Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Everdirect SH12 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5mg/plate in the absence and presence of S9 metabolic activation(OECD TG471).

Everdirect SH12 was negative effect under the condition of in vitro mammalian chromosome aberration test (OECD TG473).

Everdirect SH12 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 August 8, 2016 to November 18, 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
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:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-Aminoanthracene
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. Characteristics of five Salmonella typhimurium strains

Test strain

Histidine requirement

uvrB mutation

rfa mutation

Ampicillin resistance

TA98

+

+

+

+

TA100

+

+

+

+

TA102

+

ϴ

+

+

TA1535

+

+

+

ϴ

TA1537

+

+

+

ϴ

+ means had the characteristic; ϴ means did not have the characteristic

Table 2. Toxicity of the test article of Salmonella typhimurium TA100

Group

Test article

(mg/plate)

Reverse mutant colony number

(CFU/plate)

With S9 Mix

Negative control group

200

Positive control groupa

1292

Test group

 

5

194

2.5

242

1.25

225

0.625

221

0.3125

221

Without S9 Mix

Negative control group

218

Positive control group

955

Test group

 

5

188

2.5

193

1.25

197

0.625

172

0.3125

171

a Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: Sodium azide (5μg/plate).

Table 3. Reverse mutation test of Salmonella typhimurium TA98

Group

Test article

(mg/plate)

Reverse mutant colony number (CFU/plate)

Average of coloniesa

1

2

3

With S9 Mix

Negative control group

46

31

36

38 ± 8

Positive control groupb

262

274

323

286 ± 32*

Test group

 

 

 

 

5

29

31

25

28 ± 3

2.5

31

34

25

30 ± 5

1.25

32

26

29

29 ± 3

0.625

29

31

23

28 ± 4

0.3125

29

23

24

29 ± 6

Without S9 Mix

Negative control group

53

41

42

45 ± 7

Positive control group

298

317

291

302 ± 13*

Test group

 

 

 

 

5

45

43

48

45 ± 3

2.5

59

40

37

45 ± 12

1.25

52

50

43

48 ± 5

0.625

43

33

37

38 ± 5

0.3125

53

35

51

46 ± 10

a Average of colonies was shown as Mean ± S.D., the data were triplicate.

b Positive control group: With S9 Mix: Benzo [a] pyrene (4.0μg/plate).

Without S9 Mix: 4-Nitroquinoline-N-oxide (0.5μg/plate).

*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

Table 4. Reverse mutation test of Salmonella typhimurium TA100

Group

Test article

(mg/plate)

Reverse mutant colony number (CFU/plate)

Average of coloniesa

1

2

3

With S9 Mix

Negative control group

183

251

237

224 ± 36

Positive control groupb

1317

1301

1270

1296 ± 24*

Test group

 

 

 

 

5

213

191

231

212 ± 20

2.5

230

185

243

219 ± 30

1.25

192

191

248

210 ± 33

0.625

228

204

201

211 ± 15

0.3125

221

237

189

216 ± 24

Without S9 Mix

Negative control group

253

223

211

229 ± 22

Positive control group

947

969

891

936 ± 40*

Test group

 

 

 

 

5

209

200

196

202 ± 7

2.5

184

244

198

209 ± 31

1.25

240

197

190

209 ± 27

0.625

207

188

187

194 ± 11

0.3125

208

212

238

219 ± 16

a Average of colonies was shown as Mean ± S.D., the data were triplicate.

b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: Sodium azide (5μg/plate).

*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Table 5. Reverse mutation test of Salmonella typhimurium TA102

Group

Test article

(mg/plate)

Reverse mutant colony number (CFU/plate)

Average of coloniesa

1

2

3

With S9 Mix

Negative control group

468

395

482

448 ± 47

Positive control groupb

1010

968

997

992 ± 22*

Test group

 

 

 

 

5

392

408

451

417 ± 31

2.5

414

433

479

442 ± 33

1.25

443

417

444

435 ± 15

0.625

436

479

459

458 ± 22

0.3125

515

465

439

473 ± 39

Without S9 Mix

Negative control group

375

359

405

380 ± 23

Positive control group

976

906

945

942 ± 35*

Test group

 

 

 

 

5

482

464

494

480 ± 15

2.5

469

444

490

468 ± 23

1.25

391

489

445

442 ± 49

0.625

412

442

500

451 ± 45

0.3125

528

492

496

505 ± 20

a Average of colonies was shown as Mean ± S.D., the data were triplicate.

b Positive control group: With S9 Mix: 2-Aminoanthracene (10μg/plate).

Without S9 Mix: Mitomycin C (0.5μg/plate).

*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Table 6. Reverse mutation test of Salmonella typhimurium TA1535

Group

Test article

(mg/plate)

Reverse mutant colony number (CFU/plate)

Average of coloniesa

1

2

3

With S9 Mix

Negative control group

22

30

19

24 ± 6

Positive control groupb

229

264

243

245 ± 18*

Test group

 

 

 

 

5

26

28

25

26 ± 2

2.5

30

24

33

29 ± 5

1.25

31

26

21

26 ± 5

0.625

19

23

32

25 ± 7

0.3125

20

30

22

24 ± 5

Without S9 Mix

Negative control group

19

29

31

26 ± 6

Positive control group

261

253

266

260 ± 7*

Test group

 

 

 

 

5

26

30

26

27 ± 2

2.5

34

21

26

27 ± 7

1.25

29

21

30

27 ± 5

0.625

26

31

27

28 ± 3

0.3125

29

30

19

26 ± 6

a Average of colonies was shown as Mean ± S.D., the data were triplicate.

b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: Sodium azide (0.4μg/plate).

*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Table 7. Reverse mutation test of Salmonella typhimurium TA1537

Group

Test article

(mg/plate)

Reverse mutant colony number (CFU/plate)

Average of coloniesa

1

2

3

With S9 Mix

Negative control group

10

17

16

14 ± 4

Positive control groupb

289

333

314

312 ± 22*

Test group

 

 

 

 

5

9

15

11

12 ± 3

2.5

9

12

15

12 ± 3

1.25

11

16

14

14 ± 3

0.625

21

21

15

19 ± 3

0.3125

13

17

10

13 ± 4

Without S9 Mix

Negative control group

12

14

14

13 ± 1

Positive control group

740

529

688

652 ± 110*

Test group

 

 

 

 

5

14

21

16

17 ± 4

2.5

14

21

11

15 ± 5

1.25

21

16

11

16 ± 5

0.625

12

15

13

13 ± 2

0.3125

11

23

17

17 ± 6

a Average of colonies was shown as Mean ± S.D., the data were triplicate.

b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: 9-Aminoacridine (50.0μg/plate).

*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

Conclusions:
According to OECD 471 test method, Everdirect SH12 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5 mg/plate.
Executive summary:

This test using the procedures outlined in the SuperLub Study Plan for M62-151100084001EN which is based on the SOP for the OECD 471 (SOPF-203) and OECD 471 (OECD, 1997). The results of this OECD 471 test for Everdirect SH12 show that test validity criteria was met.

Based on the preliminary assay results, 5mg/platewas set as the highest dose in this study. In the mutagenicity assay, five doses of Everdirect SH12 at 0.3125, 0.625, 1.25, 2.5 and 5mg/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 genetoxicity was observed in all five tester strains up to 5mg/plate in the absence and presence of metabolite activations. Results showed that Everdirect SH12 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5mg/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, Everdirect SH12 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5mg/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 December 02, 2015 to December 14, 2016
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 cell micronucleus test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 1. Cell viability test

Processing Time

Groups

Absorbance

(570 nm)a

Cell Viability

(%)b

Processed for 3 hours

(with S9 Mix)

Negative controlc

0.928 ± 0.069

100.0

Positive controld

1.097 ± 0.022

118.2 ± 2.4

Test group (mg/mL)

 

 

2.0

0.457 ± 0.012

49.3 ± 1.3

1.0

1.141 ± 0.006

122.9 ± 0.7

0.5

1.034 ± 0.032

111.4 ± 3.5

0.25

1.071 ± 0.113

115.4 ± 12.1

0.125

1.233 ± 0.079

132.9 ± 8.5

Processed for 3 hours

(without S9 Mix)

Negative control

1.878 ± 0.167

100.0

Positive control

1.316 ± 0.038

70.0 ± 2.1

Test group (mg/mL)

 

 

2.0

0.571 ± 0.060

30.4 ± 3.2

1.0

1.326 ± 0.041

70.6 ± 2.1

0.5

1.270 ± 0.040

67.6 ± 2.1

0.25

1.277 ± 0.143

68.0 ± 7.6

0.125

1.352 ± 0.123

72.0 ± 6.5

Processed for 20 hours

(without S9 Mix)

Negative control

1.556 ± 0.061

100.0

Positive control

1.351 ± 0.155

86.8 ± 10.0

Test group (mg/mL)

 

 

2.0

0.266 ± 0.005

17.1 ± 0.3

1.0

0.786 ± 0.065

50.5 ± 4.2

0.5

0.922 ± 0.023

59.3 ± 1.4

0.25

1.074 ± 0.096

69.0 ± 6.1

0.125

1.207 ± 0.160

77.6 ± 10.3

a Values were expressed as Mean ± S.D., and tests were triplicate.

b Cell viability = each absorbance of the positive control or test groups / the average of absorbance of the negative control × 100%, than calculated the Mean ± S.D..

c Negative control: Ham’s F-12 medium with 1% DMSO and 10% FBS (with or without S9 Mix).

d Positive control: Group included 25μg/mL cyclophosphamide monohydrate for the cells treated with S9 Mix, and 2.5μg/mL mitomycin C for the cells treated without S9 Mix.

 

Table 2. The cell proportion with abnormal chromosome

Group

The cell proportion with abnormal chromosome (%)

Processed for

3 hours

(with S9 Mix)

Processed for

3 hours

(without S9 Mix)

Processed for

20 hours

(without S9 Mix)

Negative control

0.0

0.0

0.0

Positive control

10.3

8.0

18.0

Test group (mg/mL)

 

 

 

2.0

0.0

0.7

0.3

1.0

0.0

0.0

0.0

0.5

0.0

0.0

0.0

a Tests were repeated triplicate.

b Negative control: Ham’s F-12 medium with 1% DMSO and 10% FBS (with or without S9 Mix).

c Positive control: Group included 25μg/mL cyclophosphamide monohydrate for the cells treated with S9 Mix, and 2.5μg/mL mitomycin C for the cells treated without S9 Mix.

Conclusions:
According to OECD 473 test method, Everdirect SH12 was negative effect under the condition of in vitro mammalian chromosome aberration test.
Executive summary:

This test using the procedures outlined in the SuperLub Study Plan for M62-151100085001EN which is based on the SOP for the OECD 473 (SOPF-207) and OECD 473 (OECD, 2014). The results of this OECD 473 test for Everdirect SH12 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 chromosome aberration test, three doses of Everdirect SH12 at 0.5, 1.0 and 2.0mg/mL, negative and positive controls were tested in triplicate with or without S9 Mix. The cell proportion with abnormal chromosome was lower than 3% 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, Everdirect SH12 was negative effect in mammalian chromosome aberration test (in vitro).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 04, 2016 to December 30, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Untreated negative controls:
yes
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:
not determined
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 1. Cell viability analysis

Group

Test article

Average colony numbersa

Relative survival (%)b

With S9 Mix

Negative controlc

176.6 ± 2.7

100.0

Positive controld

166.6 ± 6.7

94.3 ± 3.8

Test groups (mg/mL)

 

 

2.0

198.0 ± 8.8

112.1 ± 5.0

1.0

218.0 ± 6.5

123.4 ± 3.7

0.5

199.2 ± 3.0

112.8 ± 1.7

0.25

203.8 ± 4.1

115.4 ± 2.3

Without S9 Mix

Negative controlc

196.0 ± 5.3

100.0

Positive controld

140.2 ± 6.5

71.5 ± 3.3

Test groups (mg/mL)

 

 

2.0

187.8 ± 6.4

95.8 ± 3.3

1.0

204.4 ± 4.0

104.3 ± 2.1

0.5

183.6 ± 2.7

93.7 ± 1.4

0.25

185.2 ± 1.6

94.5 ± 0.8

a Values were expressed as Mean ± S.D., and tests were repeated 5 times.

b Relative 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..

c Negative control: Ham’s F-12 medium with 1% DMSO and 10% FBS (S9 Mix or not).

d Positive 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

2.8 ± 2.3

5.0

Positive controld

91.6 ± 16.5

247.6*

Test groups (mg/mL)

 

 

2.0

3.2 ± 1.8

4.7

1.0

4.0 ± 2.0

8.9

0.5

3.2 ± 1.8

6.4

0.25

4.4 ± 3.0

6.1

Without S9 Mix

Negative controlc

13.0 ± 1.4

29.0

Positive controld

42.6 ± 16.9

116.1*

Test groups (mg/mL)

 

 

2.0

19.6 ± 4.5

43.9

1.0

16.6 ± 4.2

33.5

0.5

10.2 ± 1.8

14.5

0.25

10.0 ± 2.0

17.5

a Values were expressed as Mean ± S.D., and tests were repeated 5 times.

b Mutation frequency = (average colony numbers of each test groups / the number of seeding) × (1 / Colonies formation frequency).

c Negative control: Ham’s F-12 medium with 1% DMSO and 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, Everdirect SH12 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 M62-151100091001EN 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 Everdirect SH12 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 Everdirect SH12 at 0.25, 0.5, 1.0 and 2.0mg/mL, negative and positive controls were tested in five repetitions with 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, Everdirect SH12 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, 5mg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of Everdirect SH12 at 0.3125, 0.625, 1.25, 2.5 and 5mg/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 genetoxicity was observed in all five tester strains up to 5mg/plate in the absence and presence of metabolite activations. Results showed that Everdirect SH12 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5mg/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, Everdirect SH12 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5mg/plate in the absence and presence of S9 metabolic activation.

 

Mammalian chromosomal aberration test (OECD TG473)

Based on the results of the cell viability test, 2.0mg/mL was set as the highest dose in this study. In the chromosome aberration test, three doses of Everdirect SH12 at 0.5, 1.0 and 2.0mg/mL, negative and positive controls were tested in triplicate with or without S9 Mix. The cell proportion with abnormal chromosome was lower than 3% 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, Everdirect SH12 was negative effect in vitro mammalian chromosome aberration test.

 

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 Everdirect SH12 at 0.25, 0.5, 1.0 and 2.0mg/mL, negative and positive controls were tested in five repetitions with 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, Everdirect SH12 was negative effect in mammalian cell gene mutation test (in vitro).

Justification for classification or non-classification