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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CR SB32N2 was not mutagenic in the reverse mutation analysis ofSalmonellatyphimuriumup 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 December 29, 2016 to June 13, 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
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
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 CR SB32N2 in Salmonella typhimurium Strains without S9 Metabolic Activation

Treatment

(μg/plate)

Number of Revertant Colonies inSalmonella 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

29

26

75

63

95

308

235

333

20

23

15

7

17

9

Mf

28 ± 2

78 ± 16

292 ± 51

19 ± 4

11 ± 5

50

Ie

28

38

35

62

58

73

335

293

280

14

11

18

4

8

9

Mf

34 ± 5

64 ± 8

303 ± 29

14 ± 2

7 ± 3

150

Ie

31

29

33

72

58

56

288

322

299

13

12

16

12

4

7

Mf

31 ± 2

62 ± 9

303 ± 17

14 ± 2

8 ± 4

500

Ie

33

27

25

48

56

59

306

309

325

18

18

12

9

11

13

Mf

28 ± 4

54 ± 6

313 ± 10

16 ± 3

11 ± 2

1500

Ie

39

21

24

54

63

70

309

297

280

17

14

10

6

9

8

Mf

28 ± 10

62 ± 8

295 ± 15

14 ± 4

8 ± 2

5000

Ie

23

25

30

52

54

74

292

278

299

11

16

13

8

11

7

Mf

26 ± 4

60 ± 12

290 ± 11

13 ± 3

9 ± 2

Positive controlb

Ie

274

300

270

443

472

492

1382

1336

1638

336

432

409

170

165

192

Mf

281c± 16

469c±25

1452c± 163

392d± 50

176d± 14

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 CR SB32N2 in Salmonella typhimurium Strains with S9 Metabolic Activation

Treatment

(μg/plate)

Number of Revertant Colonies inSalmonella 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

46

55

44

104

104

79

439

452

364

18

19

17

25

22

25

Mf

48 ± 6

96 ± 14

418 ± 48

18 ± 1

24 ± 2

50

Ie

45

36

32

94

96

102

434

383

353

13

3

13

17

22

32

Mf

38 ± 7

97 ± 4

390 ± 41

10 ± 6

24 ± 8

150

Ie

55

41

39

87

102

108

386

332

384

11

14

9

24

20

22

Mf

45 ± 9

99 ± 11

367 ± 31

11 ± 3

22 ± 2

500

Ie

40

42

38

129

90

87

387

384

377

14

14

22

23

24

16

Mf

40 ± 2

102 ± 23

383 ± 5

17 ± 5

21 ± 4

1500

Ie

54

28

45

73

112

124

430

358

415

9

7

14

20

14

14

Mf

42 ± 13

103 ± 27

401 ± 38

10 ± 4

16 ± 3

5000

Ie

34

42

39

81

83

70

183

410

351

8

11

11

15

13

12

Mf

38 ± 4

78 ± 7

315 ± 118

10 ± 2

13 ± 2

Positive controlb

Ie

223

255

321

386

326

273

889

1015

952

445

388

379

293

246

176

Mf

266c± 50

328c± 57

952c± 63

404d± 36

238d± 59

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, CR SB32N2 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 T65316024-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 CR SB32N2 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 CR SB32N2 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 CR SB32N2 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, CR SB32N2 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.

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 CR SB32N2 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 CR SB32N2 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, CR SB32N2 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