Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 25 April 2017 to 5 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Supplied by CHIMEX SAS batch No. 0159115
- Expiration date of the lot/batch: 21 February 2018
- Purity test date: 21 February 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in the test facility in a closed vessel at room temperature (18.0 – 22.5°C) protected from light.
- Stability under test conditions: H2O: not stated; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated
- Solubility and stability of the test substance in the solvent/vehicle: H2O: unknown; EtOH: unknown; acetone: >1 g/L; CH3CN: unknown; DMSO: unknown
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
A stock solution containing 50 g/L of the test item in DMSO was freshly prepared on the day of the first and the second experiment. The test item solution was not sterile filtrated before use. The stock solution was used to prepare the geometric series of the concentrations to be tested. The following nominal concentrations were prepared for the first experiment:
5000 µg/plate, 1500 µg/plate, 500 µg/plate, 150 µg/plate and 50 µg/plate.

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: HisG46, uvrB, rfa
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: hisG428, pKM101, rfa, pAQ1
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: hisG46, uvrB, rfa, pKM101
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: hisD3052, uvrB, rfa, pKM101
Species / strain / cell type:
S. typhimurium TA 97
Remarks:
TA 97a
Additional strain / cell type characteristics:
other: hisD6610, uvrB, rfa, pKM101
Metabolic activation:
with and without
Metabolic activation system:
S9 obtained from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg bw intraperitoneally
Test concentrations with justification for top dose:
In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralized water, ethanol and dimethyl sulfoxide (DMSO).
DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
A stock solution containing 50 g/L of the test item in DMSO was freshly prepared on the day of the first and the second experiment. The test item solution was not sterile filtrated before use.
The stock solution was used to prepare the geometric series of the concentrations to be tested. The following nominal concentrations were prepared for the first experiment:
5000 µg/plate, 1500 µg/plate, 500 µg/plate, 150 µg/plate and 50 µg/plate.

The following nominal concentrations were prepared for the second experiment:
5000 µg/plate, 2500 µg/plate, 1250 µg/plate, 625 µg/plate, 313 µg/plate, 156 µg/plate and 78 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Controls
Untreated negative controls:
yes
Remarks:
Demineralized water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene diamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation) in experiment 1; preincubation in experiment 2
- Cell density at seeding (if applicable):10E9 bacteria/mL

DURATION
- Preincubation period: 20 minutes (in experiment 2)
- Exposure duration:48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): revertant bacterias were counted after the 48 hours exposure period

NUMBER OF REPLICATIONS: triplicates per bacteria strain, per concentration, without and with S9

DETERMINATION OF CYTOTOXICITY
- Method: bacteria background lawn
- Any supplementary information relevant to cytotoxicity: The test item is considered non-toxic if the quotient titre/toxicity is below 2.
Evaluation criteria:
The mean values and standard deviations of each threefold determination are calculated as well as the increase factor of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (mean revertants minus mean spontaneous revertants) is given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 or 3 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity for a global interpretation of results.
The study is considered valid if at least five analyzable concentrations are presented in all strains of the main tests.
Statistics:
No statistical analysis of results will be done and historical data are considered to improve the interpretation of results.

Results and discussion

Test resultsopen allclose all
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 97
Remarks:
TA 97a
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 102
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:
Experiment 1 :
In the first experiment, the test item showed no precipitate on the plates in any tested concentration. No sign of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Experiment 2 :
In the second experiment, the test item showed no precipitate on the plates in all tested concentrations. No sign of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.


Remarks on result:
other: For the two experiments

Any other information on results incl. tables

Table2 :Summary of the results of the experiment 1

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin.

water

Mean

102

78

11

20

120

121

324

291

24

24

sd

12.8

6.4

0.6

2.5

4.0

8.1

28.8

48.7

3.1

4.6

DMSO

Mean

88

86

13

15

115

99

335

311

31

18

sd

15.6

24.5

2.3

2.5

18.8

9.5

77.2

87.3

2.1

5.2

Positive
Controls*

Mean

580

379

407

68

441

1176

1371

1416

345

155

sd

53.8

191.8

54.3

7.9

151.4

374.2

204.6

140.6

33.5

22.0

f(I)

6.59

4.41

31.31

4.53

3.68

11.88

4.09

4.55

14.38

8.61

5000 µg/plate

Mean

98

91

11

10

100

105

259

331

17

11

sd

13.9

24.3

0.0

0.6

12.5

16.5

71.8

25.7

1.5

3.2

f(I)

1.11

1.06

0.85

0.67

0.87

1.06

0.77

1.06

0.55

0.61

1500 µg/plate

Mean

85

82

14

11

96

110

324

337

18

24

sd

5.3

13.9

2.3

2.9

11.0

13.3

64.4

102.9

1.7

2.5

f(I)

0.97

0.95

1.08

0.73

0.83

1.11

0.97

1.08

0.58

1.33

500 µg/plate

Mean

86

81

13

16

101

96

311

263

22

16

sd

5.6

10.5

2.1

2.6

6.0

20.4

40.5

16.2

5.0

3.6

f(I)

0.98

0.94

1.00

1.07

0.88

0.97

0.93

0.85

0.71

0.89

150 µg/plate

Mean

88

70

15

15

108

81

277

301

24

24

sd

6.4

17.1

1.0

5.5

21.1

4.4

36.9

63.6

1.2

5.5

f(I)

1.00

0.81

1.15

1.00

0.94

0.82

0.83

0.97

0.77

1.33

50 µg/plate

Mean

74

75

16

14

89

79

344

360

28

24

sd

6.8

13.3

3.6

3.6

1.0

4.0

59.2

38.2

1.5

6.8

f(I)

0.84

0.87

1.23

0.93

0.77

0.80

1.03

1.16

0.90

1.33

 

Table3 :Summary of the results of the experiment 2

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin.

water

Mean

84

78

18

13

95

102

313

281

15

13

sd

11.8

4.7

2.6

1.5

16.6

12.1

95.9

58.0

2.5

3.5

DMSO

Mean

85

95

17

33

81

86

320

315

15

10

sd

12.5

12.9

2.5

34.1

10.2

9.0

52.3

70.5

2.5

1.5

Positive
Controls*

Mean

389

364

407

237

628

835

1504

1267

111

81

sd

77.2

94.6

72.6

18.5

101.0

264.9

92.3

234.1

21.6

7.6

f(I)

4.58

3.83

23.94

7.18

6.61

9.71

4.70

4.02

7.40

8.10

5000 µg/plate

Mean

73

70

12

14

79

97

293

237

12

8

sd

7.6

10.6

1.5

1.0

6.2

12.2

41.6

62.1

2.0

1.2

f(I)

0.86

0.74

0.71

0.42

0.98

1.13

0.92

0.75

0.80

0.80

2500 µg/plate

Mean

79

81

14

13

77

105

227

241

13

8

sd

17.6

4.9

2.5

1.0

9.6

18.6

23.1

28.9

3.6

1.2

f(I)

0.93

0.85

0.82

0.39

0.95

1.22

0.71

0.77

0.87

0.80

1250 µg/plate

Mean

73

86

15

16

94

107

288

208

10

9

sd

11.8

3.5

6.4

7.8

7.8

21.2

24.3

94.1

0.6

0.6

f(I)

0.86

0.91

0.88

0.48

1.16

1.24

0.90

0.66

0.67

0.90

625 µg/plate

Mean

74

75

12

14

100

119

259

271

13

15

sd

13.3

4.5

1.7

1.2

22.6

5.0

12.2

42.0

3.1

3.2

f(I)

0.87

0.79

0.71

0.42

1.23

1.38

0.81

0.86

0.87

1.50

312 µg/plate

Mean

64

96

16

15

95

102

290

325

11

18

sd

2.5

23.8

1.2

0.6

15.0

15.9

45.3

84.3

1.5

5.3

f(I)

0.75

1.01

0.94

0.45

1.17

1.19

0.91

1.03

0.73

1.80

156 µg/plate

Mean

68

73

14

17

83

105

320

241

12

17

sd

10.6

8.2

4.0

3.1

7.5

15.0

52.0

76.9

2.5

4.9

f(I)

0.80

0.77

0.82

0.52

1.02

1.22

1.00

0.77

0.80

1.70

78 µg/plate

Mean

90

89

15

26

89

112

261

313

13

13

sd

17.2

14.7

1.5

2.9

12.7

29.1

55.0

75.6

2.9

3.1

f(I)

1.06

0.94

0.88

0.79

1.10

1.30

0.82

0.99

0.87

1.30

 

Table4 :Historical data of spontaneousrevertants

Strain

 

TA97a

TA98

TA100

TA102

TA1535

Induction

 

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Demin. H2O

Mean

94

98

14

17

94

97

276

296

17

16

Min

60

63

6

8

62

66

85

67

6

7

Max

144

138

35

41

141

141

425

511

30

30

SD

20

16

5

5

15

15

68

80

6

6

Exp1

102

78

11

20

120

121

324

291

24

24

Exp2

84

78

18

13

95

102

313

281

15

13

DMSO

Mean

92

102

13

15

91

94

276

288

16

16

Min

58

70

7

8

60

63

79

80

8

6

Max

135

144

46

36

136

199

393

459

33

29

SD

19

17

5

5

16

20

63

69

6

6

Exp1

88

86

13

15

115

99

335

311

31

18

Exp2

85

95

17

33

81

86

320

315

15

10

Positive Controls*

Mean

561

507

407

73

514

727

1133

1242

251

117

Min

264

241

112

39

223

273

491

408

55

45

Max

1152

1181

793

217

984

1912

2331

6083

484

712

SD

174

149

141

30

154

296

435

708

90

84

Exp1

580

379

407

68

441

1176

1371

1416

345

155

Exp2

389

364

407

237

628

835

1504

1267

111

81

 

 

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of the study, the registered item Imexine OBA/50 SEC revealed no cytotoxic effrect nor genotoxicity in the bacteria strains used. Hence, it was considered as non-mutagenic for bacteria.
Executive summary:

This GLP-compliant study was performed in order to evaluate the mutagenic potential of IMEXINE OBA/50 SEC in the Bacterial Reverse Mutation Test using five strains of Salmonella typhimurium.

The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation. In the first experiment, the test item (dissolved in DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Based on the first experiment, the test item was tested up to concentrations of 5000  µg/plate in the absence and presence of S9-mix in all bacteria strain using the pre-incubation method. The test item showed no precipitate on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in any bacteria strain. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiments showed that the test item caused no increase in the number of revertants in any bacteria strain compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in any strain, in the presence and absence of metabolic activation.

Under the experimental conditions of the study, the registered item Imexine OBA/50 SEC revealed no cytotoxic effrect nor genotoxicity in the bacteria strains used. Hence, it was considered as non-mutagenic for bacteria.