Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was found to be non mutagenic in Ames test (OECD 471, GLP, K1).

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:
May - October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine locus for Salmonella strains and tryptophan for E. coli strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
Test concentrations with justification for top dose:
Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
Experiment 2 - Pre-Incubation Method: 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate, without S9-mix for TA100, TA1535 and TA 1537 and 5, 15, 50, 150, 500, 1500 and 5000 µg/plate for TA98 without S9. 15, 50, 150, 500, 1500and 5000 μg/plate with S9 for all strains.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
In solubility checks performed in house the test item was noted as immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but fully miscible in acetone at 100 mg/mL. Acetone was therefore selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: The bacteria used in the test were obtained from:
- University of California, Berkeley, on culture discs, on 04 August 1995
- British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987

METHOD OF APPLICATION: preincubation
All testing will be performed using the pre-incubation method (20 minutes at 37 °C) except for the untreated controls.

DURATION
- Preincubation period: 20 minutes.
- Exposure duration: ca. 48 hours

CONTROLS:
- Vehicle/solvent control: Acetone
- Negative (untreated) controls were performed to assess the spontaneous revertant colony rate.
- Positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation.
- Sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and
The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).

NUMBER OF REPLICATIONS: Triplicate

- OTHER: All of the plates were incubated at 37 ± 3°C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed at 5000 µg/plate because of test item precipitation. Several further manual counts were also required due to revertant colonies spreading slightly, thus distorting the actual plate count.
Rationale for test conditions:
The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate (i.e. maximum recommended dose level). The dose range used for Experiment 2 was determined by the results of Experiment 1. Up to seven test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item.
Evaluation criteria:
Criteria for determining a positive result:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
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:
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:
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A test item precipitate (light and globular in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

MUTAGENICITY
- The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
- In the first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the bacterial tester strains from 150 µg/plate (TA100, TA 1535 and TA 1537) and 1500 µg/plate (TA98 and WP2uvrA) in the absence of S9 mix and to two bacterial strains (TA100 and TA1535) at 5000 µg/plate in the presence S9-mix. In the second mutation test, the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 150 µg/plate (TA1535, TA100 and TA1537) and 1500 µg/plate (TA98 and WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were noted to three of the bacterial tester strains at 5000 µg/plate (TA100, TA1535 and WP2uvrA).
- No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 and Experiment 2 .
- Refer Tables 7.6.1/1 to 7.6.1/5 for more details.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Refer Table 7.6.1/6
- Negative (solvent/vehicle) historical control data: Refer Table 7.6.1/6

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.
- Results for the negative controls (spontaneous mutation rates) are presented in 7.6.1/1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

Table 7.6.1/1:Spontaneous Mutation Rates (Concurrent Negative Controls)

 

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

Experiment 1

TA100

TA1535

WP2uvrA

TA98

TA1537

87

 

16

 

31

 

31

 

9

 

86

(84)

19

(17)

15

(22)

15

(24)

21

(15)

80

 

17

 

19

 

25

 

16

 

Experiment 2

TA100

TA1535

WP2uvrA

TA98

TA1537

65

 

18

 

36

 

20

 

15

 

65

(64)

23

(18)

32

(34)

15

(18)

18

(16)

61

 

14

 

34

 

19

 

16

 

 

Table 7.6.1/2:Test Results: Experiment 1 – Without Metabolic Activation

Test Period

From: 25 July 2017

To: 28 July 2018

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(Acetone)

83

81

88

(84)

3.6#

26

14

24

(21)

6.4

20

28

25

(24)

4.0

26

16

11

(18)

7.6

15

21

19

(18)

3.1

1.5 µg

90

71

84

(82)

9.7

9

16

12

(12)

3.5

25

19

24

(23)

3.2

15

21

25

(20)

5.0

18

8

16

(14)

5.3

5 µg

76

71

85

(77)

7.1

8

21

15

(15)

6.5

16

25

20

(20)

4.5

25

16

14

(18)

5.9

8

13

8

(10)

2.9

15 µg

96

94

111

(100)

9.3

12

16

9

(12)

3.5

18

24

32

(25)

7.0

10

21

17

(16)

5.6

9

9

14

(11)

2.9

50 µg

84

82

93

(86)

5.9

9

9

8

(9)

0.6

28

25

27

(27)

1.5

16

16

21

(18)

2.9

15

9

21

(15)

6.0

150 µg

86 S

79 S

80 S

(82)

3.8

8 S

6 S

20 S

(11)

7.6

25

28

12

(22)

8.5

15

13

18

(15)

2.5

3 S

3 S

3 S

(3)

0.0

500 µg

74 S

65 S

58 S

(66)

8.0

10 S

12 S

8 S

(10)

2.0

24

26

20

(23)

3.1

27

23

17

(22)

5.0

7 S

6 S

3 S

(5)

2.1

1500 µg

16 S

15 S

26 S

(19)

6.1

14 S

5 S

12 S

(10)

4.7

23 S

20 S

8 S

(17)

7.9

12 S

12 S

14 S

(13)

1.2

3 S

2 S

2 S

(2)

0.6

5000 µg

0 VP

0 VP

0 VP

(0)

0.0

0 VP

0 VP

0 VP

(0)

0.0

14 SP

14 SP

13 SP

(14)

0.6

18 SP

18 SP

17 SP

(18)

0.6

0 VP

0 VP

0 VP

(0)

0.0

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

1692

1753

1736

(1727)

31.5

596

637

489

(574)

76.4

719

710

811

(747)

55.9

311

277

286

(291)

17.6

251

111

175

(179)

70.1

ENNG:N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO:4-Nitroquinoline-1-oxide

9AA:   9-Aminoacridine

P:       Test item precipitate

#:       Standard deviation

 

Table 7.6.1/3:Test Results: Experiment 1 – With Metabolic Activation

Test Period

From: 25 July 2017

To: 28 July 2018

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(Acetone)

89

82

90

(87)

4.4#

15

17

16

(16)

1.0

32

20

41

(31)

10.5

29

40

32

(34)

5.7

15

17

20

(17)

2.5

1.5 µg

74

85

78

(79)

5.6

10

10

18

(13)

4.6

21

20

31

(24)

6.1

34

16

29

(26)

9.3

12

11

17

(13)

3.2

5 µg

97

76

91

(88)

10.8

18

15

14

(16)

2.1

24

23

30

(26)

3.8

31

44

32

(36)

7.2

12

10

12

(11)

1.2

15 µg

81

82

78

(80)

2.1

14

18

24

(19)

5.0

18

21

25

(21)

3.5

13

25

30

(23)

8.7

21

21

20

(21)

0.6

50 µg

68

90

94

(84)

14.0

20

13

12

(15)

4.4

31

31

33

(32)

1.2

25

23

31

(26)

4.2

14

12

20

(15)

4.2

150 µg

96

95

84

(92)

6.7

12

21

14

(16)

4.7

34

26

24

(28)

5.3

35

36

27

(33)

4.9

17

13

17

(16)

2.3

500 µg

92

72

105

(90)

16.6

9

23

15

(16)

7.0

31

33

19

(28)

7.6

25

28

23

(25)

2.5

21

20

13

(18)

4.4

1500 µg

90

82

84

(85)

4.2

16

26

12

(18)

7.2

29

25

24

(26)

2.6

23

29

33

(28)

5.0

12

13

21

(15)

4.9

5000 µg

64 SP

66 SP

71 SP

(67)

3.6

10 SP

14 SP

10 SP

(11)

2.3

39 P

18 P

15 P

(24)

13.1

23 P

34 P

20 P

(26)

7.4

7 P

11 P

11 P

(10)

2.3

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1170

1028

1286

(1161)

129.2

288

320

285

(298)

19.4

168

168

166

(167)

1.2

144

158

170

(157)

13.0

467

454

475

(465)

10.6

2AA:   2-Aminoanthracene

BP:     Benzo(a)pyrene

P:        Test item precipitate

#:       Standard deviation

 

 

Table 7.6.1/4:Test Results: Experiment 2 – Without Metabolic Activation

Test Period

From: 14 August 2017

To: 17 August 2017

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(Acetone)

78

65

61

(68)

8.9#

19

19

20

(19)

0.6

20

23

23

(22)

1.7

20

21

22

(21)

1.0

19

16

5

(13)

7.4

0.5 µg

64

70

83

(72)

9.7

17

19

14

(17)

2.5

N/T

N/T

19

11

14

(15)

4.0

1.5 µg

79

78

63

(73)

9.0

13

23

19

(18)

5.0

N/T

N/T

13

17

5

(12)

6.1

5 µg

88

65

74

(76)

11.6

22

21

16

(20)

3.2

20

20

29

(23)

5.2

13

25

21

(20)

6.1

16

9

11

(12)

3.6

15 µg

62

71

73

(69)

5.9

22

17

16

(18)

3.2

28

33

28

(30)

2.9

20

28

29

(26)

4.9

13

18

15

(15)

2.5

50 µg

64

68

74

(69)

5.0

18

31

18

(22)

7.5

29

18

21

(23)

5.7

17

20

25

(21)

4.0

9

9

10

(9)

0.6

150 µg

63 S

67 S

60 S

(63)

3.5

12 S

17 S

21 S

(17)

4.5

28

19

22

(23)

4.6

29

15

17

(20)

7.6

10 S

5 S

14 S

(10)

4.5

500 µg

58 S

41 S

51 S

(50)

8.5

14 S

15 S

13 S

(14)

1.0

21

20

26

(22)

3.2

14

16

12

(14)

2.0

5 S

7 S

6 S

(6)

1.0

1500 µg

N/T

N/T

17 S

17 S

21 S

(18)

2.3

14 S

12 S

26 S

(17)

7.6

N/T

5000 µg

N/T

N/T

31 SP

20 SP

28 SP

(26)

5.7

9 SP

11 SP

11 SP

(10)

1.2

N/T

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

807

620

724

(717)

93.7

1500

1528

1598

(1542)

50.5

668

627

485

(593)

96.0

175

205

180

(187)

16.1

347

152

193

(231)

102.8

ENNG:N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO:4-Nitroquinoline-1-oxide

9AA:   9-Aminoacridine

P:       Test item precipitate

#:       Standard deviation

Table 7.6.1/5:Test Results: Experiment 2 – With Metabolic Activation

Test Period

From: 14 August 2017

To: 17 August 2017

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(Acetone)

71

67

85

(74)

9.5#

16

20

22

(19)

3.1

35

24

33

(31)

5.9

34

24

38

(32)

7.2

13

14

7

(11)

3.8

15 µg

61

75

89

(75)

14.0

26

22

22

(23)

2.3

40

36

31

(36)

4.5

26

32

39

(32)

6.5

13

15

13

(14)

1.2

50 µg

74

75

76

(75)

1.0

21

24

13

(19)

5.7

29

41

33

(34)

6.1

40

36

24

(33)

8.3

17

13

14

(15)

2.1

150 µg

86

82

75

(81)

5.6

11

18

29

(19)

9.1

24

45

31

(33)

10.7

36

36

26

(33)

5.8

20

15

15

(17)

2.9

500 µg

79

79

75

(78)

2.3

17

17

33

(22)

9.2

27

34

31

(31)

3.5

44

13

42

(33)

17.3

11

18

21

(17)

5.1

1500 µg

62

65

65

(64)

1.7

22

21

31

(25)

5.5

33

35

26

(31)

4.7

23

32

33

(29)

5.5

11

13

8

(11)

2.5

5000 µg

64 SP

60 SP

55 SP

(60)

4.5

26 SP

31 SP

29 SP

(29)

2.5

19 SP

23 SP

23 SP

(22)

2.3

30 P

29 P

34 P

(31)

2.6

15 P

19 P

18 P

(17)

2.1

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1029

1096

1037

(1054)

36.6

233

212

200

(215)

16.7

139

117

157

(138)

20.0

137

107

70

(105)

33.6

394

453

413

(420)

30.1

2AA  2-Aminoanthracene

BP          Benzo(a)pyrene

P                Test item precipitate

S                Sparse bacterial background lawn

#        Standard deviation

Table 7.6.1/6:History Profile of Vehicle and Positive Control Values

OMBINED VEHICLE AND UNTREATED CONTROL VALUES 2015

Strain

S9-Mix

TA100

TA1535

TA102

WP2uvrA

TA98

TA1537

WP2uvrA

pKM101

WP2pKM101

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Values†

274

278

504

285

26

13

461

229

526

299

506

282

42

51

39

49

Min

60

61

7

7

222

278

10

12

11

10

4

6

87

98

89

93

Max

166

175

31

29

376

388

58

43

45

46

27

27

237

254

174

177

Mean

91

95

16

14

286

333

24

27

21

24

12

13

156

164

123

137

SD

19.3

19.1

4.5

4.0

48.7

37.6

5.6

5.9

6.2

6.1

3.8

3.4

42.2

35.6

23.1

21.2

POSITIVE CONTROL VALUES 2015

 

Strain

S9-Mix

TA100

TA1535

TA102

WP2uvrA

TA98

TA1537

WP2uvrA

pKM101

WP2pKM101

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

 

Values

276

280

252

264

13

13

231

227

262

276

253

261

20

35

20

35

 

Min

222

250

79

118

953

673

116

103

100

78

164

97

430

494

745

325

 

Max

2266

2402

2779

457

3140

1655

2769

550

502

705

2318

823

1696

2264

3662

1174

 

Mean

614

927

472

246

2303

1093

792

266

222

218

911

336

761

1461

2257

569

 

SD

260.6

452.5

434.8

55.7

815.2

376.5

342.1

97.7

70.2

107.6

412.4

135.7

350.0

382.0

790.7

220.3

 

COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2016

Strain

S9-Mix

TA100

TA1535

TA102

WP2uvrA

TA98

TA1537

WP2uvrA

pKM101

WP2pKM101

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Values

399

401

758

393

60

30

690

345

788

415

762

398

32

32

16

24

Min

63

66

8

8

216

221

10

13

8

12

3

4

97

104

78

52

Max

154

156

34

39

340

375

53

53

49

51

24

23

268

243

148

166

Mean

90

93

15

15

268

310

22

27

21

25

12

13

161

159

118

110

SD

14.5

14.3

4.5

5.2

26.4

31.1

5.8

6.3

4.8

5.7

3.5

3.5

39.2

32.3

17.0

29.3

POSITIVE CONTROL VALUES 2016

 

Strain

S9-Mix

TA100

TA1535

TA102

WP2uvrA

TA98

TA1537

WP2uvrA

pKM101

WP2pKM101

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

 

Values

409

406

381

386

30

28

341

335

388

385

379

381

14

24

8

16

 

Min

221

284

84

92

897

629

107

102

100

96

95

101

445

574

1674

372

 

Max

2222

2863

2994

879

2326

2140

1611

637

449

4357

1413

639

1117

1855

2823

945

 

Mean

724

1264

854

240

1633

950

718

240

186

188

406

290

743

1271

2379

535

 

SD

320.4

562.9

664.9

62.1

564.5

382.7

338.6

98.2

49.8

230.8

227.0

92.7

214.6

326.5

426.2

143.3

 

SD: Standard deviation

Min: Minimum value

Max: Maximum value

†: No. of mean values used to create dataset

Conclusions:
Under the test conditions, the test item is not considered as mutagenic in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA were exposed to the test item at the following concentrations:

- Experiment 1 - Pre incubation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix

- Experiment 2 - Pre-Incubation Method: 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate,  without S9-mix for TA100, TA1535 and TA 1537 and 5, 15, 50, 150, 500, 1500 and 5000 µg/plate for TA98 without S9. 15, 50, 150, 500, 1500and  5000 μg/plate with S9 for all strains.

Rat liver homogenate (10% liver S9 in standard co-factors) was used as a metabolizing system. Vehicle control, negative (untreated) and positive control groups were also included in mutagenicity tests.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range.  All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation.  Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

In the first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the bacterial tester strains from 150 µg/plate (TA100, TA 1535 and TA 1537) and 1500 µg/plate (TA98 and WP2uvrA) in the absence of S9 mix and to two bacterial strains (TA100 and TA1535) at 5000 µg/plate in the presence S9-mix. In the second mutation test, the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 150 µg/plate (TA1535, TA100 and TA1537) and 1500 µg/plate (TA98 and WP2uvrA).  In the presence of S9-mix, weakened bacterial background lawns were noted to three of the bacterial tester strains at 5000 µg/plate (TA100, TA1535 and WP2uvrA).  

A test item precipitate (light and globular in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 and Experiment 2 .

Under the test conditions, the test item is not considered as mutagenic in these bacterial systems.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA were exposed to the test item at the following concentrations:

- Experiment 1 - Pre incubation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix

- Experiment 2 - Pre-Incubation Method: 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate,  without S9-mix for TA100, TA1535 and TA 1537 and 5, 15, 50, 150, 500, 1500 and 5000 µg/plate for TA98 without S9. 15, 50, 150, 500, 1500and  5000 μg/plate with S9 for all strains.

Rat liver homogenate (10% liver S9 in standard co-factors) was used as a metabolizing system. Vehicle control, negative (untreated) and positive control groups were also included in mutagenicity tests.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range.  All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation.  Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

In the first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the bacterial tester strains from 150 µg/plate (TA100, TA 1535 and TA 1537) and 1500 µg/plate (TA98 and WP2uvrA) in the absence of S9 mix and to two bacterial strains (TA100 and TA1535) at 5000 µg/plate in the presence S9-mix. In the second mutation test, the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 150 µg/plate (TA1535, TA100 and TA1537) and 1500 µg/plate (TA98 and WP2uvrA).  In the presence of S9-mix, weakened bacterial background lawns were noted to three of the bacterial tester strains at 5000 µg/plate (TA100, TA1535 and WP2uvrA).  

A test item precipitate (light and globular in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 and Experiment 2 .

Under the test conditions, the test item is not considered as mutagenic in these bacterial systems.

Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding germ cell mutagenicity according to the Regulation (EC) No. 1272/2008 (CLP)and to the Globally Harmonised System of classification and labelling of chemicals (GHS).