Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: non mutagenic (OECD 471, GLP, K, rel. 1).

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:
04 August - 01 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD Guideline 471 without any deviation
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
dated 21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
dated 30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
dated August 1998
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
28 October 2016
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1003396875
- Expiration date of the lot/batch: 18 May 2021
- Purity test date: 13 July 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark (closed containers)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions were prepared in acetone by mixing on a vortex mixer. The test item was confirmed as a UVCB product and, therefore, no purity correction was required. Acetone is toxic to the bacterial cells at 0.1 mL (100 µL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 µL) aliquots (Maron et al., 1981). Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x 10^-4 microns. All formulations were used within four hours of preparation and were assumed to be stable for this period.
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: 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: In solubility checks performed in house, the test item was noted as insoluble in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully soluble in acetone at 100 mg/mL. Therefore, acetone was selected as the vehicle. Following solubility information provided by the Sponsor, sterile distilled water was not evaluated as a potential vehicle in this test system.
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
with metabolic activation
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: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 minutes in Experiment 2.
- Exposure duration: ca. 48 hours for both experiment

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). Several manual counts were 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 experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of Experiment 1, and was 15 to 5000 µg/plate. Six 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 potential toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
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.
- If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537, which have relatively low spontaneous reversion rates) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it will be considered to exhibit mutagenic activity in this test system. (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.
Statistics:
None
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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
- Precipitation: A test item film (creamy in appearance) was noted from 1500 μg/plate in both experiments in the absence and presence of S9, 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.
- No visible reduction in the growth of the bacterial background lawn was noted at any dose level, either in the presence or absence of metabolic activation (S9-mix), in Experiment 1 (plate incorporation method) and in Experiment 2 (pre incubation method).
- 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 (plate incorporation method) and Experiment 2 (pre incubation method).
- 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

94

 

29

 

27

 

31

 

12

 

87

(91)

35

(30)

22

(25)

35

(28)

14

(14)

91

 

25

 

27

 

19

 

17

 

Experiment 2

TA100

TA1535

WP2uvrA

TA98

TA1537

88

 

27

 

22

 

38

 

10

 

77

(82)

29

(27)

22

(22)

20

(28)

10

(11)

81

 

24

 

19

 

25

 

13

 

 

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

 

Test Period

From: 17 August 2017

To: 20 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)

97

108

119

(108)

11.0#

26

32

31

(30)

3.2

26

24

28

(26)

2.0

24

25

19

(23)

3.2

17

12

15

(15)

2.5

1.5 µg

111

105

102

(106)

4.6

30

30

28

(29)

1.2

36

19

33

(29)

9.1

19

15

16

(17)

2.1

16

17

15

(16)

1.0

5 µg

105

129

107

(114)

13.3

28

24

25

(26)

2.1

29

34

26

(30)

4.0

27

30

19

(25)

5.7

9

19

19

(16)

5.8

15 µg

108

104

90

(101)

9.5

30

25

23

(26)

3.6

38

27

34

(33)

5.6

14

24

17

(18)

5.1

13

15

16

(15)

1.5

50 µg

92

102

105

(100)

6.8

24

27

28

(26)

2.1

25

30

21

(25)

4.5

18

17

20

(18)

1.5

19

9

8

(12)

6.1

150 µg

108

101

125

(111)

12.3

34

29

29

(31)

2.9

28

32

28

(29)

2.3

17

20

11

(16)

4.6

21

18

14

(18)

3.5

500 µg

100

106

117

(108)

8.6

32

31

31

(31)

0.6

24

38

23

(28)

8.4

11

24

19

(18)

6.6

14

18

13

(15)

2.6

1500 µg

92 F

102 F

93 F

(96)

5.5

26 F

36 F

31 F

(31)

5.0

27 F

26 F

28 F

(27)

1.0

28 F

24 F

18 F

(23)

5.0

15 F

16 F

14 F

(15)

1.0

5000 µg

111 F

108 F

100 F

(106)

5.7

33 F

36 F

28 F

(32)

4.0

30 F

33 F

29 F

(31)

2.1

25 F

21 F

28 F

(25)

3.5

18 F

11 F

17 F

(15)

3.8

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

587

505

553

(548)

41.2

447

460

462

(456)

8.1

907

958

789

(885)

86.7

187

143

166

(165)

22.0

309

315

348

(324)

21.0

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

4NQO:4-Nitroquinoline-1-oxide

9AA:   9-Aminoacridine

F:       Film

#:       Standard deviation

 

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

 

Test Period

From: 17 August 2017

To: 20 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)

119

90

79

(96)

20.7#

32

29

26

(29)

3.0

32

45

44

(40)

7.2

35

32

38

(35)

3.0

15

19

10

(15)

4.5

1.5 µg

84

76

106

(89)

15.5

28

32

21

(27)

5.6

47

38

32

(39)

7.5

27

35

32

(31)

4.0

13

8

20

(14)

6.0

5 µg

100

91

100

(97)

5.2

31

30

24

(28)

3.8

37

36

37

(37)

0.6

26

27

26

(26)

0.6

10

15

10

(12)

2.9

15 µg

98

101

102

(100)

2.1

31

34

25

(30)

4.6

39

41

35

(38)

3.1

30

40

31

(34)

5.5

21

13

13

(16)

4.6

50 µg

85

129

95

(103)

23.1

33

29

30

(31)

2.1

41

38

46

(42)

4.0

28

29

29

(29)

0.6

12

12

6

(10)

3.5

150 µg

80

77

110

(89)

18.2

26

27

33

(29)

3.8

52

41

38

(44)

7.4

29

24

32

(28)

4.0

13

18

14

(15)

2.6

500 µg

109

104

94

(102)

7.6

32

33

35

(33)

1.5

36

36

35

(36)

0.6

33

29

40

(34)

5.6

9

13

10

(11)

2.1

1500 µg

90 F

83 F

92 F

(88)

4.7

24 F

31 F

30 F

(28)

3.8

37 F

41 F

37 F

(38)

2.3

29 F

37 F

35 F

(34)

4.2

12 F

17 F

10 F

(13)

3.6

5000 µg

92 F

91 F

80 F

(88)

6.7

28 F

31 F

25 F

(28)

3.0

40 F

38 F

44 F

(41)

3.1

29 F

38 F

25 F

(31)

6.7

15 F

12 F

13 F

(13)

1.5

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

1093

1101

1181

(1125)

48.7

264

270

262

(265)

4.2

193

194

194

(194)

0.6

161

246

234

(214)

46.0

495

476

495

(489)

11.0

2AA:   2-Aminoanthracene

BP:     Benzo(a)pyrene

F:        Film

#:       Standard deviation

 

 

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

 

Test Period

From: 29 August 2017

To: 01 September 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)

79

89

89

(86)

5.8#

11

24

14

(16)

6.8

29

19

28

(25)

5.5

32

24

36

(31)

6.1

6

7

14

(9)

4.4

15 µg

70

99

90

(86)

14.8

15

15

18

(16)

1.7

13

16

23

(17)

5.1

33

32

26

(30)

3.8

13

11

9

(11)

2.0

50 µg

80

91

90

(87)

6.1

18

18

19

(18)

0.6

20

25

21

(22)

2.6

38

30

36

(35)

4.2

9

15

6

(10)

4.6

150 µg

79

99

86

(88)

10.1

17

11

21

(16)

5.0

20

13

28

(20)

7.5

34

29

31

(31)

2.5

12

7

8

(9)

2.6

500 µg

96

80

84

(87)

8.3

17

14

15

(15)

1.5

29

28

26

(28)

1.5

28

34

27

(30)

3.8

11

9

9

(10)

1.2

1500 µg

81 F

92 F

70 F

(81)

11.0

21 F

17 F

18 F

(19)

2.1

27 F

20 F

29 F

(25)

4.7

32 F

28 F

26 F

(29)

3.1

7 F

8 F

10 F

(8)

1.5

5000 µg

94 F

80 F

86 F

(87)

7.0

20 F

18 F

17 F

(18)

1.5

26 F

23 F

27 F

(25)

2.1

34 F

36 F

30 F

(33)

3.1

7 F

8 F

6 F

(7)

1.0

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

612

641

1156

(803)

306.1

453

463

498

(471)

23.6

773

751

702

(742)

36.3

335

359

393

(362)

29.1

171

164

206

(180)

22.5

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

4NQO:4-Nitroquinoline-1-oxide

9AA:   9-Aminoacridine

F:        Film

#:       Standard deviation

 

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

 

Test Period

From: 29 August 2017

To: 01 September 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)

104

85

115

(101)

15.2#

23

23

26

(24)

1.7

29

29

28

(29)

0.6

29

32

44

(35)

7.9

10

7

14

(10)

3.5

15 µg

83

88

102

(91)

9.8

25

26

13

(21)

7.2

30

28

20

(26)

5.3

33

39

27

(33)

6.0

6

11

11

(9)

2.9

50 µg

104

124

83

(104)

20.5

21

26

20

(22)

3.2

34

18

25

(26)

8.0

34

38

35

(36)

2.1

12

14

8

(11)

3.1

150 µg

109

125

125

(120)

9.2

19

22

21

(21)

1.5

26

28

24

(26)

2.0

27

35

32

(31)

4.0

11

9

9

(10)

1.2

500 µg

109

104

117

(110)

6.6

19

23

28

(23)

4.5

35

31

39

(35)

4.0

31

36

25

(31)

5.5

10

14

7

(10)

3.5

1500 µg

95 F

107 F

114 F

(105)

9.6

21 F

19 F

18 F

(19)

1.5

28 F

31 F

30 F

(30)

1.5

39 F

35 F

36 F

(37)

2.1

7 F

14 F

12 F

(11)

3.6

5000 µg

100 F

95 F

104 F

(100)

4.5

20 F

19 F

24 F

(21)

2.6

35 F

24 F

32 F

(30)

5.7

33 F

34 F

29 F

(32)

2.6

11 F

13 F

11 F

(12)

1.2

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

998

967

1254

(1073)

157.5

230

200

193

(208)

19.7

243

273

267

(261)

15.9

148

150

134

(144)

8.7

369

349

375

(364)

13.6

2AA:   2-Aminoanthracene

BP:      Benzo(a)pyrene

F:       Film

#:       Standard deviation

 

 

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

 

COMBINED 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

 

 

 

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 - 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: 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix

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.

No visible reduction in the growth of the bacterial background lawn was noted at any dose level, either in the presence or absence of metabolic activation (S9-mix), in Experiment 1 (plate incorporation method) and in Experiment 2 (pre incubation method).

A test item film (creamy in appearance) was noted from 1500 μg/plate in both experiments in the absence and presence of S9, 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 (plate incorporation method) and Experiment 2 (pre incubation method).

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

Table 7.6/1: Summary of genotoxicity test

Test n°

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration

Statement

1

 

Thompson, 2017

Ames Test

(OECD 471)

K, rel. 1

Gene mutation

TA 1535,

TA 1537,

TA 98,

TA 100,

WP2 uvrA-

-S9

+S9

Up to cytotoxic or highest recommended concentration

-S9 : non mutagenic

+S9 : non mutagenic

 

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

- 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: 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix

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.

No visible reduction in the growth of the bacterial background lawn was noted at any dose level, either in the presence or absence of metabolic activation (S9-mix), in Experiment 1 (plate incorporation method) and in Experiment 2 (pre incubation method).

A test item film (creamy in appearance) was noted from 1500 μg/plate in both experiments in the absence and presence of S9, 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 (plate incorporation method) and Experiment 2 (pre incubation method).

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 registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information, no classification is proposed.