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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CJ302 was not mutagenic in the reverse mutation analysis of Salmonella typhimuriumup 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 October 22, 2015 to June 14, 2016
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
Target gene:
Bacterial gene reverse mutation
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 Aroclor 1254-induced Sprague-Dawley rats
Untreated 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
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
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:
not specified
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:
not specified
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:
not specified
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:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid

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

+

+

+

+

+

rfamutation

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 CJ302 inSalmonella typhimuriumStrains 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

29

37

43

97

74

89

313

395

326

23

18

17

15

11

9

Mf

36 ± 7

87 ± 12

345 ± 44

19 ± 3

12 ± 3

50

Ie

39

37

47

78

78

72

355

348

355

15

21

25

15

8

12

Mf

41 ± 5

76 ± 3

353 ± 4

20 ± 5

12 ± 4

150

Ie

39

48

45

81

87

77

298

344

400

22

27

26

10

12

8

Mf

44 ± 5

82 ± 5

347 ± 51

25 ± 3

10 ± 2

500

Ie

49

30

43

90

68

87

235

359

297

13

21

20

6

8

10

Mf

41 ± 10

82 ± 12

297 ± 62

18 ± 4

8 ± 2

1500

Ie

41

33

31

77

76

99

268

310

369

27

18

27

8

6

13

Mf

35 ± 5

84 ± 13

316 ± 51

24 ± 5

9 ± 4

5000

Ie

23

34

42

86

61

72

338

343

364

17

19

22

9

9

13

Mf

33 ± 10

73±13

348 ± 14

19 ± 3

10 ± 2

Positive controlb

Ie

310

221

252

529

546

545

1069

975

1012

339

336

331

302

380

351

Mf

261c± 45

540c± 10

1019c± 47

345d± 18

344d± 39

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 CJ302 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

27

29

27

106

122

80

388

357

366

16

13

18

13

18

11

Mf

28 ± 1

103 ± 21

370 ± 16

16 ± 3

14 ± 4

50

Ie

39

22

32

120

71

145

323

333

360

16

14

9

14

10

8

Mf

31 ± 9

112 ± 38

339 ± 19

13 ± 4

11 ± 3

150

Ie

29

35

34

97

60

83

349

318

325

16

18

9

10

19

24

Mf

33 ± 3

80 ± 19

331 ± 16

14 ± 5

18 ± 7

500

Ie

20

34

22

77

152

122

245

354

298

14

13

12

10

17

13

Mf

 25 ± 8

117 ± 38

299 ± 55

13 ± 1

13 ± 4

1500

Ie

28

37

39

101

124

88

361

345

350

17

14

16

14

8

7

Mf

35 ± 6

104 ± 18

352 ± 8

16 ± 2

10 ± 4

5000

Ie

37

27

26

128

100

125

275

362

314

24

16

16

13

14

15

Mf

30 ± 6

118 ± 15

317 ± 44

19 ± 5

14 ± 1

Positive controlb

Ie

162

201

173

655

711

668

975

930

1274

203

213

201

283

323

334

Mf

179c± 20

678c± 29

1060c± 187

206d± 6

313d± 27

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

Conclusions:
According to OECD 471 test method, CJ302 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 T65315026-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 CJ302 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 CJ302 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 CJ302 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, CJ302 was not mutagenic in the reverse mutation analysis of Salmonella typhimuriumup to 5000 μg/plate in the absence and presence of S9 metabolic activation.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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 CJ302 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. The results of concurrent positive and negative controls and three non-cytotoxic dose levels obtained supported the validity of the assay.

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 CJ302 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, CJ302 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