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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 April 20, 2016 to October 14, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
5-({4-chloro-6-[ethyl(phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(1-sulfo-2-naphthyl)diazenyl]naphthalene-2,7-disulfonic acid, lithium sodium salts
EC Number:
942-803-7
Molecular formula:
Not applicable; this UVCB substance contains: C31H21ClN7O10S3.xLi.yNa, (x + y) = 3; 0 < (x,y) < 3 with 804.0 < MW < 852.1 g/mol (UVCB substance), C31H22N7O11S3.xLi.yNa, (x + y) = 3; 0 < (x,y) < 3 with 785.5 < MW < 833.7 g/mol (UVCB substnace), and traces of NaCl and Na2SO4.
IUPAC Name:
5-({4-chloro-6-[ethyl(phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(1-sulfo-2-naphthyl)diazenyl]naphthalene-2,7-disulfonic acid, lithium sodium salts
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 Aroclor 1254-induced Sprague-Dawley rats
Controls
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

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:
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

Any other information on results incl. tables

Table 1. Genotype Confirmation Tests of Salmonella typhimuriumTester 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 CJ306 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

13

41

17

62

80

65

326

312

374

19

10

13

8

9

6

Mf

24 ± 15

69 ± 10

337 ± 33

14 ± 5

8 ± 2

50

Ie

25

41

38

66

67

54

317

247

274

13

11

12

12

6

4

Mf

35 ± 9

62 ± 7

279 ± 35

12 ± 1

7 ± 4

150

Ie

29

32

20

83

80

64

218

274

400

11

12

19

10

4

6

Mf

27 ± 6

76 ± 10

297 ± 93

14 ± 4

7 ± 3

500

Ie

19

27

22

62

67

68

257

309

302

20

17

15

4

11

12

Mf

23 ± 4

66 ± 3

289 ± 28

17 ± 3

9 ± 4

1500

Ie

22

30

25

69

50

72

286

247

191

9

8

8

10

10

8

Mf

26 ± 4

64 ± 12

241 ± 48

8 ± 1

9 ± 1

5000

Ie

21

27

24

66

50

68

249

272

251

9

15

13

9

6

5

Mf

24 ± 3

61 ± 10

257 ± 13

12 ± 3

7 ± 2

Positive controlb

Ie

190

220

205

584

483

589

1061

1259

1060

370

425

410

78

90

110

Mf

205c± 15

552c± 60

1127c± 115

402d± 28

93d± 16

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 CJ306 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

21

18

29

89

78

90

453

461

416

11

13

9

10

5

10

Mf

23 ± 6

86 ± 7

443 ± 24

11 ± 2

8 ± 3

50

Ie

23

16

12

103

82

87

385

418

385

9

6

9

6

7

12

Mf

17 ± 6

91 ± 11

396 ± 19

8 ± 2

8 ± 3

150

Ie

26

20

21

98

81

80

409

396

431

9

8

7

9

9

14

Mf

22 ± 3

86 ± 10

412 ± 18

8 ± 1

11 ± 3

500

Ie

23

22

22

85

91

89

391

392

408

14

14

12

16

10

4

Mf

22 ± 1

88 ± 3

397 ± 10

13 ± 1

10 ± 6

1500

Ie

15

26

17

73

71

88

333

365

361

13

7

7

4

9

10

Mf

19 ± 6

77 ± 9

353 ± 17

9 ± 3

8 ± 3

5000

Ie

17

24

23

74

71

84

329

308

306

13

13

9

6

11

7

Mf

21 ± 4

76 ± 7

314 ± 13

12 ± 2

8 ± 3

Positive controlb

Ie

174

162

141

597

573

609

1968

2178

2076

152

167

180

205

183

209

Mf

159c± 17

593c± 18

2074c± 105

166d± 14

199d±14

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

Applicant's summary and conclusion

Conclusions:
According to OECD 471 test method, CJ306 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 T65315028-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 CJ306 show that test validity criteria was met.

Based on the preliminary assay results, 5000μg/platewas set as the highest dose in this study. In the mutagenicity assay, five doses of CJ306 at 50, 150, 500, 1500 and 5000μg/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 5000μg/plate in the absence and presence of metabolite activations. Results showed that CJ306 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5000μg/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, CJ306 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000μg/plate in the absence and presence of S9 metabolic activation.

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