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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria: Key study. Test method according to OECD 471, GLP study. The test item did not induce any significant increase in the number of revertants in any of the strains tested, with and without metabolic activation, up to 5000 µg/plate. Based on the available data, the test item is not mutagenic.

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:
15/10/2018 - 31/10/2018
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
Target gene:
his D (S. typhimurium TA 98); his C (S. typhimurium TA 1537); his G (S. typhimurium TA 100 and TA1535); tryp E (E. coli WP2 uvrA pKM101)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: ΔuvrB and rfa mutated
Remarks:
(TA 98 and TA 100: pKM 101)
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: uvrA, pKM 101 mutated
Metabolic activation:
with and without
Metabolic activation system:
S9 mix of liver rats
Test concentrations with justification for top dose:
Initial mutation test (plate incorporation) / Confirmatory mutation test (pre-incubation): 50, 150, 500, 1500 and 5000 µg/plate.
The maximum test concentration was 5000 μg test item/plate since in the preliminary test the numbers of revertant colonies were mostly in the normal range, no citotoxicity was observed and there was no precipitation up to the highest dose tested (TA100 without metabolic activation).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: the test item was found to be soluble in Dimethyl sulfoxide (DMSO) at 100 mg/mL.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO), acetone, NaCl 0.15M
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene (1, 2 μg/plate; S. typhimurium strains, + S9), cis-Platinum (II) Diammine Dichloride (1 μg/plate; E.coli, - S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
1.Plate incorporation (initial mutation test): A stock solution of the test item was prepared at 100 mg / mL. In a test tube, 0.1 mL of the bacterial suspension containing 1-9E09 bacteria/mL and 0.1 mL of each dilution of the original solution and 0.5 mL of sterile phosphate buffer are successively added to 2 mL of overlay agar maintained super cooled at 45ºC containing 10% (v/v) of a L-Histidine-D-Biotine solution (0.5 mM) for Salmonella Typhimurium strains, or containing 5% (v/v) of nutrient broth nº2 to which are added 5 μL of a L-Tryptophane solution at 2 mg/mL for Escherichia coli strain. In the assay with metabolic activation, either a standard plate incorporation method where the protocol is similar to the described above, except that, 500 μL of S9-mix fraction is quickly added, before pouring the mixture onto the plates. After a 48-72 hour incubation period at 37ºC, revertant colonies are counted in each plate.
2.Pre-incubation (confirmatory mutation test): Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer were added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes were gently mixed and incubated for 30 min at 37ºC in a shaking incubator. After the incubation period, molten top agar was added to the tubes; the content was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. After preparation, the plates were incubated at 37ºC for 48 hours.

DURATION
- Preincubation period: 30 minutes (confirmatory mutation test)
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): The lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3 (test item, negative and positive controls).

DETERMINATION OF CYTOTOXICITY
- Method: other: relative total growth.
- Preliminary cytotoxicity test (Strain TA100): In a test tube 0.1 mL of the bacterial suspension (1-9E 03 bacteria /mL) and 0.1 mL of the stock solution of L-Histidine-D-Biotine (2.5 mM). After homogenization, the content of the tube is poured onto a Petri plate (90 mm in diameter) containing minimal agar (20 mL). 3 plates per concentration are incubated for 48-72 h at 37ºC, and the colonies counted. A negative control containing the blank alone is run in parallel. In case of bacteriostatic activity is detected, the highest concentration to be retined is that exhibiting a bacteriostatic activity of 75% or less. The precipitate, if present, should not interfere with the scoring. The following four dilutions studied are distributed according to a semi-logarithmic progression.

OTHER:
- Sterility test: Test item and the corresponding dilutions are added to 2 mL of top agar maintained at 45ºC, and poured after homogenization on the bottom agar (20 mL) onto a Petri plate (90 mm in diameter) (n=3). Plates are incubated for 48-72 hours at 37ºC and then examined. There should be no bacterial growth on any plate. S9-mix sterility is checked using the same protocol.
- Preparation of the metabolic activation system (S9 fraction): Obtention of S9 fraction: S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, is provided by MOLTOX (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 - USA) (S9 Moltox-11101-5-3805 validated on 7.2017 - expiry date: 25.05.2019). Preparation of S9-mix 10% (v/v): The final concentration of co-factors and salts is as follows: S9 fraction 10%; MgCl2-6H2O 8 mM; KCl 33 mM; Glucose-6-Phophate Na2 5 mM; NADP Na2 4mM; Phosphate buffer pH 7.4 0.1 M.
Rationale for test conditions:
Results of sterility controls show the absence of any bacterial growth in the presence of test item S9-mix. Results of the bacteriostatic activity control show no toxicity. Values and frequency are within the laboratory's historical control ranges.
Evaluation criteria:
The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101) strains without and with metabolic activation.
The result of the test is considered positive if a dependent relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is at least two fold that of spontaneous revertant colonies for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101), and three fold for TA 1535 and TA 1537.
All results must be confirmed in an independent experiment.
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:
valid
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:
valid
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:
valid
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:
valid
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS : None observed.

RANGE-FINDING/SCREENING STUDIES: No precipitate and no inhibitory, cytotoxic effect of the test item was observed up to the highest dose tested (5000 µg/plate).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data (- S9): values for spontaneous revertants (revertants/plate) in the period of 2008 - 2017 were as follows: Salmonella typhimurium TA98: 496.3 ± 219.9, TA100: 991.8 ± 331.2, TA1535: 731.5 ± 220.2, TA1537: 876.5 ± 448.3, Escherichia coli WP2 uvrA: 484.6 ± 168.2.
- Positive historical control data (+ S9): values with metabolic activation in the period of 2008 - 2017 were: Salmonella typhimurium TA98: 572.9 ± 222.1, TA100: 846.8 ± 359.5, TA1535: 109.2 ± 56.0, TA1537: 55.1 ± 24.7, Escherichia coli WP2 uvrA: 686.5 ± 253.3.
- Negative (solvent/vehicle) historical control data (- S9): values for untreated control sample without metabolic activation in the period of 2008 - 2017 were as follows: Salmonella typhimurium TA98: 16.0 ± 3.9, TA100: 59.8 ± 11.8, TA1535: 11.0 ± 3.6, TA1537: 6.0 ± 2.5, Escherichia coli WP2 uvrA: 48.8 ± 7.8.
- Negative (solvent/vehicle) historical control data (+ S9): values with metabolic activation in the period of 2008 - 2017 were: Salmonella typhimurium TA98: 23.2 ± 5.0, TA100: 96.2 ± 22.3, TA1535: 12.2 ± 4.1, TA1537: 8.1 ± 3.5, Escherichia coli WP2 uvrA: 156.8 ± 34.6.
- There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental “historical” values obtained in the laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxic effects of the test item were observed in any of the five tested strains used up to the highest dose (with and without metabolic activation) in assays 1 and 2.

Table 3. Sterility control.

Serie

Doses

Colony number/plate

Control n° 1

 

1

2

3

Solution of

 

test item

 

LEMI code :

18/0260-221018-S1

5000 µg /plate

0

0

0

1500 µg /plate

0

0

0

500 µg /plate

0

0

0

150 µg /plate

0

0

0

50 µg /plate

0

0

0

S9-mix

500 µL/plate

0

0

0

Control n° 2

 

1

2

3

Solution of

 

test item

 

 

LEMI code :

 18/0260-291018-S1

5000 µg /plate

0

0

0

1500 µg /plate

0

0

0

500 µg /plate

0

0

0

150 µg /plate

0

0

0

50 µg /plate

0

0

0

S9-mix

500 µL/plate

0

0

0

Table 4. Bacteriostatic activity controls.

 

 

 

 

 

Doses (/plate)

 

 

 

0

(neg. Ctrl.)

DMSO

50 µg

150 µg

500 µg

1 500 µg

2 500 µg

5 000 µg

Control

nº1

N1

N2

N3

N

%

508

549

504

520 ± 25

-

510

489

515

505 ± 14

97%

504

523

515

514 ± 10

99%

509

511

520

513 ± 6

99%

522

516

508

515 ± 7

99%

489

503

517

503 ± 14

97%

499

515

516

510 ± 10

98%

526

489

420

478 ± 54

92%

Control

Nº2

N1

N2

N3

N

%

495

464

486

482 ± 16

-

475

463

487

475 ± 12

99%

474

485

502

487 ± 14

101%

519

466

491

492 ± 27

102%

484

497

469

483 ± 14

100%

505

495

477

492 ± 14

102%

 

492

491

457

480 ± 20

100%

Table 5. Mutagenic activity tests: assay 1 (plate incorporation).

Assay 1 – TA 1535 (+S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

21

16

22

19.67

3.21

_

Positive control solvent

5 µL

16

16

11

14.33

2.89

_

Positive control :

Sodium azide

5 µg in 5 µL

728

903

750

793.67

95.32

55.37

Vehicle

50µL

19

16

9

14.67

5.13

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

17

10

21

12

17

9

16

19

14

19

12

8

17

20

15

12.67

11.33

19.00

15.33

17.00

4.04

4.16

2.00

4.16

2.00

0.86

0.77

1.30

1.05

1.16

Assay 1 – TA 1535 (-S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

19

11

11

13.67

4.62

_

Positive control solvent

20 µL

13

13

15

13.67

1.15

_

Positive control :

2-Anthramine

2 µg in 20 µL

203

187

214

201.33

13.58

14.73

Vehicle

50µL

16

21

19

18.67

2.52

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

12

12

13

19

10

17

14

23

15

24

13

14

12

16

19

14.00

13.33

16.00

16.67

17.67

2.65

1.15

6.08

2.08

7.09

0.75

0.71

0.86

0.89

0.95

Assay 1 – TA 1537 (+S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

10

5

5

6.67

2.89

_

Positive control solvent

20 µL

8

8

12

9.33

2.31

_

Positive control :

9-Aminoacridine

50 µg in 20 µL

1817

1962

1901

1893.33

72.80

202.86

Vehicle

50µL

4

9

4

5.67

2.89

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

6

5

3

4

9

12

4

5

7

7

8

8

4

4

10

8.67

5.67

4.00

5.00

8.67

3.06

2.08

1.00

1.73

1.53

1.53

1.00

0.71

0.88

1.53

Assay 1 – TA 1537 (-S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

10

11

6

9.00

2.65

_

Positive control solvent

20 µL

13

11

14

12.67

1.53

_

Positive control :

2-Anthramine

2 µg in 20 µL

48

61

48

52.33

7.51

4.13

Vehicle

50µL

13

5

9

9.00

4.00

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

15

13

14

15

8

6

10

12

12

14

13

11

10

8

12

11.33

11.33

12.00

11.67

11.33

4.73

1.53

2.00

3.51

3.06

1.26

1.26

1.33

1.30

1.26

Assay 1 – TA 98 (+S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

19

9

12

13.33

5.13

_

Positive control solvent

20 µL

14

9

12

11.67

2.52

_

Positive control :

2-Nitrofluorene

2 µg in 20 µL

583

573

551

569.00

16.37

48.77

Vehicle

50µL

13

12

18

14.33

3.21

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

16

23

11

11

16

19

10

14

18

8

7

11

20

9

11

14.00

14.67

15.00

12.67

11.67

6.24

7.23

4.58

4.73

4.04

0.98

1.02

1.05

0.88

0.81

Assay 1 – TA 98 (-S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

12

29

20

20.33

8.50

_

Positive control solvent

20 µL

22

18

12

17.33

5.03

_

Positive control :

2-Anthramine

2 µg in 20 µL

569

576

489

544.67

48.34

31.42

Vehicle

50µL

14

17

23

18.00

4.58

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

16

31

21

21

21

20

12

28

25

23

15

22

22

25

27

17.00

21.67

23.67

23.67

23.67

2.65

9.50

3.79

2.31

3.06

0.94

1.20

1.31

1.31

1.31

Assay 1 – TA 100 (+S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

93

71

72

78.67

12.42

_

Positive control solvent

20 µL

73

82

69

74.67

6.66

_

Positive control :

Sodium azide

20 µg in 20 µL

1563

1498

1561

1540.67

36.96

20.63

Vehicle

50µL

70

73

65

69.33

4.04

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

83

86

78

64

75

79

79

84

86

70

64

70

70

72

88

75.33

78.33

77.33

74.00

77.67

10.02

8.02

7.02

11.14

9.29

1.09

1.13

1.12

1.07

1.12

Assay 1 – TA 100 (-S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

80

79

104

87.67

14.15

_

Positive control solvent

20 µL

108

86

94

96.00

11.14

_

Positive control :

2-Anthramine

2 µg in 20 µL

1068

1153

854

1025.00

154.07

10.68

Vehicle

50µL

90

112

84

95.33

14.74

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

84

88

86

103

98

95

91

110

102

105

79

92

107

118

100

86.00

90.33

101.00

107.67

101.00

8.19

2.08

13.08

8.96

3.61

0.90

0.95

1.06

1.13

1.06

Assay 1 – E. coli (+S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

116

129

117

120.67

7.23

_

Positive control solvent

10 µL

112

129

102

114.33

13.65

_

Positive control : cis-Platinum (II)

1 µg in 10 µL

629

722

811

720.67

91.01

6.30

Vehicle

50µL

126

116

107

116.33

9.50

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

104

107

105

108

111

108

116

119

113

108

120

124

115

126

107

110.67

115.67

113.00

115.67

108.67

8.33

8.50

7.21

9.29

2.08

0.95

0.99

0.97

0.99

0.93

Assay 1 – E. coli (-S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

180

181

188

183.00

4.36

_

Positive control solvent

5 µL

178

187

195

186.67

8.50

_

Positive control :

Dimethylbenzanthracene

5 µg in 5 µL

1204

1008

1220

1144.00

118.05

6.13

Vehicle

50µL

180

171

219

190.00

25.51

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

184

179

177

207

190

170

182

174

193

181

183

198

179

184

183

179.00

186.33

176.67

194.67

184.67

7.81

10.21

2.52

11.59

4.73

0.94

0.98

0.93

1.02

0.97

Table 6. Mutagenic activity tests: assay 2 (pre-incubation).

Assay 2 – TA 1535 (+S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

15

12

9

12.00

3.00

_

Positive control solvent

5 µL

14

11

10

11.67

2.08

_

Positive control :

Sodium azide

5 µg in 5 µL

889

962

848

899.67

57.74

77.11

Vehicle

50µL

11

9

7

9.00

2.00

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

9

7

11

10

7

8

9

11

13

12

12

16

7

14

12

9.67

10.67

9.67

12.33

10.33

2.08

4.73

2.31

2.08

2.89

1.07

1.19

1.07

1.37

1.15

Assay 2 – TA 1535 (-S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

10

13

12

11.67

1.53

_

Positive control solvent

10 µL

13

13

9

11.67

2.31

_

Positive control :

2-Anthramine

1 µg in 10 µL

151

164

152

155.67

7.23

13.34

Vehicle

50µL

11

7

10

9.33

2.08

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

9

16

16

11

17

12

15

13

13

10

9

24

14

16

8

10.00

18.33

14.33

13.33

11.67

1.73

4.93

1.53

2.52

4.73

1.07

1.96

1.54

1.43

1.25

Assay 2 – TA 1537 (+S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

3

4

6

4.33

1.53

_

Positive control solvent

20 µL

5

7

7

6.33

1.15

_

Positive control :

9-Aminoacridine

50 µg in 20 µL

1564

1756

1361

1560.33

197.53

246.37

Vehicle

50µL

1

5

7

4.33

3.06

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

4

7

4

2

4

4

8

3

1

6

7

4

6

5

3

5.00

6.33

4.33

2.67

4.33

1.73

2.08

1.53

2.08

1.53

1.15

1.46

1.00

0.62

1.00

Assay 2 – TA 1537 (-S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

6

15

1

7.33

7.09

_

Positive control solvent

10 µL

4

7

5

5.33

1.53

_

Positive control :

2-Anthramine

1 µg in 10 µL

51

61

47

53.00

7.21

9.94

Vehicle

50µL

7

4

7

6.00

1.73

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

7

7

6

8

4

4

9

5

5

10

6

4

1

5

4

5.67

6.67

4.00

6.00

6.00

1.53

2.52

2.65

1.73

3.46

0.94

1.11

0.67

1.00

1.00

Assay 2 – TA 98 (+S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

14

21

23

19.33

4.73

_

Positive control solvent

20 µL

21

20

17

19.33

2.08

_

Positive control :

2-Nitrofluorene

2 µg in 20 µL

441

508

621

523.33

90.97

27.07

Vehicle

50µL

12

24

17

17.67

6.03

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

22

22

26

21

20

21

16

22

23

27

18

21

20

14

19

20.33

19.67

22.67

19.33

22.00

2.08

3.21

3.06

4.73

4.36

1.15

1.11

1.28

1.09

1.25

Assay 2 – TA 98 (-S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

34

32

32

32.67

1.15

_

Positive control solvent

10 µL

32

30

35

32.33

2.52

_

Positive control :

2-Anthramine

1 µg in 10 µL

328

427

500

418.33

86.33

12.94

Vehicle

50µL

32

38

34

34.67

3.06

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

26

27

31

40

25

33

26

38

27

28

31

29

30

34

37

30.00

27.33

33.00

33.67

30.00

3.61

1.53

4.36

6.51

6.24

0.87

0.79

0.95

0.97

0.87

Assay 2 – TA 100 (+S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

66

57

65

62.67

4.93

_

Positive control solvent

20 µL

63

60

61

61.33

1.53

_

Positive control :

Sodium azide

20 µg in 20 µL

1452

1388

1411

1417.00

32.42

23.10

Vehicle

50µL

60

65

58

61.00

3.61

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

58

61

58

60

63

67

62

61

62

60

69

64

66

67

71

64.67

62.33

61.67

63.00

64.67

5.86

1.53

4.04

3.61

5.69

1.06

1.02

1.01

1.03

1.06

Assay 2 – TA 100 (-S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

102

95

95

97.33

4.04

_

Positive control solvent

10 µL

87

92

83

87.33

4.51

_

Positive control :

2-Anthramine

1 µg in 10 µL

371

522

419

437.33

77.15

5.01

Vehicle

50µL

71

79

80

76.67

4.93

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

89

96

101

87

91

93

92

82

92

85

90

85

95

81

94

90.67

91.00

92.67

86.67

90.00

2.08

5.57

9.71

5.51

4.58

1.18

1.19

1.21

1.13

1.17

Assay 2 – E. coli (+S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

76

82

83

80.33

3.79

_

Positive control solvent

10 µL

81

85

89

85.00

4.00

_

Positive control : cis-Platinum (II)

1 µg in 10 µL

817

583

756

718.67

121.39

8.45

Vehicle

50µL

67

90

75

77.33

11.68

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

88

87

83

74

76

86

76

79

80

89

77

75

81

87

79

83.67

79.33

81.00

80.33

81.33

5.86

6.66

2.00

6.51

6.81

1.08

1.03

1.05

1.04

1.05

Assay 2 – E. coli (-S9)

Dose/Plate

 

Plate

 

Mean

Standard deviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

140

139

129

136.00

6.08

_

Positive control solvent

5 µL

134

118

135

129.00

9.54

_

Positive control :

Dimethylbenzanthracene

2.5 µg

in 5 µL

1045

992

1156

1064.33

83.69

8.25

Vehicle

50µL

119

108

104

110.33

7.77

_

Test item

5000 µg

1500 µg

500 µg

150 µg

50 µg

122

123

137

109

112

137

115

105

117

124

112

113

110

120

94

123.67

117.00

117.33

115.33

110.00

12.58

5.29

17.21

5.69

15.10

1.12

1.06

1.06

1.05

1.00

Conclusions:
The test item did not induce any significant increase in the number of revertants in any of the strains tested, with and without metabolic activation, up to 5000 µg/plate. Based on the available data, the test item is not mutagenic.
Executive summary:

A bacterial reverse mutation test was conducted on the test substance according to OECD guideline 471 under GLP conditions. Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA were exposed to concentrations of the test substance ranging from 50 to 5000 µg/plate in DMSO, with and without metabolic activation, according to preliminary assays. The metabolic activation system (S9 fraction) prepared from Sprague Dawley rat liver homogenate was provided by MOLTOX. Two independent assays were performed: an initial mutation test (plate incorporation method) and a confirmatory mutation test (pre-incubation method) were carried out. Untreated, solvent controls and strain specific positive controls were included in the assays and were within the historical control range in all strains. All validity criteria were fulfilled. The test item did not induce any significant increase in the number of revertants in any of the strains tested, with and without metabolic activation, up to 5000 µg/plate. Based on the available data, the test item is not mutagenic.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the available data (negative Ames test), the test substance is not classified in accordance with CLP Regulation (EC) No. 1272/2008.