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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicty in vitro:

Key study: OECD 471, GLP study. The test item at tested doses do not induce significant increase in the number of revertants in Salmonella typhimurium and in Escherichia coli, either with or without metabolic activation. Therefore the test item was 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:
December 8, 2016 - January 5, 2017
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
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes


Target gene:
Salmonella typhimurium - histidine
Escherichia coli - tryptophane
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: delta uvr B and rfa
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
other: delta uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prerpared from rat liver homogenate
Test concentrations with justification for top dose:
ASSAY 1: 5 000, 4000, 1 500, 500, 150 and 50 μg/plate - the highest concetration showed one toxicity consistent with the maximum tolerated 75%. A supplementary dose at 4000 μg/plate was tested to be sure this hight toxicity no hindering the scoring of the revertants.
ASSAY 2: 5 000, 1 500, 500, 150 and 50 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no react with test item and is compatible with the survival of the bacteria and S9 activity.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
NaCL 0.15 M, Acetone, DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-Anthramine, cis-Platinum (II) Diammine Dichloride
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- plate incorporation method: ASSAY 1- Performed with and without metabolic activation. Test item tested at dose level (5 000, 4000, 1 500, 500, 150 and 50 μg/plate). The test item diluted in DMSO or controls (0.1 mL) and other components (overnight culture of test strain(0.1mL), phosphate buffer or S9 mix (0.5mL)) were prepared freshly and added to 2 mL of overlay agar, maintained supercooled at 45° C, containing 10 % (v/v) of a L-Histidine-D-Biotine solution (0.5 mM) in case of salmonella strain or containing 5% (v/v) of nutrient broth n° 2 to which are added 5 μL of a L-Tryptophane solution at 2 mg/mL in case of escherichia coli strain.
This solution was mixed and poured on the surface of minimal agar plates. For the assay with metabolic activation 500 μL of S9-mix fraction were quickly added, before pouring the mixture onto the plates. After preparation, the plates were incubated at 37°C for 48 hours.

- pre-incubation: ASSAY 2- Performed with and without metabolic activation with dose level (5 000, 1 500, 500, 150 and 50 μg/plate.) Solution of the test item with the test strain were preincubated with shaking for 30 min., at 37° C prior to mixing with the overlay agar and pouring onto the minimal agar plate. When metabolic activation is used, 500 μL of S9-mix fraction are added before the pre-incubation.
- Cell density at seeding (if applicable):1-9 x109 bacteria/mL

If the first assay is positive, the second one is performed in the same manner.
If the first assay, in presence of test item, is negative, the pre-incubation test is performed for the second assay.

DURATION
- Preincubation period:30 min at 37ºC (Assay 2)
- Exposure duration:48-72 hours at 37°C

SELECTION AGENT (mutation assays): absence histidine or tryptophane,the lack of amino-acid in the medium.
Mutants can grow only if they can produce essential amino acid.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY (Strain TA100)
- Method: relative total growth
A preliminary cytotoxicity test was performed with the strain TA100 to determine any sign of cytotoxicity. Test substance was tested at dose level 5 000, 4000, 1 500, 500, 150 and 50 μg/plate. Suplementary dose 4000 μg/plate tested on ASSAY 1 because of sign of toxicity at the highest dose level.
In a test tube 0.1 mL of the bacterial suspension (1-9 x 103 bacteria/mL) and 0.1 mL of the stock solution and dilutions, are successively added to 2 mL of top agar at 45°C, containing 10 % (v/v) of a 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). The three plates per concentration are incubated for 48-72 hours at 37°C, and the colonies counted. A negative control containing the blank alone is run in parallel. In case bacteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75 % or less.

OTHER :
Sterility tests:
Test item and the corresponding dilutions are added to 2 mL of top agar maintained at 45°C, 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 37°C and then examined. There should be no bacterial growth on any plate. S9-mix sterility is checked using the same protocol. See table No.3
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.
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:
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:
E. coli WP2 uvr A pKM 101
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: No precipitation observed

RANGE-FINDING/SCREENING STUDIES: The preliminary cytotoxicity testing on strain TA100 showed in presence of 5 000 μg/plate one toxicity consistent with the maximum tolerated 75%. A supplementary dose at 4 000 μg/plate (80 % of the maximal dose) was tested on the first mutagenic assay to be sure this hight toxicity no hindering the scoring of the revertants.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
without metabolic activation:
TA 1535 Sodium Azide, N=975, Mean=702.3+-203.4, (min/max=190.0/1481.0)
TA 1537 9-Aminoacridine, N=975, Mean=851.2+-428.1 (min/max=219.0/1967.0)
TA 98 2-Nitrofluorene N=975, Mean=512.6+-219.5 (min/max=187.0/1667.0)
TA100 Sodium Azide, N=975, Mean=934.4+-325.2 (min/max=381.0/1690.0)
E.coli WP2 CIS-PLATINUM (II) DIAMINUM DICHLORIDE N=717, Mean=502.8+-168.1 (min/max=248.0/1089.0)

with metabolic activation, without pre-incubation
TA 1535 2-Anthramine, N=525, Mean=12.3+-4.0, (min/max=3.0/23.0)
TA 1537 2-Anthramine, N=525, Mean=55.8+-23.4 (min/max=24.0+-170.0)
TA 98 2-Anthramine, N=525, Mean=574.5+-209.5 (min/max=219.0/1499.0)
TA 100 2-Anthramine, N=522, Mean=862.1+-359.1 (min/max=361.0/2163.0)
E.coli WP2 dimethyl benzoanthracane N=372, Mean=707.3+-248.0 (min/max=378.0/1680.0)

with metabolic activation, with pre-incubation
TA 1535 2-Anthramine, N=519, Mean=73.2+-34.9, (min/max=25.0/185.0)
TA 1537 2-Anthramine, N=519, Mean=49.5+-22.5 (min/max=21.0/182.0)
TA 98 2-Anthramine, N=519 Mean=474.6+-196.3, (min/max=174.0/1370.0)
TA 100 2-Anthramine, N=519, Mean=682.4+-290.0 (min/max=309.0/1839.0)
E.coli WP2 dimethyl benzoanthracane N=372, Mean=701.8+-229.7 (min/max=397.0/1680.0)

- Negative (solvent/vehicle) historical control data (with ranges, means and standard deviation and confidence interval (e.g. 95%):
without metabolic activation
TA1535 N=975, Mean= 10.9+-3.6 (min/max=40/230)
TA1537 N=975, Mean= 6.0+-2.4 (min/max=10/140)
TA 98,N=975, Mean= 16.0+-3.8 (min/max=60/290)
TA 100 N=975, Mean= 62.2+-14.3 (min/max=410/126.0)
E.coli WP2 N=717, Mean= 75.0+-29.2 (min/max=410/188.0)

with metabolic activation, without pre-incubation
TA1535 N=525, Mean= 12.3+-4.0 (min/max=30/23.0)
TA1537 N=525, Mean= 8.0+-3.1 (min/max=1.0/24.0)
TA 98,N=525, Mean= 23.2+-4.8 (min/max=12.0/38.0)
TA 100 N=522 Mean= 101.7+-22.9 (min/max=58.0/189.0)
E.coli WP2 N=372, Mean= 154.8+-33.6 (min/max=80.0/264.0)

with metabolic activation, with pre-incubation
TA1535 N=519, Mean= 12.7+-4.2 (min/max=5.0/25.0)
TA1537 N=519, Mean= 8.3+-3.2 (min/max=1.0/19.0)
TA 98,N=519, Mean= 23.3+-5.2 (min/max=11.0/36.0)
TA 100 N=519, Mean= 101.3+-24.8(min/max=51.0/189.0)
E.coli WP2 N=372, Mean= 157.5+-35.4 (min/max=69.0/250.0),

Table No.3: Sterility control

Serie

Doses

                      Colony number/plate

Control nº1

 

1

2

3

Solution of test item

Lemi code :

GGA191216-S2

 

5000ug/plate

0

0

0

4000ug/plate

0

0

0

1500ug/plate

0

0

0

500ug/plate

0

0

0

150ug/plate

0

0

0

 

50ug/plate

 

 

 

S9-mix

500ul/plate

0

0

0

Control nº2

 

1

2

3

Solution of test item

Lemi code :

GGA020117-S2

 

5000ug/plate

0

0

0

1500ug/plate

0

0

0

500ug/plate

0

0

0

150ug/plate

0

0

0

50ug/plate

0

0

0

S9-mix

500ul/plate

0

0

0

Table No. 4: Bacteriostatic activity

 

 

0

Negative control

DMSO

50ug

150ug

500ug

1500ug

2500ug

5000ug

Solution of test item

Lemi code :

GGA081216-S2

 

N1

259

311

279

304

298

261

279

68

N2

288

307

311

309

297

272

278

74

N3

304

255

301

287

284

304

302

76

N4

284+-23

291+-31

297+-16

300+-12

293+-8

279+-22

286+-14

73+-4

%

   -

103

105

106

103

98

101

26

 

 

0

Negative control

DMSO

2500ug

3000ug

4000ug

5000ug

 

Solution of test item

Lemi code : GGA121216-S1

 

N1

388

428

341

358

205

89

 

N2

330

329

334

301

229

88

 

N3

394

401

370

326

158

95

 

N4

371+-35

386+-51

348+-19

328+-29

197+-36

91+-4

 

%

   -

104

94

89

53

24

 

 

0

Negative control

DMSO

50ug

150ug

500ug

1500ug

4000ug

5000ug

Solution of test item

Lemi code :

GGA191216-S2

 

N1

221

220

263

232

248

178

150

59

N2

241

241

246

234

245

218

148

64

N3

223

248

238

198

231

168

139

66

N4

228+-11

236+-15

249+-13

221+-20

241+-9

188+-26

146+-6

63+-4

%

   -

104

109

97

106

82

64

28

N1 Number of colonies in plate 1

N2 Number of colonies in plate 2

N3 Number of colonies in plate 3

N Mean per plate

% Percent of survival compared to negative control

Table No.5 : TA 1535, ASSAY 1 without metabolic activation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

7

6

7

6.67

0.58

  -

Positive control solvent

5uL

6

8

6

6.67

1.15

  -

Positive control

Sodium Azide

5ug

In 5uL

513

616

583

570.67

52.60

85.6

Vehicle

50uL

8

10

5

7.67

2.52

  -

Solution of test item

Lemi code GGA191216-S2

5000ug

2

2

2

2.00

0.00

0.26

4000ug

10

18

5

11.00

6.56

1.43

1500ug

9

8

6

7.67

1.53

1.00

500ug

4

11

6

7.00

3.61

0.91

150ug

14

8

8

10.00

3.46

1.30

50ug

6

14

10

10.00

4.00

1.30

Table No.6: TA 1535 Assay 1 – with metabolic activation (10 % S9-mix) – without pre-incubation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

14

8

5

9.00

4.58

  -

Positive control solvent

20uL

7

6

5

6.00

1.00

  -

Positive control

2-Anthramine

 

2ug

In 20uL

90

71

61

74.00

14.73

12.33

Vehicle

50uL

15

8

11

11.33

3.51

  -

Solution of test item

Lemi code GGA191216-S2

5000ug

5

5

5

5.00

0.00

0.44

4000ug

5

6

5

5.33

0.58

0.47

1500ug/

5

13

9

9.00

4.00

0.79

500ug

10

9

10

9.67

0.58

0.85

150ug

15

21

16

17.33

3.21

1.53

50ug

17

13

6

12.00

5.57

1.06

Table No.7: TA 1535 Assay 2 – without metabolic activation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

14

16

7

12.33

4.73

  -

Positive control solvent

5uL

11

12

12

11.67

0.58

  -

Positive control

Sodium azide

5 ug

In 5uL

735

642

692

689.67

46.54

59.11

Vehicle

50uL

14

14

16

14.67

1.15

  -

Solution of test item

Lemi code :GGA020117-S2

 

5000ug

7

12

15

11.33

4.04

0.77

1500ug

15

10

11

12.00

2.65

0.82

500ug

8

7

13

9.33

3.21

0.64

150ug

14

13

12

13.00

1.00

0.89

50ug

17

8

12

12.33

4.51

0.84

Table No.8: TA 1535 Assay 2 – with metabolic activation (10 % S9-mix) – with pre-incubation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

13

17

11

13.67

3.06

  -

Positive control solvent

10uL

12

12

10

11.33

1.15

  -

Positive control

2-Anthranine

1 ug

In 10uL

64

49

67

60.00

9.64

5.29

Vehicle

50uL

14

16

14

14.67

1.15

  -

Solution of test item

Lemi code :GGA020117-S2

 

5000ug*

6

10

1

5.67

4.51

0.39

1500ug*

5

3

3

3.67

1.15

0.25

500ug

10

15

20

15.00

5.00

1.02

150ug

23

14

14

17.00

5.20

1.16

50ug

9

10

10

9.67

0.58

0.66

* Moderate thinning of the background bacterial lawn.

Table No.9: TA 1537 Assay 1 – without metabolic activation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

5

5

7

5.67

1.15

  -

Positive control solvent

20uL

4

3

6

4.33

1.53

  -

Positive control

9-aminoacridine

 

50ug

In 20uL

1055

1110

933

1032.67

90.59

238.31

Vehicle

50uL

4

5

2

3.67

1.53

  -

Solution of test item

Lemi code GGA191216-S2

5000ug

2

2

1

1.67

0.58

0.45

4000ug

2

5

5

4.00

1.73

1.09

1500ug

4

3

4

3.67

0.58

1.00

500ug

5

5

5

5.00

0.00

1.36

150ug

6

6

3

5.00

1.73

1.36

50ug

6

3

2

3.67

2.08

1.00

Table No.10: TA 1537, Assay 1 – with metabolic activation (10 % S9-mix) – without pre-incubation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

7

4

6

5.67

1.53

  -

Positive control solvent

20uL

12

8

10

10.00

2.00

  -

Positive control

2-Anthramine

 

2ug

In 20uL

25

39

30

31.33

7.09

3.13

Vehicle

50uL

5

6

6

5.67

0.58

  -

Solution of test item

Lemi code GGA191216-S2

5000ug

2

3

1

2.00

1.00

0.35

4000ug

1

1

2

1.33

0.58

0.24

1500ug

3

5

3

3.67

1.15

0.65

500ug

3

6

9

6.00

3.00

1.06

150ug

9

10

8

9.00

1.00

1.59

50ug

13

9

10

10.67

2.08

1.88

Table No.11, TA 1537, Assay 2 – without metabolic activation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

7

4

4

5.00

1.73

  -

Positive control solvent

20uL

3

2

4

3.00

1.00

  -

Positive control

9-aminoacridine

50 ug

In 20 uL

1716

1860

1415

1663.67

227.07

554.56

Vehicle

50uL

3

2

2

2.33

0.58

  -

Solution of test item

Lemi code :GGA020117-S2

 

5000ug

5

4

3

4.00

1.00

1.71

1500ug

5

5

3

4.33

1.15

1.86

500ug

3

2

1

2.00

1.00

0.86

150ug

2

2

2

2.00

0.00

0.86

50ug

3

2

4

3.00

1.00

1.29

Table No.12: TA 1537, Assay 2 – Assay 2 – with metabolic activation (10% S9-mix) – with pre-incubation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

8

5

9

7.33

2.08

  -

Positive control solvent

10uL

2

3

5

3.33

1.53

  -

Positive control

2-Anthramine

 

1 ug

In 10 uL

28

25

22

25.00

3.00

7.50

Vehicle

50uL

5

4

3

4.00

1.00

  -

Solution of test item

Lemi code :GGA020117-S2

 

5000ug

5

5

4

4.67

0.58

1.17

1500ug

7

5

6

6.00

1.00

1.5

500ug

7

4

5

5.33

1.53

1.33

150ug

7

6

6

6.33

0.58

1.58

50ug

4

5

7

5.33

1.53

1.33

Table No.13: TA 98 Assay 1 – without metabolic activation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

22

11

22

18.33

6.35

  -

Positive control solvent

20uL

22

25

18

21.67

3.51

  -

Positive control

2-Nitrofluorene

 

2ug

In 20uL

191

229

217

212.33

19.43

9.80

Vehicle

50uL

35

24

16

25.00

9.54

  -

Solution of test item

Lemi code GGA191216-S2

5000ug*

27

23

24

24.67

2.08

0.99

4000ug*

15

25

19

19.67

5.03

0.79

1500ug

22

26

24

24.00

2.00

0.96

500ug

11

24

32

22.33

10.60

0.89

150ug

26

19

28

24.33

4.73

0.97

50ug

18

18

21

19.00

1.73

0.76

Table No.14: TA 98 Assay 1 – with metabolic activation (10 % S9-mix) – without pre-incubation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

18

22

23

21.00

2.65

  -

Positive control solvent

20uL

30

23

34

29.00

5.57

  -

Positive control

2-Anthramine

 

2ug

In 20uL

253

233

239

241.67

10.26

8.33

Vehicle

50uL

24

26

19

23.00

3.61

  -

Solution of test item

Lemi code GGA191216-S2

5000ug*

26

16

22

21.33

5.03

0.93

4000ug*

26

15

19

20.00

5.57

0.87

1500ug

18

16

18

17.33

1.15

0.75

500ug

30

33

26

29.67

3.51

1.29

150ug

31

36

24

30.33

6.03

1.32

50ug

35

31

26

30.67

4.51

1.33

* Moderate thinning of the background bacterial lawn.

Table No.15: TA 98 Assay 2 – without metabolic activation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

27

13

22

20.67

7.09

  -

Positive control solvent

20uL

17

17

16

16.67

0.58

  -

Positive control

2-Nitrofluorene

 

2 ug

In 20 uL

252

266

312

276.67

31.39

16.60

Vehicle

50uL

21

20

20

20.33

0.58

  -

Solution of test item

Lemi code :GGA020117-S2

 

5000ug*

7

19

15

13.67

6.11

0.67

1500ug*

16

16

14

15.33

1.15

0.75

500ug

14

17

14

15.00

1.73

0.74

150ug

28

18

13

19.67

7.64

0.97

50ug

22

13

17

17.33

4.51

0.85

Table No.16, TA 98 Assay 2 – with metabolic activation (10 % S9-mix) – with pre-incubation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

28

26

26

26.67

1.15

  -

Positive control solvent

10uL

19

16

32

22.33

8.50

  -

Positive control

2-Anthramine

 

1 ug

In 10 uL

254

259

237

250.00

11.53

11.19

Vehicle

50uL

31

24

26

27.00

3.61

  -

Solution of test item

Lemi code :GGA020117-S2

 

5000ug*

8

9

6

7.67

1.53

0.28

1500ug*

12

9

13

11.33

2.08

0.42

500ug

14

29

16

19.67

8.14

0.73

150ug

11

18

22

17.00

5.57

0.63

50ug

12

14

23

16.33

5.86

0.60

* Moderate thinning of the background bacterial lawn.

Table No.17: TA 100, Assay 1 – without metabolic activation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

54

53

51

52.67

1.53

  -

Positive control solvent

20uL

57

52

50

53.00

3.61

  -

Positive control

Sodium azide

 

20ug

In 20uL

1334

1134

1256

1241.33

100.80

23.42

Vehicle

50uL

74

65

53

64.00

10.54

  -

Solution of test item

Lemi code GGA191216-S2

5000ug*

42

47

30

39.67

8.74

0.62

4000ug*

29

30

26

28.33

2.08

0.44

1500ug

49

52

65

55.33

8.50

0.86

500ug

72

62

73

69.00

6.08

1.08

150ug

76

60

67

67.67

8.02

1.06

50ug

68

55

73

65.33

9.29

1.02

Table No.18: TA 100, Assay 1 – with metabolic activation (10 % S9-mix) – without pre-incubation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

71

72

91

78.00

11.27

  -

Positive control solvent

20uL

72

73

72

72.33

0.58

  -

Positive control

2-Anthramine

 

2 ug

In 20uL

392

373

395

386.67

11.93

5.35

Vehicle

50uL

81

85

75

80.33

5.03

  -

Solution of test item

Lemi code GGA191216-S2

5000ug*

27

21

30

26.00

4.58

0.32

4000ug*

54

24

46

41.33

15.53

0.51

1500ug

71

87

72

76.67

8.96

0.95

500ug

84

79

70

77.67

7.09

0.97

150ug

84

72

70

75.33

7.57

0.94

50ug

72

79

70

73.67

4.73

0.92

* Moderate thinning of the background bacterial lawn.

Table No.19: TA 100, Assay 2 – without metabolic activation (-S9-mix)

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

54

67

60

60.33

6.51

  -

Positive control solvent

20uL

60

51

60

57.00

5.20

  -

Positive control

Sodium Azide

 

20 ug

In 20 uL

1531

1505

1569

1535.00

32.19

26.93

Vehicle

50uL

46

48

55

49.67

4.73

  -

Solution of test item

Lemi code :GGA020117-S2

 

5000ug*

46

48

42

45.33

3.06

0.91

1500ug

57

57

48

54.00

5.20

1.09

500ug

43

62

65

56.67

11.93

1.14

150ug

66

60

58

61.33

4.16

1.23

50ug

64

56

49

56.33

7.51

1.13

Table No.20: TA 100, Assay 2 – with metabolic activation (10 % S9-mix) – with pre-incubation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

120

95

88

101.00

16.82

  -

Positive control solvent

10uL

76

78

75

76.33

1.53

  -

Positive control

2-Anthramine

 

1 ug

In 10 uL

356

390

370

372.00

17.09

4.87

Vehicle

50uL

73

72

79

74.67

3.79

  -

Solution of test item

Lemi code :GGA020117-S2

 

5000ug*

10

15

18

14.33

4.04

0.19

1500ug

65

70

65

66.67

2.89

0.89

500ug

69

81

69

73.00

6.93

0.98

150ug

82

75

71

76.00

5.57

1.02

50ug

83

73

72

76.00

6.08

1.02

* Moderate thinning of the background bacterial lawn.

Table No.21: E. COLI, Assay 1 – without metabolic activation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

107

109

105

107.00

2.00

  -

Positive control solvent

10uL

131

109

99

113.00

16.37

  -

Positive control

cis-Platinum (II)

 

1ug

In 10uL

310

324

341

325.00

15.52

2.88

Vehicle

50uL

92

102

105

99.67

6.81

  -

Solution of test item

Lemi code GGA191216-S2

5000ug

52

35

30

39.00

11.53

0.39

4000ug

52

55

59

55.33

3.51

0.56

1500ug

73

70

90

77.67

10.79

0.78

500ug

98

113

120

110.33

11.24

1.11

150ug

134

129

144

135.67

7.64

1.36

50ug

132

131

119

127.33

7.23

1.28

Table No.22: E. COLI, Assay 1 – with metabolic activation (10 % S9-mix) – without pre-incubation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

182

158

171

170.33

12.01

  -

Positive control solvent

5uL

152

124

140

138.67

14.05

  -

Positive control

DimethylbenzanthracEne

 

5ug

In 5uL

424

503

436

454.33

42.57

3.28

Vehicle

50uL

123

128

143

131.33

10.41

  -

Solution of test item

Lemi code GGA191216-S2

5000ug

62

53

42

52.33

10.02

0.40

4000ug

115

84

76

91.67

20.60

0.70

1500ug

105

106

124

111.67

10.69

0.85

500ug

161

162

158

160.33

2.08

1.22

150ug

180

171

169

173.33

5.86

1.32

50ug

176

160

147

161.00

14.53

1.23

Table No.23: E. COLI, Assay 2 – without metabolic activation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

137

144

122

134.33

11.24

  -

Positive control solvent

10uL

125

156

128

136.33

17.10

  -

Positive control

cis-Platinum (II)

 

1 ug

In 10 uL

345

332

328

335.00

8.89

2.46

Vehicle

50uL

108

129

99

112.00

15.39

  -

Solution of test item

Lemi code :GGA020117-S2

 

5000ug

134

113

131

126.00

11.36

1.13

1500ug

123

133

131

129.00

5.29

1.15

500ug

133

134

138

135.00

2.65

1.21

150ug

133

144

118

131.67

13.05

1.18

50ug

141

126

116

127.67

12.58

1.14

Table No.24: E. COLI, Assay 2 – with metaboblic activation (10 % S9-mix) – with pre-incubation

Serie

Dose/plate

Nº1

Nº2

Nº3

mean

Standart deviation

R

Negative control

100uL

215

198

186

199.67

14.57

  -

Positive control solvent

5uL

211

192

204

202.33

9.61

  -

Positive control

Dimethylbenzanthracene 

2.5 ug

In 5 uL

447

438

506

463.67

36.94

2.29

Vehicle

50uL

204

181

187

190.67

11.93

  -

Solution of test item

Lemi code :GGA020117-S2

 

5000ug*

42

39

31

37.33

5.69

0.20

1500ug*

85

65

91

80.33

13.61

0.42

500ug

187

175

169

177.00

9.17

0.93

150ug

182

172

170

174.67

6.43

0.92

50ug

221

220

166

202.33

31.47

1.06

* Moderate thinning of the background bacterial lawn.

Conclusions:
The test item tested at doses 5 000, 1 500, 500, 150 and 50 μg/plate do not induce significant increase in the number of revertants in Salmonella typhimurium and in Escherichia coli, either with or without metabolic activation. Therefore the test item was not mutagenic.
Executive summary:

In accordance with OECD guideline 471 and GLP study, solutions of test item have ben tested for their capacity to induce reverse mutation in four strain of Salmonella typhimurium and one strain of Escherichia coli. This study was performed in absence and in presence of metabolic activation system S9 obtained from rat livery. Assay 1 (direct incorporation) and Assay 2 (pre-incubation) consist of various concentrations of stock solution were tested. Stock solution was prepared at 100 mg/ml in DMSO. Negative and positive controls were carried out as well. Based of the preliminary test on strain TA100 which showed in presence of 5 000 μg/plate one toxicity consistent with the maximum tolerated 75%, a supplementary dose at 4 000 μg/plate was added to be sure this hight toxicity no hindering the scoring of the revertants. Assay 1 was tested in doses 5 000, 4000, 1 500, 500, 150 and 50 μg/plate and Assay 2 at doses 5 000, 1 500, 500, 150 and 50 μg/plate. Plates were incubated at 37ºC for 48-72 h and after this period colonies were counted. There was no evidence of any increase in the number of revertant colonies in the presence of the test item stock solution and dilutions without and with metabolic activation. In the presence of the highest dose tested a moderate thinning of the background was observed in strains (TA1535,TA98, TA100). It was consistent with the toxicity measured at this dose. The test item at tested doses do not induce significant increase of revertants in Salmonella typhimurium nor in Escherichia coli, either with and without metabolic activation. 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

Genetic toxicity in vitro:

Key study: In accordance with OECD guideline 471 and GLP studies, solutions of test item have ben tested for their capacity to induce reverse mutation in four strain of Salmonella typhimurium and one strain of Escherichia coli. This study was performed in absence and in presence of metabolic activation system S9 obtained from rat livery. Assay 1 and Assay 2 consist of various concentrations of stock solution were tested. Stock solution was prepared at 100 mg/ml in DMSO. Negative and positive controls were carried out. Based of the preliminary test on strain TA100 which showed in presence of 5 000 μg/plate one toxicity consistent with the maximum tolerated 75%, a supplementary dose at 4 000 μg/plate was added to be sure this hight toxicity no hindering the scoring of the revertants. Assay 1 was tested in doses 5 000, 4000, 1 500, 500, 150 and 50 μg/plate and Assay 2 at doses 5 000, 1 500, 500, 150 and 50 μg/plate. The test item at tested doses do not induce do not induce significant increase in the number of revertants in Salmonella typhimurium and in Escherichia coli and it can be assumed as no mutagenic.

Justification for classification or non-classification

Based on the available information, the negative results from in vitro Genetic toxicity study lead to the conclusion, that the test item is not classified as mutagenic according to CLP Regulation (EC) no.1272/2008.