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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 November 28, 2016 to June 02, 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
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
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

+

+

+

+

+

rfa mutation

inhibition zone of crystal violet

+

+

+

+

+

ΔuvrB 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 CJ321 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

35

29

89

79

74

410

345

358

18

16

17

15

8

10

Mf

31 ± 3

81 ± 8

371 ± 34

17 ± 1

11 ± 4

50

Ie

28

23

38

79

63

82

348

345

327

14

16

19

15

17

18

Mf

30 ± 8

75 ± 10

340 ± 11

16 ± 3

17 ± 2

150

Ie

24

37

26

63

68

66

354

305

325

21

17

24

18

19

12

Mf

 29 ± 7

66 ± 3

328 ± 25

21 ± 4

16 ± 4

500

Ie

24

36

32

43

59

54

347

356

309

16

10

20

6

16

11

Mf

31 ± 6

52 ± 8

337 ± 25

15 ± 5

11 ± 5

1500

Ie

32

38

30

48

48

57

329

309

323

15

14

20

14

14

12

Mf

33 ± 4

51 ± 5

320 ± 10

 16 ± 3

13 ± 135

5000

Ie

35

32

28

56

70

38

269

274

283

16

16

16

5

11

13

Mf

32 ± 4

55 ± 16

275 ± 7

16 ± 0

 10 ± 4

Positive controlb

Ie

193

278

208

572

539

568

1966

1772

1896

472

489

419

172

168

151

Mf

226c± 45

560c±18

 1878c± 98

460d± 37

164d± 11

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 CJ321 in Salmonella typhimurium Strains with S9 Metabolic Activation

Treatment

(μg/plate)

Number of Revertant Colonies in Salmonella typhimurium

TA98

TA100

TA102

TA1535

TA1537

TA98

(repeat)

Replicate

1

2

3

1

2

3

1

2

3

1

2

3

1

2

3

1

2

3

Negative controla

Ie

61

63

52

105

107

89

393

382

422

14

10

4

19

20

26

46

41

43

Mf

59 ± 6

100 ± 10

399 ± 21

9 ± 5

22 ± 4

 43 ± 3

50

Ie

66

69

52

105

79

95

194

361

359

13

11

15

25

18

33

66

37

48

Mf

62 ± 9

93 ± 13

305 ± 96

13 ± 2

25 ± 8

50 ± 15

150

Ie

73

99

134

102

99

101

386

371

238

19

13

12

20

28

29

107

77

145

Mf

102 ± 31

101 ± 2

332 ± 81

15 ± 4

 26 ± 5

110c± 34

500

Ie

198

195

175

139

134

104

437

385

312

11

14

15

35

24

41

156

133

179

Mf

189c± 13

126 ± 19

378 ± 63

13 ± 2

33 ± 9

 156c± 23

1500

Ie

84

91

96

102

110

80

444

271

294

17

12

9

35

29

38

76

79

93

Mf

90 ± 6

97 ± 16

336 ± 94

13 ± 4

34 ± 5

83 ± 9

5000

Ie

52

54

55

72

92

83

386

328

342

13

15

13

22

19

21

31

40

30

Mf

54 ± 2

82 ± 10

352 ± 30

14 ± 1

21 ± 2

34 ± 6

Positive controlb

Ie

277

272

277

229

297

399

1093

1256

1402

306

303

392

386

303

244

233

278

212

Mf

275c± 3

308c± 86

1250c± 155

334d± 51

311d± 71

241c± 34

a: Negative control was sterile deionized water.

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

1 μg/plate 2-aminofluorene for TA100

2 μg/plate 2-aminoanthracene for TA102

0.5 μg/plate 2-aminoanthracene for TA1535

2 μg/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, CJ321 was considered a suspect or equivocal results in the reverse mutation analysis of Salmonella typhimurium.
Executive summary:

This test using the procedures outlined in the QPS Taiwan Study Plan for T65316021-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 CJ321 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 CJ321 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. Results showed that CJ321 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5000 μg/plate in the absence of metabolite activation. However,treatment with CJ321 in the presence of S9 metabolic activation increased the number of revertants in tester strain of Salmonella typhimurium TA98 at the dose 500μg/plate. The increase was not dose-responsiveness which led to an equivocal result. An equivocal result was also shown in the repeat (confirmation) study.

Based on the data obtained from these studies, it was concluded that under the test condition, CJ321 was considered a suspect or equivocal results in the reverse mutation analysis of Salmonella typhimurium.