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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 April 20, 2016 to August 29, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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:
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 Aroclor 1254-induced Sprague-Dawley rat
Controls
Negative controls:
yes
Remarks:
sterile deionized water
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
mitomycin C
other: Acridine mutagen ICR 191 2-Aminofluorene 2-Aminoanthracene
Evaluation criteria:
Acceptable ranges of background revertants for five tester strains are:
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:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Negative controls valid:
yes
Positive controls valid:
yes

Any other information on results incl. tables

Table 1. Genotype Confirmation Tests 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

+

+

+

+

+

rfa mutation

Inhibition zone of crystal violet

+

+

+

+

+

uvrB mutation

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 CJ304 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

28

27

26

76

72

79

318

321

301

16

15

15

10

8

10

Mf

27 ± 1

76 ± 4

313 ± 11

15 ± 1

9 ± 1

50

Ie

30

36

28

72

71

70

363

234

218

17

9

10

10

13

7

Mf

31 ± 4

71 ± 1

272 ± 80

12 ± 4

10 ± 3

150

Ie

15

29

22

66

56

54

267

221

348

14

12

16

5

8

6

Mf

22 ± 7

59 ± 6

279 ± 64

14 ± 2

6 ± 2

500

Ie

31

33

23

80

55

76

316

302

285

16

15

22

11

11

10

Mf

29 ± 5

70 ± 13

301 ± 16

18 ± 4

11 ± 1

1500

Ie

26

18

20

75

67

63

310

305

333

19

17

21

11

9

10

Mf

21 ± 4

68 ± 6

316 ± 15

19 ± 2

10 ± 1

5000

Ie

24

21

24

45

58

51

314

238

254

11

13

14

10

12

4

Mf

23 ± 2

51±7

269 ± 40

13 ± 2

9 ± 4

Positive controlb

Ie

152

154

170

403

401

538

732

834

824

440

406

417

87

90

63

Mf

159c± 10

447c± 79

797c± 56

421d± 17

80d± 15

a: Negative control was sterile deionized water.

b: Positive controls: 1μg/plate 2-nitrofluorene for TA98        0.5 μg/plate sodium azide for TA100

  62.5μg/plate mitomycin C for TA102     0.1 μg/plate sodium azide for TA1535

  0.5μg/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 CJ304 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

25

33

31

73

72

89

312

355

386

4

13

5

14

13

12

Mf

30 ± 4

85 ± 11

351 ± 37

7 ± 5

13 ± 1

50

Ie

39

33

30

87

84

77

319

288

307

8

11

6

13

12

11

Mf

34 ± 5

83 ± 5

305 ± 16

8 ± 3

12 ± 1

150

Ie

31

34

25

76

83

83

277

300

231

14

6

10

16

17

9

Mf

30 ± 5

81 ± 4

269 ± 35

 10 ± 4

14 ± 4

500

Ie

23

34

29

75

67

93

319

337

332

10

12

19

21

11

20

Mf

 29 ± 6

78 ± 13

329 ± 9

14 ± 5

17 ± 6

1500

Ie

39

24

22

81

63

59

335

286

336

9

12

11

16

11

10

Mf

28 ± 9

68 ± 12

319 ± 29

11 ± 2

12 ± 3

5000

Ie

14

27

25

78

60

60

245

276

285

6

8

7

8

10

7

Mf

22 ± 7

66 ± 10

269 ± 21

7 ± 1

8 ± 2

Positive controlb

Ie

210

207

184

667

676

698

1439

1685

1594

204

218

183

287

278

279

Mf

200c± 14

680c± 16

1573c± 124

202d± 18

281d± 5

a: Negative control was sterile deionized water.

b: Positive controls: 0.5μg/plate 2-aminofluorene for TA98    4 μg/plate 2-aminofluorene for TA100

  4μg/plate 2-aminoanthracene for TA102   1 μg/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, CJ304 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 T65315027-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 CJ304 show that test validity criteria was met.

Based on the preliminary assay results, 5000μg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CJ304 at 50, 150, 500, 1500 and 5000μg/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 cytotoxicity was observed in all five tester strains up to 5000μg/plate in the absence and presence of metabolite activations. Results showed that CJ304 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5000μg/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, CJ304 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000μg/plate in the absence and presence of S9 metabolic activation.