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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was tested in the Bacterial reverse mutation assay with five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537). The study procedures described in this report were based on the most recent Guideline OECD 471 (2020) and EU Method B.13/14 (2008). The test was performed with +S9 standing for the presence of a metabolic activation, and -S9 standing for absence of metabolic activation.

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:
2020-2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
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:
Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GmbH, Gießen. Produced from the livers of male Sprague-Dawley rats which were treated with Phenobarbital/5,6-Benzoflavone
- concentration or volume of S9 mix and S9 in the final culture medium: Phosphate buffer 22.5 mL; 0.1M NADP-solution 1.0 mL; 1M G6P-solution 0.125 mL; Salt solution 0.5 mL; Rat liver S9 1.0 mL
Test concentrations with justification for top dose:
Experiment 1: Test concentrations 5, 1.5, 0.5, 0.15, 0.05 µL/plate.
Experiment 1b: Test concentrations 5, 1.5, 0.5, 0.15, 0.05, 0.015, 0.005 µL/plate.
Experiment 2: Test concentrations 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08 µL/plate.

S. typhimurium TA 102 was not tested in Experiment 1.
Top dose of 5 µL/plate as recommended on the OECD Guideline
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The test material was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations

- Justification for percentage of solvent in the final culture medium: The test material is soluble in a concentration of 50 mL/L in DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
mitomycin C
other: 4-Nitro-1,2-phenylene diamine
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
Number of replicates: 3 replicates per bacteria strain with (+S9) and 3 replicates per bacteria strain without metabolic activation(-S9)
- Number of independent experiments: 3, Experiment 1, 1b and 2

Three valid experiments were performed; the initial experiment had to be repeated with ad-ditional lower concentrations due to an insufficient number of analyzable non-toxic concentrations as indicated in the guideline.
For TA100 (+/-S9) experiment 1 was invalid, because the values of spontaneous revertants of the solvent controls demin. water and DMSO did not meet the historical control data range.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; Agar. Experiment 1 and Experiment 1b used the plate incorporation method. Experiment 2 used the pre-incubation method

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes
- Exposure duration/duration of treatment: 48h
Rationale for test conditions:
The test was conducted in compliance with the following guidelines:
- OECD Guidelines for the Testing of Chemicals Part 471, adopted 26. Jun. 2020 “Bacterial Reverse Mutation Test“
- Commission Regulation (EC) No. 440 2008, EU-Method B.13/14 adopted 30. May 2008 “Mutagenicity –Reverse mutation test using bacteria”.


Three valid experiments were performed; the initial experiment had to be repeated with additional lower concentrations due to an insufficient number of analyzable non-toxic concentrations as indicated in the guideline.
Evaluation criteria:
A result is considered positive if 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 concentrations occurs in at least one tested strain with or without metabolic activation.
A biologically relevant increase is described as follows:
• if in the bacteria strains S. typhimurium TA98, TA100, TA102 the number of revertants is at least twice as high than the reversion rate of the negative controls (increase fac-tor of at least 2.0)
• if in the bacteria strains S. typhimurium TA1535 and TA1537 the number of revertants is at least three times higher than the reversion rate of the negative controls (increase factor of at least 3.0).


A substance is not mutagenic if it does not meet the criteria above.
If the criteria listed above are not clearly met, the results will be assessed as equivocal and will be discussed.
Statistics:
Statistical analysis was not performed for this test
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
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
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
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:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid

Mean Revertants Experiment 1

Strain

TA98

TA100

TA102

TA1535

TA1537

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin.

water

Mean

12

14

iv

iv

-

-

6

7

7

4

sd

1.0

1.5

iv

iv

-

-

1.0

2.1

0.0

1.5

DMSO

Mean

9

16

iv

iv

-

-

9

6

5

5

sd

1.0

2.6

iv

iv

-

-

2.1

1.0

2.0

1.7

Positive
Controls*

Mean

576

128

iv

iv

-

-

309

297

59

17

sd

16.0

7.6

iv

iv

-

-

14.0

8.3

1.0

6.0

f(I)

64.00

8.00

iv

iv

-

-

51.50

49.50

11.80

3.40

5

µL/plate

Mean

0

3

iv

iv

-

-

3

10

0

0

sd

0.0

3.0

iv

iv

-

-

2.0

3.5

0.0

0.0

f(I)

0.00

0.19

iv

iv

-

-

0.33

1.67

0.00

0.00

1.5

µL/plate

Mean

12

11

iv

iv

-

-

19

19

5

7

sd

2.5

3.8

iv

iv

-

-

7.0

1.5

1.5

1.7

f(I)

1.33

0.69

iv

iv

-

-

2.11

3.17

1.00

1.40

0.5

µL/plate

Mean

13

8

iv

iv

-

-

12

17

7

4

sd

3.8

2.6

iv

iv

-

-

3.6

2.1

0.6

3.5

f(I)

1.44

0.50

iv

iv

-

-

1.33

2.83

1.40

0.80

0.15 µL/plate

Mean

9

15

iv

iv

-

-

6

9

3

4

sd

1.5

4.6

iv

iv

-

-

4.0

3.1

2.5

1.7

f(I)

1.00

0.94

iv

iv

-

-

0.67

1.50

0.60

0.80

0.05 µL/plate

Mean

9

12

iv

iv

-

-

10

4

3

5

sd

0.6

3.2

iv

iv

-

-

4.4

0.6

1.7

2.1

f(I)

1.00

0.75

iv

iv

-

-

1.11

0.67

0.60

1.00

sd = standard deviation±

* Different positive controls were used, see table under 'Any other information on materials and methos incl tables' section

f(I) = increase factor, calculation [mean revertants divide by means spontaneous revrtants]

iv = invalid

- = not tested

Mean Revertants Experiment 1b

Strain

TA98

TA100

TA102

TA1535

TA1537

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin.

water

Mean

19

22

73

82

260

247

5

6

3

6

sd

3.1

1.5

7.0

5.7

18.3

12.2

1.5

2.5

2.5

0.6

DMSO

Mean

14

16

74

76

247

243

6

5

3

3

sd

1.5

3.0

13.6

13.7

15.1

22.0

1.5

1.5

1.5

2.1

0.9% NaCl

Mean

-

-

-

-

245

-

-

-

-

-

sd

-

-

-

-

8.3

-

-

-

-

-

Positive
Controls*

Mean

601

149

573

2003

581

737

356

145

33

88

sd

28.1

4.2

31.1

164.2

24.4

31.1

22.3

10.0

14.2

7.2

f(I)

42.93

9.31

7.85

26.36

2.37

3.03

71.20

29.00

11.00

29.33

5

µL/plate

Mean

3

6

0

81

233

249

7

10

1

1

sd

2.5

1.2

0.0

9.3

6.1

16.7

2.1

1.5

1.2

1.0

f(I)

0.21

0.38

0.00

1.07

0.94

1.02

1.17

2.00

0.33

0.33

1.5

µL/plate

Mean

11

10

85

93

241

233

13

12

3

3

sd

2.1

4.6

12.7

6.7

15.1

19.7

3.0

3.2

2.1

1.7

f(I)

0.79

0.63

1.15

1.22

0.98

0.96

2.17

2.40 

1.00

1.00

0.5

µL/plate

Mean

12

14

89

84

224

233

13

10

2

5

sd

2.0

4.0

9.0

9.7

6.9

6.1

3.1

1.0

1.7

3.2

f(I)

0.86

0.88

1.20

1.11

0.91

0.96

2.17

2.00

0.67

1.67

0.15 µL/plate

Mean

12

13

82

78

244

233

8

7

2

2

sd

3.2

1.7

11.1

8.7

14.4

9.2

0.6

2.6

1.5

2.1

f(I)

0.86

0.81

1.11

1.03

0.99

0.96

1.33

1.40

0.67

0.67

0.05 µL/plate

Mean

15

16

78

84

255

232

7

4

2

4

sd

2.6

2.6

3.1

13.0

4.6

6.9

2.1

1.2

0.6

2.1

f(I)

1.07

1.00

1.05

1.11

1.03

0.95

1.17

0.80

0.67

1.33

0.015 µL/plate

Mean

14

18

76

75

257

260

3

5

2

4

sd

3.6

4.9

7.2

1.5

18.0

10.6

2.0

1.2

1.5

2.6

f(I)

1.00

1.13

1.03

0.99

1.04

1.07

0.50

1.00

0.67

1.33

0.005 µL/plate

Mean

14

18

86

86

241

240

8

5

3

4

sd

1.7

1.5

5.5

9.6

2.3

10.6

2.5

2.3

1.0

1.5

f(I)

1.00

1.13

1.16

1.13

0.98

0.99

1.33

1.00

1.00

1.33

sd = standard deviation±

* Different positive controls were used, see 'Any other information on materials and methos incl tables' section

f(I) = increase factor, calculation [mean revertants divide by means spontaneous revrtants]

- = not tested

Mean Revertants Experiment 2

Strain

TA98

TA100

TA102

TA1535

TA1537

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin.

water

Mean

38

38

63

59

244

232

8

9

5

5

sd

6.0

5.9

5.1

3.5

8.0

17.4

1.0

1.0

1.0

1.0

DMSO

Mean

33

38

58

57

236

249

8

7

6

5

sd

3.2

0.6

2.0

3.5

10.6

8.3

0.6

0.6

1.0

0.0

0.9% NaCl

Mean

-

-

-

-

235

-

-

-

-

-

sd

-

-

-

-

8.3

-

-

-

-

-

Positive
Controls*

Mean

1096

118

416

1720

809

996

359

231

56

79

sd

52.5

4.7

18.3

38.2

18.9

32.7

32.3

12.2

4.6

2.6

f(I)

33.21

3.11

6.60

30.18

3.44

4.00

44.88

33.00

9.33

15.80

5

µL/plate

Mean

0

0

0

0

165

219

1

3

0

0

sd

0.0

0.0

0.0

0.0

16.2

8.3

0.6

1.5

0.0

0.0

f(I)

0.00

0.00

0.00

0.00

0.70

0.88

0.13

0.43

0.00

0.00

2.5

µL/plate

Mean

6

17

0

61

241

239

12

18

0

4

sd

3.1

1.2

0.0

3.2

12.9

16.2

5.8

4.0

0.0

2.0

f(I)

0.18

0.45

0.00

1.07

1.02

0.96

1.50

2.57

0.00

0.80

1.25

µL/plate

Mean

32

29

60

126

240

240

11

12

5

5

sd

4.4

1.5

1.0

8.7

8.0

12.0

3.2

3.2

2.5

2.3

f(I)

0.97

0.76

1.03

2.21

1.02

0.96

1.38

1.71

0.83

1.00

0.63 µL/plate

Mean

34

31

138

80

235

239

13

5

4

6

sd

1.5

4.6

2.0

8.5

12.2

8.3

3.5

2.5

1.0

0.6

f(I)

1.03

0.82

2.38

1.40

1.00

0.96

1.63

0.71

0.67

1.20

0.31 µL/plate

Mean

33

38

121

70

237

237

7

8

4

5

sd

7.6

4.9

3.0

1.5

8.3

6.1

4.9

2.9

0.6

0.6

f(I)

1.00

1.00

2.09

1.23

1.00

0.95

0.88

1.14

0.67

1.00

0.16 µL/plate

Mean

32

37

74

65

236

243

11

5

5

5

sd

4.4

1.5

0.0

1.5

4.0

10.1

2.9

1.5

1.2

2.5

f(I)

0.97

0.97

1.28

1.14

1.00

0.98

1.38

0.71

0.83

1.00

0.08 µL/plate

Mean

27

39

58

59

237

237

5

7

4

6

sd

3.0

2.6

3.2

1.5

9.2

14.0

0.6

2.1

1.0

0.0

f(I)

0.82

1.03

1.00

1.04

1.00

0.95

0.63

1.00

0.67

1.20

sd = standard deviation±

* Different positive controls were used, see 'Any other information on materials and methos incl tables' section

f(I) = increase factor, calculation [mean revertants divide by means spontaneous revrtants]

- = not tested

Conclusions:
Based on the results of this study it is concluded that the test item is mutagenic in the Salmonella typhimurium strains TA100 in the presence and absence of metabolic activation and TA1535 in the presence of metabolic activation under the experimental conditions in this study.
Executive summary:

The test item was tested in the Bacterial reverse mutation assay with five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537). The study procedures described in this report were based on the most recent Guideline OECD 471 (2020) and EU Method B.13/14 (2008). The test was performed with +S9 standing for the presence of a metabolic activation, and -S9 standing for absence of metabolic activation. Three valid experiments were performed; the initial experiment had to be repeated with additional lower concentrations due to an insufficient number of analyzable non-toxic concentrations as indicated in the guideline. It was concluded that the test item is mutagenic in the Salmonella typhimuriumstrains TA100 in the presence and absence of metabolic activation and TA1535 in the presence of metabolic activation.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Justification for classification or non-classification

It was concluded that the test item is mutagenic in the Salmonella typhimurium strains TA100 in the presence and absence of metabolic activation and TA1535 in the presence of metabolic activation.