Registration Dossier

Administrative data

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).

CJ302 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation (OECD TG473).

CJ302 was negative effect under the condition of in vitro mammalian cell gene mutation test (OECD TG476).

Link to relevant study records

Referenceopen allclose all

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:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 08, 2016 to May 16, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1
Metabolic activation:
with and without
Metabolic activation system:
The post-mitochondrial fraction (S9) of liver from Aroclor 1254 induced Sprague- Dawley rats
Untreated negative controls:
yes
Remarks:
Sterile deionized water
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1
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. Karyology Analysis of Chinese Hamster Ovary Cells

No. of chromosome

<18

18

19

20

21

22

>22

No. of cells

0

1

20

25

4

0

0

Table 2. Concurrent Cytotoxicity Analysis of CJ302 in Chinese Hamster Ovary Cells

Concentration

(µg/mL)

Cell Number

(× 105cells)

ICCa

N-N0

RICCb

(%)

Cytotoxicityc

(%)

Before Treatment

Untreated (-9S)

21.4

 

 

 

Untreated (+9S)

28.0

 

 

 

After Treatment

Scheme I (-S9, 3h)

Negative Control

61.6

40.2

100.0

0.0

51.2

56.8

35.4

88.1

11.9

128

57.8

36.4

90.5

9.5

320

54.6

33.2

82.6

17.4

800

55.2

33.8

84.1

15.9

2000

53.8

32.4

80.6

19.4

Positive Controld

47.0

25.6

63.7

36.3

Scheme II (+S9, 3h)

Negative Control

58.2

30.2

100.0

0.0

51.2

74.2

46.2

153.0

0.0

128

52.4

24.4

80.8

19.2

320

71.8

43.8

145.0

0.0

800

55.0

27.0

89.4

10.6

2000

56.2

28.2

93.4

6.6

Positive Controle

49.0

21.0

69.5

30.5

Scheme I (-S9, 20h)

Negative Control

56.2

34.8

100.0

0.0

51.2

57.0

35.6

102.3

0.0

128

54.6

33.2

95.4

4.6

320

57.8

36.4

104.6

0.0

800

91.8

70.4

202.3

0.0

2000

47.2

25.8

74.1

25.9

Positive Controlf

61.4

40.0

114.9

0.0

a: ICC: increased in cell counts = Cell No.After treatment(N) - Cell No.Before treatment(N0)

b: RICC: relative increase in cell counts; RICC = (ICCtreatment/ICCcontrol) × 100

c: Cytotoxicity (%) = 100 – RICC

d: Positive control was 0.33 µg/mL mitomycin C (MMC)

e: Positive control was 11.2 µg/mL cyclophosphamide (CPP)

f: Positive control was 0.2 µg/mL mitomycin C (MMC)

 

Table 3. Summary of Chromosome Aberrations in Chinese Hamster Ovary Cells for CJ302

Treatment

 

Concentration

(µg/mL)

Treating Hour

S9

(-/+)

Aberrant Cells

(%)

Scheme I (-S9, 3h)

Negative Control

0

3

-

0.00

Test Article

51.2

3

-

0.00

128

3

-

0.00

320

3

-

1.00

800

3

-

0.67

2000

3

-

0.67

Positive Control(MMC)

0.33

3

-

21.00*

Scheme II (+S9, 3h)

Negative Control

0

3

+

0.00

Test Article

51.2

3

+

0.00

128

3

+

0.00

320

3

+

0.33

800

3

+

0.00

2000

3

+

1.00

Positive Control (CPP)

11.2

3

+

22.67*

Scheme I (-S9, 20h)

Negative Control

0

20

-

0.00

Test Article

51.2

20

-

0.00

128

20

-

0.67

320

20

-

1.00

800

20

-

1.00

2000

20

-

0.33

Positive Control(MMC)

0.2

20

-

32.00*

*: The frequency of aberrant cells was significantly higher than that of the negative control (One-tailed binomial test, α = 0.01).
Conclusions:
According to OECD 473 test method, CJ302 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation.
Executive summary:

This test using the procedures outlined in the QPS Study Plan for T65316026-GT and OECD 473 (OECD, 2016). The results of this OECD 473 test for CJ302 show that test validity criteria was met.

Based on the cytotoxicity result, five concentrations of 51.2, 128, 320, 800 and 2000 µg/mL were used for all three schemes in the assay. All tests were conducted in duplicate with concurrent negative and positive controls. Results showed that the percentage of aberrant cells of negative controls in all three test schemes was 0%. The positive controls induced significant increases in percentages (21.00%, 22.67% and 32.00%, respectively) of aberrant cells as compared to the corresponding negative control. There were at least three analyzable concentrations obtained for each test scheme, which met the requirements for a valid test. In all three schemes, the test article treated cell cultures did not show more than 3% of the frequencies of structural chromosome aberration. Therefore,CJ302 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 15, 2018 to January 2, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) (migrated information)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
It was no cytotoxic effect (the cell survival rate was >50 %) in 2 mg/mL with and without S9 Mix.
True negative controls:
yes
Remarks:
Ham's F-12
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
at the highest dosage (2.0 mg/mL)
Positive controls validity:
valid

Table 1. Cell viability analysis

Group

Test article

Average colony numbersa

Relative survival (%)b

With S9 Mix

Negative controlc

74.5 ± 12.0

100.00 ± 0.00

Positive controld

65.0 ± 5.7

85.98 ± 0.07

Test groups (mg/mL)

 

 

2.0

46.0 ± 9.9

60.85 ± 0.13

1.0

46.0 ± 2.8

60.85 ± 0.04

0.5

50.5 ± 9.2

66.80 ± 0.12

0.25

57.5 ± 7.8

76.06 ± 0.10

Without S9 Mix

Negative controlc

60.0 ± 7.1

100.00 ± 0.00

Positive controld

42.5 ± 13.4

63.43 ± 0.20

Test groups (mg/mL)

 

 

2.0

50.0 ± 8.5

74.63 ± 0.13

1.0

45.5 ± 4.9

67.91 ± 0.07

0.5

53.5 ± 6.4

79.85 ± 0.09

0.25

56.0 ± 4.2

83.58 ± 0.06

aValues were expressed as Mean ± S.D., and tests were repeated two times.

bRelative survival = each colony numbers of the positive control or test groups / the average of colony numbers in the negative control × 100%, then calculated the Mean ± S.D..

cNegative control: Ham’s F-12 medium with 10% FBS (S9 Mix or not).

dPositive control: 4μg/mL B[a]P for the cell treated with S9 Mix, and 0.25μg/mL 4-NQO for the cells treated without S9 Mix.

 

Table 2. Mutation frequency analysis

Group

Test article

Average colony numbersa

Mutation frequency (× 10-6)b

With S9 Mix

Negative controlc

13.3 ± 7.0

19.0

Positive controld

30.0 ± 4.4

51.8

Test groups (mg/mL)

 

 

2.0

28.7 ± 4.5

37.3

1.0

22.7 ± 6.5

36.7

0.5

18.3 ± 7.4

39.9

0.25

12.3 ± 4.7

25.5

Without S9 Mix

Negative controlc

15..3 ± 7.4

17.1

Positive controld

25.0 ± 15.1

44.8

Test groups (mg/mL)

 

 

2.0

13.7 ± 3.5

21.1

1.0

13.0 ± 2.6

21.4

0.5

11.0 ± 2.0

20.9

0.25

9.7 ± 2.5

18.0

aValues were expressed as Mean ± S.D., and tests were repeated three times.

bMutation frequency = (numbers of colonies /number of seeding) × (1 / Colonies formation frequency).

cNegative control: Ham’s F-12 medium with 10% FBS (S9 Mix or not).

dPositive control: 4μg/mL B[a]P for the cell treated with S9 Mix, and 0.25μg/mL 4-NQO for the cells treated without S9 Mix.

* Significantly different from the negative control group (ρ < 0.005).

Conclusions:
According to OECD 476 test method, CJ302 was negative effect under the condition of in vitro mammalian cell gene mutation test.
Executive summary:

This test using the procedures outlined in the SuperLub Study Plan for M62-180800004001EN which is based on the SOP for the OECD 476 (SOPF-240) and OECD 476 (OECD, 2015).The results of this OECD 476 test for CJ302 show that test validity criteria was met.

Based on the results of the cell viability test, 2.0mg/mL was set as the highest dose in this study. In the gene mutation test, four doses of CJ302 at 0.25, 0.5, 1.0 and 2.0mg/mL, negative and positive controls were tested infive repetitionswith or without S9 Mix. The mutation frequency was no significantly different from the negative control group for all test groups in the absence and presence of S9 Mix. Based on the data obtained from this study, it was concluded that under the test condition, CJ302 was negative effect in mammalian cell gene mutation test (in vitro).

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation test (OECD TG471)

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.

Mammalian chromosomal aberration test (OECD TG473)

Based on the cytotoxicity result, five concentrations of 51.2, 128, 320, 800 and 2000µg/mL were used for all three schemes in the assay. All tests were conducted in duplicate with concurrent negative and positive controls. Results showed that the percentage of aberrant cells of negative controls in all three test schemes was 0%. The positive controls induced significant increases in percentages (21.00%, 22.67% and 32.00%, respectively) of aberrant cells as compared to the corresponding negative control. There were at least three analyzable concentrations obtained for each test scheme, which met the requirements for a valid test. In all three schemes, the test article treated cell cultures did not show more than 3% of the frequencies of structural chromosome aberration. Therefore, CJ302 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation.

Mammalian cell gene mutation tests (OECD TG476)

Based on the results of the cell viability test, 2.0mg/mL was set as the highest dose in this study. In the gene mutation test, four doses of CJ302 at 0.25, 0.5, 1.0 and 2.0mg/mL, negative and positive controls were tested infive repetitionswith or without S9 Mix. The mutation frequency was no significantly different from the negative control group for all test groups in the absence and presence of S9 Mix. Based on the data obtained from this study, it was concluded that under the test condition, CJ302 was negative effect in mammalian cell gene mutation test (in vitro).

 

Justification for classification or non-classification