Registration Dossier

Administrative data

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 500 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:
09 September 2019 - 26 September 2019
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:
Type and composition of metabolic activation system:
- source of S9 : S9 fraction prepared from Sprague Dawley rat liver homogenate and provided by MOLTOXTM (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 - USA).
- method of preparation of S9 mix : 10% S9 fraction, 8 mM MgCL2-6H2O, 33 mM KCl, 5 mM Glucose-6-Phosphate Na2, 4 mM NADP Na2 and 0.1 M Phosphate buffer pH 7.4.
- concentration or volume of S9 mix and S9 in the final culture medium: 500 μL of S9-mix.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Sterility test: 500 μL of S9-mix were 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 were incubated for 48 - 72 hours at 37°C and then examined.
Test concentrations with justification for top dose:
Initial mutation test (plate incorporation) / Confirmatory mutation test (pre-incubation +S9): 5, 15, 50, 150, 250 and 500 µg/plate.
In the preliminary cytotoxicity test (strain TA100) a very high toxicity was found for doses from 700 to 5000 μg/plate. Therefore, the test item was tested at the highest dose of 500 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: the test item was found soluble in water at 5 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) Diamine Dichloride (1 μg/plate; E.coli, - S9)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
1. Plate incorporation (initial mutation test): A stock solution of the test item was prepared at 5 mg/mL. In a test tube, 0.1 mL of the bacterial suspension containing 1-9 E09 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, 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 +S9): The test item solution with the test strain, and 500 μL of S9-mix fraction are preincubated with shaking for 30 min., at 37ºC prior to mixing with the overlay agar and pouring onto the minimal agar plate.. After a 48-72 hour incubation period at 37ºC, revertant colonies are counted in each plate.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 minutes (confirmatory mutation test)
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Preliminary cytotoxicity test (Strain TA100): In a test tube 0.1 mL of the bacterial suspension (1-9 E03 bacteria /mL) and 0.1 mL of the stock solution and dilutions were successively added to 2 mL of top agar at 45ºC, containing 10 % (v/v) of a solution of L-Histidine-D-Biotine (2.5 mM). After homogenization, the content of the tube was poured onto a Petri plate (90 mm in diameter) containing minimal agar (20 mL). 3 plates per concentration were incubated for 48-72 h at 37ºC, and the colonies counted. A negative control containing the blank alone was run in parallel. In case of bacteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75% or less. The precipitate, if present, should not interfere with the scoring.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
In the bacterial reverse mutation test, mutations are detected which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain.
After a 48-72-hour incubation period at 37ºC, revertant colonies were manually counted in each plate. The following ratio was calculated per plate: R = Number of revertant colonies in the presence of the test item / Number of revertant colonies in the absence of the test item.

- 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.



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:
cytotoxicity
Remarks:
Slight thinning or thinning of the bacterial lawn for doses of 250 and 500 µg/plate.
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
Remarks:
Slight to high thinning of the bacterial lawn for doses of 150 to 500 µg/plate.
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:
cytotoxicity
Remarks:
Slight to high thinning of the bacterial lawn for doses of 150 to 500 µg/plate.
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
Remarks:
Slight thinning or thinning of the bacterial lawn for doses of 250 and 500 µg/plate.
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:
cytotoxicity
Remarks:
Slight thinning or thinning of the bacterial lawn for doses of 250 and 500 µg/plate.
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: In the preliminary cytotoxicity test (strain TA100) a very high toxicity was found for doses from 700 to 5000 μg/plate. Therefore, the test item was tested at the highest dose of 500 μg/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: see tables below.

Ames test:
- Signs of toxicity: see table 5 below.
- Individual plate counts: see table 5 below.
- Mean number of revertant colonies per plate and standard deviation: see table 5 below.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see table 6 below
- Negative (solvent/vehicle) historical control data: see table 6 below
- 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.


Table 3. Sterility control.

Series

 

Doses

Colony number/plate

Control n° 1

1

2

3

 

500 µg /plate

0

0

0

Solution of

250 µg /plate

0

0

0

 

VIOLET LCC6D dried BATCH: 3/19

150 µg /plate

0

0

0

50 µg /plate

0

0

0

(LEMI code : 19/0180-160919-S1)

15 µg /plate

0

0

0

 

5 µg /plate

0

0

0

S9-mix

500 µL/plate

0

0

0

Control n° 2

Doses

1

2

3

 

500 µg /plate

0

0

0

Solution of

250 µg /plate

0

0

0

 

VIOLET LCC6D dried BATCH: 3/19

150 µg /plate

0

0

0

50 µg /plate

0

0

0

(LEMI code : 19/0180-230919-S1)

15 µg /plate

0

0

0

 

5 µg /plate

0

0

0

S9-mix

500 µL/plate

0

0

0

Table 4. Bacteriostatic activity controls.

 

 

0

(negative control)

Water

150µg

250µg

350µg

500µg

700µg

1000µg

2500µg

5000µg

Solution of

VIOLET LCC6D dried BATCH: 3/19

 

LEMI code : 19/0180-110919-S1

N1

776

759

432

370

184

186

2

0

0

0

N2

674

725

382

329

181

156

9

0

0

0

N3

758

711

438

368

194

177

8

0

0

0

N

736±54

732±25

417±31

356±23

186±7

173±15

6±4

0±0

0±0

0±0

%

-

99%

57%

48%

25%

24%

1%

0%

0%

0%

 

 

0

(negative control)

Water

50 µg

150 µg

250 µg

500 µg

700 µg

1000 µg

Solution of

VIOLET LCC6D dried BATCH: 3/19

 

LEMI code : 19/0180-130919-S1

N1

623

633

631

396

240

180

0

0

N2

667

641

645

198

256

138

0

0

N3

616

652

634

139

314

191

0

0

N

635±28

642±10

637±7

244± 135

270±39

170± 28

0±0

0±0

%

-

101%

100%

38%

42%

27%

0%

0%

 

 

0

(negative control)

Water

5 µg

15 µg

50 µg

150 µg

250 µg

500 µg

Solution of

VIOLET LCC6D dried BATCH: 3/19

 

LEMI code : 19/0180-160919-S1

N1

669

657

751

678

583

314

280

181

N2

718

671

683

712

588

293

258

133

N3

704

709

711

659

592

339

256

201

N

697±25

679±27

715 ± 34

683±27

588±5

315 ± 23

265±13

172±35

%

-

97%

103%

98%

84%

45%

38%

25%

 

 

0

(negative control)

Water

5 µg

15 µg

50 µg

150 µg

250 µg

500 µg

Solution of

VIOLET LCC6D dried BATCH: 3/19

 

LEMI code : 19/0180-230919-S1

N1

671

636

629

613

526

402

301

195

N2

687

644

675

671

584

356

259

123

N3

699

686

681

643

601

333

236

215

N

686±14

655±27

662±28

642±29

570±39

364 ± 35

265±33

178±48

%

-

96%

96%

94%

83%

53%

39%

26%

Table 5. Result tables.

TA1535 Assay n°1 – without metabolic activation (-S9-mix)

 

Serie

 

Dose/Plate

Plate

 

Mean

 

Standarddeviation

 

R

n° 1

n° 2

n° 3

Negative control

100 µL

13

11

13

12.33

1.15

_

Positive control solvent

5 µL

12

12

12

12.00

0.00

_

Positive control :

Sodium azide

5 µg

in 5 µL

1023

987

1058

1022.67

35.50

85.22

Vehicle

100 µL

9

10

10

9.67

0.58

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-160919-S1

500 µg***

2

4

1

2.33

1.53

0.24

250 µg**

8

7

15

10.00

4.36

1.03

150 µg*

16

10

14

13.33

3.06

1.38

50 µg

18

12

15

15.00

3.00

1.55

15 µg

12

14

18

14.67

3.06

1.52

5 µg

17

12

18

15.67

3.21

1.62

TA1535 Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

12

11

16

13.00

2.65

_

Positive control solvent

20 µL

17

15

17

16.33

1.15

_

Positive control :

2-Anthramine

2 µg

in 20 µL

87

85

95

89.00

5.29

5.45

Vehicle

100 µL

13

10

15

12.67

2.52

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-160919-S1

500 µg***

4

5

3

4.00

1.00

0.32

250 µg*

7

23

3

11.00

10.58

0.87

150µg

10

17

13

13.33

3.51

1.05

50 µg

18

14

7

13.00

5.57

1.03

15 µg

18

10

5

11.00

6.56

0.87

5 µg

10

18

17

15.00

4.36

1.18

TA1535 Assay n°2 – without metabolic activation (-S9-mix)

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

23

11

14

16.00

6.24

_

Positive control solvent

5 µL

15

12

18

15.00

3.00

_

Positive control :

Sodium azide

5 µg

in 5 µL

1175

1147

1245

1189.00

50.48

79.27

Vehicle

100 µL

15

12

11

12.67

2.08

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-230919-S1

500 µg***

1

8

3

4.00

3.61

0.32

250 µg**

8

3

12

7.67

4.51

0.61

150 µg*

11

8

17

12.00

4.58

0.95

50 µg

8

13

8

9.67

2.89

0.76

15 µg

4

16

13

11.00

6.24

0.87

5 µg

8

15

14

12.33

3.79

0.97

TA1535 Assay n°2 – with metabolic activation (10 % S9-mix) – with pre-incubation

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

21

18

11

16.67

5.13

_

Positive control solvent

10 µL

19

13

8

13.33

5.51

_

Positive control :

2-Anthramine

1 µg

in 10 µL

77

80

99

85.33

11.93

6.40

Vehicle

100 µL

16

13

19

16.00

3.00

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-230919-S1

500 µg***

6

9

3

6.00

3.00

0.38

250 µg*

15

14

12

13.67

1.53

0.85

150 µg

13

17

13

14.33

2.31

0.90

50 µg

10

9

19

12.67

5.51

0.79

15 µg

17

16

10

14.33

3.79

0.90

5 µg

10

19

10

13.00

5.20

0.81

TA1537 Assay n°1 – without metabolic activation (-S9-mix)

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

6

16

4

8.67

6.43

_

Positive control solvent

20 µL

8

5

5

6.00

1.73

_

Positivecontrol:

9-Aminoacridine

50 µg

in 20 µL

1856

1958

1482

1765.33

250.62

294.22

Vehicle

100 µL

12

2

8

7.33

5.03

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-160919-S1

500 µg**

3

4

5

4.00

1.00

0.55

250 µg*

12

8

10

10.00

2.00

1.36

150 µg

12

15

8

11.67

3.51

1.59

50 µg

9

10

11

10.00

1.00

1.36

15 µg

7

3

8

6.00

2.65

0.82

5 µg

8

6

7

7.00

1.00

0.95

TA1537 Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

16

6

8

10.00

5.29

_

Positive control solvent

20 µL

10

11

9

10.00

1.00

_

Positive control :

2-Anthramine

2 µg

in 20 µL

26

38

39

34.33

7.23

3.43

Vehicle

100 µL

12

7

11

10.00

2.65

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-160919-S1

500 µg**

4

4

5

4.33

0.58

0.43

250 µg*

6

9

9

8.00

1.73

0.80

150µg

12

9

11

10.67

1.53

1.07

50 µg

15

9

19

14.33

5.03

1.43

15 µg

9

15

19

14.33

5.03

1.43

5 µg

6

10

12

9.33

3.06

0.93

TA1537 Assay n°2 – without metabolic activation (-S9-mix)

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

19

11

15

15.00

4.00

_

Positive control solvent

20 µL

8

8

14

10.00

3.46

_

Positivecontrol:

9-Aminoacridine

50 µg

in 20 µL

1058

1042

1312

1137.33

151.48

113.73

Vehicle

100 µL

18

16

12

15.33

3.06

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-230919-S1

500 µg**

4

1

6

3.67

2.52

0.24

250 µg*

7

10

7

8.00

1.73

0.52

150 µg

13

8

15

12.00

3.61

0.78

50 µg

7

11

11

9.67

2.31

0.63

15 µg

15

7

15

12.33

4.62

0.80

5 µg

10

15

10

11.67

2.89

0.76

TA1537 Assay n°2 – with metabolic activation (10% S9-mix) – with pre-incubation

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

19

20

15

18.00

2.65

_

Positive control solvent

10 µL

14

8

13

11.67

3.21

_

Positive control :

2-Anthramine

1 µg

in 10 µL

45

32

49

42.00

8.89

3.60

Vehicle

100 µL

14

21

18

17.67

3.51

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-230919-S1

500 µg**

6

6

3

5.00

1.73

0.28

250 µg*

17

15

19

17.00

2.00

0.96

150 µg

13

12

11

12.00

1.00

0.68

50 µg

15

12

20

15.67

4.04

0.89

15 µg

20

16

18

18.00

2.00

1.02

5 µg

17

13

11

13.67

3.06

0.77

TA98 Assay n°1 – without metabolic activation (-S9-mix)

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

12

17

18

15.67

3.21

_

Positive control solvent

20 µL

17

15

14

15.33

1.53

_

Positive control :

2-Nitrofluorene

2 µg

in 20 µL

383

402

379

388.00

12.29

25.30

Vehicle

100 µL

17

21

15

17.67

3.06

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-160919-S1

500 µg**

10

9

16

11.67

3.79

0.66

250 µg*

21

25

38

28.00

8.89

1.58

150 µg

38

34

30

34.00

4.00

1.92

50 µg

26

26

29

27.00

1.73

1.53

15 µg

23

23

15

20.33

4.62

1.15

5 µg

16

18

20

18.00

2.00

1.02

TA98 Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

17

28

19

21.33

5.86

_

Positive control solvent

20 µL

28

15

19

20.67

6.66

_

Positive control :

2-Anthramine

2 µg

in 20 µL

342

436

416

398.00

49.52

19.26

Vehicle

100 µL

15

24

19

19.33

4.51

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-160919-S1

500 µg**

24

28

16

22.67

6.11

1.17

250 µg*

27

37

27

30.33

5.77

1.57

150 µg

12

24

33

23.00

10.54

1.19

50 µg

28

28

22

26.00

3.46

1.34

15 µg

30

29

24

27.67

3.21

1.43

5 µg

30

32

23

28.33

4.73

1.47

TA98 Assay n°2 – without metabolic activation (-S9-mix)

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

24

16

11

17.00

6.56

_

Positive control solvent

20 µL

18

18

11

15.67

4.04

_

Positive control :

2-Nitrofluorene

2 µg

in 20 µL

388

338

410

378.67

36.90

24.17

Vehicle

100 µL

18

18

26

20.67

4.62

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-230919-S1

500 µg**

15

13

17

15.00

2.00

0.73

250 µg*

38

31

26

31.67

6.03

1.53

150 µg

31

27

22

26.67

4.51

1.29

50 µg

29

23

18

23.33

5.51

1.13

15 µg

21

14

13

16.00

4.36

0.77

5 µg

15

10

14

13.00

2.65

0.63

TA98 Assay n°2 – with metabolic activation (10 % S9-mix) – with pre-incubation

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

23

33

28

28.00

5.00

_

Positive control solvent

10 µL

28

17

18

21.00

6.08

_

Positive control :

2-Anthramine

1 µg

in 10 µL

237

264

272

257.67

18.34

12.27

Vehicle

100 µL

31

31

29

30.33

1.15

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-230919-S1

500 µg**

32

26

35

31.00

4.58

1.02

250 µg*

24

17

33

24.67

8.02

0.81

150 µg

26

27

24

25.67

1.53

0.85

50 µg

20

25

23

22.67

2.52

0.75

15 µg

21

25

26

24.00

2.65

0.79

5 µg

34

19

20

24.33

8.39

0.80

TA100 Assay n°1 – without metabolic activation (-S9-mix)

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

82

93

70

81.67

11.50

_

Positive control solvent

20 µL

91

82

85

86.00

4.58

_

Positive control :

Sodium azide

20 µg

in 20 µL

1590

1460

1149

1399.67

226.61

16.28

Vehicle

100 µL

80

79

90

83.00

6.08

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-160919-S1

500 µg***

5

5

3

4.33

1.15

0.05

250 µg**

99

94

89

94.00

5.00

1.13

150 µg*

100

106

97

101.00

4.58

1.22

50 µg

90

95

98

94.33

4.04

1.14

15 µg

84

101

106

97.00

11.53

1.17

5 µg

60

75

77

70.67

9.29

0.85

TA100 Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

108

113

125

115.33

8.74

_

Positive control solvent

20 µL

70

114

94

92.67

22.03

_

Positive control :

2-Anthramine

2 µg

in 20 µL

1047

696

792

845.00

181.40

9.12

Vehicle

100 µL

79

118

95

97.33

19.60

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-160919-S1

500 µg***

6

3

2

3.67

2.08

0.04

250 µg*

132

150

123

135.00

13.75

1.39

150 µg

124

118

95

112.33

15.31

1.15

50 µg

110

107

94

103.67

8.50

1.07

15 µg

98

103

99

100.00

2.65

1.03

5 µg

79

94

104

92.33

12.58

0.95

TA100 Assay n°2 – without metabolic activation (-S9-mix)

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

105

53

63

73.67

27.59

_

Positive control solvent

20 µL

98

84

76

86.00

11.14

_

Positive control :

Sodium azide

20 µg

in 20 µL

1510

1413

1577

1500.00

82.46

17.44

Vehicle

100 µL

61

78

62

67.00

9.54

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-230919-S1

500 µg***

9

7

6

7.33

1.53

0.11

250 µg**

95

95

74

88.00

12.12

1.31

150 µg*

107

83

94

94.67

12.01

1.41

50 µg

91

80

97

89.33

8.62

1.33

15 µg

95

85

79

86.33

8.08

1.29

5 µg

98

90

84

90.67

7.02

1.35

TA100 Assay n°2 – with metabolic activation (10 % S9-mix) – with pre-incubation

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

104

93

79

92.00

12.53

_

Positive control solvent

10 µL

109

98

111

106.00

7.00

_

Positive control :

2-Anthramine

1 µg

in 10 µL

383

420

552

451.67

88.84

4.26

Vehicle

100 µL

100

102

108

103.33

4.16

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-230919-S1

500 µg***

9

10

2

7.00

4.36

0.07

250 µg*

69

99

58

75.33

21.22

0.73

150 µg

76

84

90

83.33

7.02

0.81

50 µg

92

93

85

90.00

4.36

0.87

15 µg

105

95

111

103.67

8.08

1.00

5 µg

112

103

90

101.67

11.06

0.98

E. COLI Assay n°1 – without metabolic activation (-S9-mix)

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

87

111

82

93.33

15.50

_

Positive control solvent

10 µL

120

89

115

108.00

16.64

_

Positivecontrol:

cis-Platinum(II)

1 µg

in 10 µL

244

328

356

309.33

58.29

2.86

Vehicle

100 µL

97

127

121

115.00

15.87

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-160919-S1

500 µg**

115

101

128

114.67

13.50

1.00

250 µg*

121

162

153

145.33

21.55

1.26

150 µg

190

183

182

185.00

4.36

1.61

50 µg

173

164

175

170.67

5.86

1.48

15 µg

179

123

159

153.67

28.38

1.34

5 µg

147

161

153

153.67

7.02

1.34

E. COLI Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

262

241

216

239.67

23.03

_

Positive control solvent

5 µL

229

231

214

224.67

9.29

_

Positive control :

Dimethylbenzanthracene

5 µg

in 5 µL

471

510

499

493.33

20.11

2.20

Vehicle

100 µL

253

227

242

240.67

13.05

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-160919-S1

500 µg**

235

242

252

243.00

8.54

1.01

250 µg*

218

239

263

240.00

22.52

1.00

150 µg

227

200

214

213.67

13.50

0.89

50 µg

225

211

225

220.33

8.08

0.92

15 µg

236

194

236

222.00

24.25

0.92

5 µg

258

236

253

249.00

11.53

1.03

E. COLI Assay n°2 – without metabolic activation (-S9-mix)

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

132

133

130

131.67

1.53

_

Positive control solvent

10 µL

140

132

129

133.67

5.69

_

Positivecontrol:

cis-Platinum(II)

1 µg

in 10 µL

410

350

423

394.33

38.94

2.95

Vehicle

100 µL

121

115

126

120.67

5.51

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-230919-S1

500 µg**

88

105

131

108.00

21.66

0.90

250 µg*

161

159

163

161.00

2.00

1.33

150 µg

142

138

170

150.00

17.44

1.24

50 µg

153

171

135

153.00

18.00

1.27

15 µg

174

140

181

165.00

21.93

1.37

5 µg

135

162

158

151.67

14.57

1.26

E. COLI Assay n°2 – with metabolic activation (10 % S9-mix) – with pre-incubation

 

Serie

 

Dose/Plate

Plate

Mean

Standarddeviation

R

n° 1

n° 2

n° 3

Negative control

100 µL

183

188

198

189.67

7.64

_

Positive control solvent

5 µL

210

190

175

191.67

17.56

_

Positive control :

Dimethylbenzanthracene

2.5 µg

in 5 µL

485

516

537

512.67

26.16

2.67

Vehicle

100 µL

207

236

221

221.33

14.50

_

Solution of

VIOLET LCC6D dried BATCH: 3/19

LEMI code : 19/0180-230919-S1

500 µg**

211

192

195

199.33

10.21

0.90

250 µg*

180

194

224

199.33

22.48

0.90

150 µg

206

193

209

202.67

8.50

0.92

50 µg

212

196

197

201.67

8.96

0.91

15 µg

208

207

218

211.00

6.08

0.95

5 µg

216

213

204

211.00

6.24

0.95

* slight thinning of the bacterial lawn

** thinning of the bacterial lawn

*** high thinning of the bacterial lawn

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 500 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 5 to 500 µg/plate in water, with and without metabolic activation, according to preliminary assays. The metabolic activation system (S9 fraction) was prepared from Sprague Dawley rat liver homogenate (provided by MOLTOX). Two independent assays were performed: an initial mutation test (plate incorporation method) and a confirmatory mutation test (plate incorporation method without S9 and pre-incubation method with S9) 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 500 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 for mutagenecity in accordance with CLP Regulation (EC) No. 1272/2008.