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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 29, 2016 to June 13, 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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
The post-mitochondrial fraction (S9) prepared from uninduced male Golden Syrian Hamster liver
Controls
Untreated negative controls:
yes
Remarks:
sterile deionized water
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
congo red
mitomycin C
other: Acridine mutagen ICR 191, 2-Aminofluorene, 2-Aminoanthracene
Evaluation criteria:
Tester Strain Revertants
TA98 10-60
TA100 50-240
TA102 180-480
TA1535 5-45
TA1537 2-25

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1. Genotype Confirmation Test of Salmonella typhimurium Tester Strains

Genotype

character

Phenotypic observation

Tester Strains

TA98

TA100

TA102

TA1535

TA1537

Histidine requirement

growing on biotin plate

growing on histidine/biotin plate

+

+

+

+

+

rfamutation

inhibition zone of crystal violet

+

+

+

+

+

ΔuvrBmutation

growing on non UV-irradiated plate

+

+

+

+

+

growing on UV-irradiated plate

+

R-factor

ampicillin resistance

+

+

+

Genotype confirmed

Passed

Passed

Passed

Passed

Passed

+: the presence

: the absence

 

Table 2. Mutagenicity Test of CJ308 in Salmonella typhimurium Strains without S9 Metabolic Activation

Treatment

(μg/plate)

Number of Revertant Colonies in Salmonella typhimurium

TA98

TA100

TA102

TA1535

TA1537

Replicate

1

2

3

1

2

3

1

2

3

1

2

3

1

2

3

Negative controla

Ie

30

20

33

61

98

79

271

270

341

15

17

19

11

13

13

Mf

28 ± 7

79 ± 19

294 ± 41

17 ± 2

12 ± 1

50

Ie

13

21

19

66

82

68

305

319

328

17

20

18

17

5

10

Mf

18 ± 4

72 ± 9

317 ± 12

18 ± 2

11 ± 6

150

Ie

17

18

16

72

71

61

256

270

334

14

23

18

15

5

11

Mf

17 ± 1

68 ± 6

287 ± 42

18 ± 5

10 ± 5

500

Ie

25

15

18

68

92

60

275

241

300

11

17

9

7

12

7

Mf

19±5

73 ± 17

272 ± 30

12 ± 4

9 ± 3

1500

Ie

16

23

21

51

91

60

250

265

319

12

11

13

9

6

8

Mf

20 ± 4

67 ± 21

278 ± 36

12 ± 1

8 ± 2

5000

Ie

17

22

23

61

96

85

232

268

319

8

16

16

13

15

7

Mf

21 ± 3

81 ± 18

273 ± 44

13 ± 5

12 ± 4

Positive controlb

Ie

266

275

238

421

481

509

1996

1962

2130

374

424

235

180

153

146

Mf

260c± 19

470c± 45

2029c± 89

344d± 98

160d± 18

a: Negative control was sterile deionized water.

b: Positive controls: 1 μg/plate 2-nitrofluorene for TA98

0.5 μg/plate sodium azide for TA100

62.5 ng/plate mitomycin C for TA102

0.1 μg/plate sodium azide for TA1535

0.3 μg/plate acridine mutagen ICR 191 for TA1537

c: Greater than 2-fold negative control spontaneous revertants

d: Greater than 3-fold negative control spontaneous revertants

e: I: Number of revertants/plate is shown for each individual plate.

f: M: The value of mean ± S.D. from triplicate plates of each treatment was calculated.

 

Table 3. Mutagenicity Test of CJ308 in Salmonella typhimurium Strains with S9 Metabolic Activation

Treatment

(μg/plate)

Number of Revertant Colonies in Salmonella typhimurium

TA98

TA100

TA102

TA1535

TA1537

Replicate

1

2

3

1

2

3

1

2

3

1

2

3

1

2

3

Negative controla

Ie

56

44

41

104

121

104

545

463

609

9

18

15

28

34

26

Mf

47 ± 8

110 ± 10

539 ± 73

14 ± 5

29 ± 4

50

Ie

58

37

38

83

97

103

567

423

495

9

19

11

26

38

33

Mf

44 ± 12

94 ± 10

495 ± 72

13 ± 5

32 ± 6

150

Ie

38

54

41

97

98

104

582

563

574

7

19

10

28

23

24

Mf

44 ± 9

100 ± 4

573 ± 10

12 ± 6

25 ± 3

500

Ie

42

33

53

97

67

78

570

574

565

17

14

16

27

26

29

Mf

43 ± 10

81 ± 15

570 ± 5

16 ± 2

27 ± 2

1500

Ie

36

31

25

79

99

96

590

410

526

11

7

15

29

25

20

Mf

31 ± 6

91 ± 11

509 ± 91

11 ± 4

25 ± 5

5000

Ie

33

31

20

65

65

92

516

393

506

12

7

8

20

24

17

Mf

28 ± 7

74 ± 16

472 ± 68

9 ± 3

20 ± 4

Positive controlb

Ie

277

226

189

299

320

343

1518

1644

1930

254

254

293

319

292

351

Mf

231c± 44

321c± 22

1697c± 211

267d± 23

321d± 30

a: Negative control was sterile deionized water.

b: Positive controls: 60μg/plate Congo Red for TA98

1μg/plate 2-aminofluorene for TA100

2 μg/plate 2-aminoanthracene for TA102

0.5μg/plate 2-aminoanthracene for TA1535

2μg/plate 2-aminoanthracene for TA1537

c: Greater than 2-fold negative control spontaneous revertants

d: Greater than 3-fold negative control spontaneous revertants

e: I: Number of revertants/plate is shown for each individual plate.

f: M: The value of mean ± S.D. from triplicate plates of each treatment was calculated.

Applicant's summary and conclusion

Conclusions:
According to OECD 471 test method, CJ308 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000 μg/plate.
Executive summary:

This test using the procedures outlined in the QPS Taiwan Study Plan for T65316019-GT which is based on the SOP for the OECD 471 (CTPS-TE00201) and OECD 471 (OECD, 1997). The results of this OECD 471 test for CJ308 show that test validity criteria was met.

Based on the preliminary assay results, 5000 μg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CJ308 at 50, 150, 500, 1500 and 5000 μg/plate, concurrent negative and strain-specific positive controls were tested in tester strains TA98, TA100, TA102, TA1535 and TA1537 in triplicate with or without S9 Mix activation. No toxicity was observed in all five tester strains up to 5000 μg/plate in the absence and presence of metabolite activations. Results showed that CJ308 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5000 μg/plate either in the absence or in the presence of metabolite activation.

Based on the data obtained from this study, it was concluded that under the test condition, CJ308 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000 μg /plate in the absence and presence of S9 metabolic activation.