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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 January 17, 2017 to July 03, 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-Amoinoanthracene
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 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 102
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 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 μg/plate
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
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 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 μg/plate
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The substance was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 1500 ug/plate.

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 CJ313 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

27

25

31

50

53

70

288

322

268

16

6

13

21

14

15

Mf

28 ± 3

58 ± 11

293 ± 27

12 ± 5

17 ± 4

50

Ie

29

23

30

56

61

53

322

336

318

18

12

16

4h

9

12

Mf

27 ± 4

57 ± 4

325 ± 9

15 ± 3

8g± 4

150

Ie

14

18

18

58

71

52

216

350

280

13

16

22

17

11

14

Mf

17 ± 2

60 ± 10

282 ± 67

17 ± 5

14 ± 3

500

Ie

18

17

26

57

49

80

332

256

204

12

15

16

6

12

16

Mf

20 ± 5

62 ± 16

261 ± 59

14 ± 2

11 ± 5

1500

Ie

23

25

23

70

61

58

294

294

278

14

17

16

17

10

9

Mf

24 ± 1

63 ± 6

289 ± 9

16 ± 2

12 ± 4

5000

Ie

25

22

17

42

60

57

258

176

292

13

11

18

10

13

16

Mf

21 ± 4

53 ± 10

242 ± 60

14 ± 4

13 ± 3

Positive controlb

Ie

181

255

266

374

374

430

1411

1229

1376

439

428

349

191

181

174

Mf

234c± 46

393c±32

 1339c± 97

405d± 49

182d± 9

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.

g: Cytotoxicty: a > 50% reduction when compared to the mean colony number of negative control revertants.

h: Extreme low data may consider as an outlier (out of range of historical data).

 

Table 3. Mutagenicity Test of CJ313 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

38

41

38

86

75

89

515

486

562

15

11

13

29

33

32

Mf

39 ± 2

83 ± 7

521 ± 38

13 ± 2

31 ± 2

50

Ie

28

40

37

63

108

70

337

414

483

12

14

18

28

22

19

Mf

35 ± 6

80 ± 24

411 ± 73

15 ± 3

23 ± 5

150

Ie

43

38

47

61

76

88

422

416

131

7

15

11

26

24

31

Mf

43 ± 5

75 ± 14

384 ± 61

11 ± 4

27 ± 4

500

Ie

23

36

41

84

88

67

296

316

390

15

16

17

29

35

34

Mf

33 ± 9

80 ± 11

334 ± 50

16 ± 1

33 ± 3

1500

Ie

36

35

33

93

77

90

294

367

391

12

12

11

18

18

13

Mf

35 ± 2

87 ± 9

351 ± 51

12 ± 1

16 ± 3

5000

Ie

27

27

17

65

54

80

343

179

136

10

11

7

10

8

16

Mf

24 ± 6

66 ± 13

219g± 109

9 ± 2

11g± 4

Positive controlb

Ie

263

329

312

254

298

346

1133

1336

1086

300

307

459

146

132

110

Mf

301c± 34

299c± 46

1185c± 133

355d± 90

129d± 18

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.

g: Cytotoxicty: a > 50% reduction when compared to the mean colony number of negative control revertants.

Applicant's summary and conclusion

Conclusions:
According to OECD 471 test method, CJ313 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 1500 μg/plate.
Executive summary:

This test using the procedures outlined in the QPS Taiwan Study Plan for T65316020-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 CJ313 show that test validity criteria was met.

Based on the preliminary assay results, 5000 μg/platewas set as the highest dose in this study. In the mutagenicity assay, five doses of CJ313 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.Cytotoxicity was observed in tester strains TA102 and TA1537 at 5000 μg/plate in the presence of metabolite activation. Unexpected greater than 50% reduction of negative control revertants was observed in tester strain TA1537 at 50 μg/plate but would not consider cytotoxicity in the absence of metabolite activation. Results showed that CJ313 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 in the absence of metabolite activation, three tester strains TA98, TA100 and TA1535 in the presence of metabolite activation up to 5000 μg/plate; two tester strains TA102 and TA1537 in the presence of metabolite activation up to 1500 μg/plate. Based on the data obtained from this study, it was concluded that under the test condition, CJ313 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 1500 μg/plate in the absence and presence of S9 metabolic activation.