13

sd

1.5

1.2

f(I)

1.00

1.08

Background lawn

++

++

++= background lawn, + = background lawn reduced, - = no background lawn, sd = standard deviation, f(I) = increase factor,* Different positive controls were used

 

Conclusions:
The test item was considered to be non-mutagenic with and without metabolic activation in bacteria.
Executive summary:

A study according to OECD guideline 471 was performed to assess the genotoxic potential of the test item. The test item was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in three experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

In the experiment 1a, the test item (dissolved in DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method. The test item showed no precipitates on the plates at any of the concentrations. Signs of toxicity towards the bacteria strains (no bacterial background lawn and/or no revertant growth) were observed in both the absence and presence of metabolic activation from 150 µg/plate upwards depending on the strain used. Due to the toxicity results in the experiment 1a, in the experiemnt 1b and 2the test item (dissolved in DMSO) was tested up to the following concentrations in the absence and presence of S9-mix: 150 µg/plate (TA98, TA100), 1500 µg/plate (TA1535) and 5000 µg/plate (TA97a, TA102). The test item showed no precipitates on the plates at any of the concentrations. At the concentration 150 µg/plate and upwards (depending on the strain used), signs of toxicity were observed towards the respective bacteria strain with and without metabolic activation. The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A study according to OECD guideline 471 was performed to assess the genotoxic potential of the test item. The test item was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in three experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

In the experiment 1a, the test item (dissolved in DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method. The test item showed no precipitates on the plates at any of the concentrations. Signs of toxicity towards the bacteria strains (no bacterial background lawn and/or no revertant growth) were observed in both the absence and presence of metabolic activation from 150 µg/plate upwards depending on the strain used. Due to the toxicity results in the experiment 1a, in the experiemnt 1b and 2the test item (dissolved in DMSO) was tested up to the following concentrations in the absence and presence of S9-mix: 150 µg/plate (TA98, TA100), 1500 µg/plate (TA1535) and 5000 µg/plate (TA97a, TA102). The test item showed no precipitates on the plates at any of the concentrations. At the concentration 150 µg/plate and upwards (depending on the strain used), signs of toxicity were observed towards the respective bacteria strain with and without metabolic activation. The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The results indicate that the substance is non-mutagenic. Based on available data on genetic toxicity, the test item is not classified for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EC) No 2017/776.