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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CJ305 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(OECD TG471).

 

Link to relevant study records
Reference
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 April 6, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
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:
S9 mix activation
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:
Acceptable ranges of background revertants from the mean value of triplicateplates for five tester strains without S9 metabolic activation are:
Tester Strain Revertants
TA98 10-60
TA100 50-240
TA102 180-480
TA1535 5-45
TA1537 2-25
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

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 CJ305 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

23

26

67

85

70

365

385

378

22

15

17

19

14

14

Mf

26 ± 4

74 ± 10

376 ± 10

18 ± 4

16 ± 3

50

Ie

36

20

20

52

74

60

394

384

357

16

17

17

20

19

18

Mf

25 ± 9

62 ± 11

378 ± 19

17 ± 1

19 ± 1

150

Ie

35

23

13

57

68

71

397

367

362

12

19

21

12

19

14

Mf

24 ± 11

65 ± 7

375 ± 19

17 ± 5

15 ± 4

500

Ie

15

24

35

65

69

75

378

339

370

12

11

17

10

17

22

Mf

25 ± 10

70 ± 5

362 ± 21

13 ± 3

16 ± 6

1500

Ie

16

18

20

73

73

80

350

378

384

14

12

17

15

16

23

Mf

18 ± 2

75 ± 4

371 ± 18

14 ± 3

18 ± 4

5000

Ie

19

10

19

74

55

71

380

332

393

28

18

16

20

23

22

Mf

16 ± 5

67 ± 10

367 ± 32

21 ± 6

22 ± 2

Positive controlb

Ie

193

254

214

395

438

416

2292

1890

1870

379

352

304

58

52

40

Mf

220c± 31

416c±22

2017c± 238

345d± 38

50d± 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.

 

Table 3. Mutagenicity Test of C305 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

43

39

52

90

105

93

495

270

626

14

10

11

35

25

29

Mf

45 ± 7

96 ± 8

464 ± 180

12 ± 2

30 ± 5

50

Ie

39

38

35

82

84

91

406

555

527

11

16

14

19

34

8

Mf

37 ± 2

86 ± 5

496 ± 79

14 ± 3

20 ± 13

150

Ie

45

35

38

92

89

99

506

213

427

10

13

12

31

22

16

Mf

39 ± 5

93 ± 5

382 ± 152

12 ± 2

23 ± 8

500

Ie

33

35

31

106

92

93

492

492

471

14

8

14

28

34

19

Mf

33 ± 2

97 ± 8

485 ± 12

12 ± 3

27 ± 8

1500

Ie

31

40

39

99

83

71

561

506

486

10

17

12

23

28

24

Mf

37 ± 5

84 ± 14

518 ± 39

13 ± 4

25 ± 3

5000

Ie

29

23

26

61

90

89

407

475

452

10

14

13

16

23

24

Mf

26 ± 3

80 ± 16

445 ± 35

12 ± 2

21 ± 4

Positive controlb

Ie

376

310

301

314

351

347

1570

1152

1362

406

315

300

387

309

240

Mf

329c± 41

337c± 20

1361c± 209

340d± 57

312d± 74

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.

Conclusions:
According to OECD 471 test method, CJ305 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 T65316018-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 CJ305 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 CJ305 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 cytotoxicity was observed in all five tester strains up to 5000 μg/plate in the absence and presence of metabolite activations. Results showed that CJ305 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, CJ305 was not mutagenic in the reverse mutation analysis of Salmonellatyphimurium up to 5000 μg/plate in the absence and presence of S9 metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

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 CJ305 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 cytotoxicity was observed in all five tester strains up to 5000 μg/plate in the absence and presence of metabolite activations. Results showed that CJ305 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, CJ305 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.

Justification for classification or non-classification