Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Everlan SL65 was mutagenic in the reverse mutation analysis of Salmonella typhimurium (OECD TG471).

Everlan SL65 was positive effect under the condition of in vitro mammalian chromosome aberration test (OECD TG473).

Everlan SL65 was positive 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:
Sep. 20, 2016 - Dec 8, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD 471:1997
Deviations:
no
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
Details on mammalian cell type (if applicable):
Source of Cell: MOLTOX, molecular Toxicology, Inc.
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-Aminoanthracene
Evaluation criteria:
for the groups treated with an without S9 mix, the range of natural reverse mutant colony number were as following: (CFU/plate)
Tester Strains With S9 mix without S9 mix
TA98 32-51 22-51
TA100 197-386 166-326
TA102 378-487 262-407
TA1535 18-41 17-43
TA1537 9-26 6-25
Statistics:
statistic via SPSS software, p < 0.05 (One-Way ANOVA).
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
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. 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

213

Positive control groupa

1228

Test group

 

5

348

2.5

716

1.25

415

0.625

353

0.3125

282

Without S9 Mix

Negative control group

202

Positive control group

781

Test group

 

5

486

2.5

446

1.25

350

0.625

302

0.3125

228

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

43

32

51

42 ± 10

Positive control groupb

353

378

347

359 ± 16*

Test group

 

 

 

 

5

102

109

92

101 ± 9*

2.5

96

98

85

93 ± 7*

1.25

101

87

130

106 ± 22*

0.625

87

94

100

94 ± 7*

0.3125

49

56

71

59 ± 11

Without S9 Mix

Negative control group

22

27

28

26 ± 3

Positive control group

373

345

400

373 ± 28*

Test group

 

 

 

 

5

37

39

63

46 ± 14

2.5

90

76

86

84 ± 7*

1.25

59

67

76

67 ± 9*

0.625

54

56

59

56 ± 3*

0.3125

46

54

64

55 ± 9*

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

171

191

235

199 ± 33

Positive control groupb

1553

1647

1498

1566 ± 75*

Test group

 

 

 

 

5

485

362

414

420 ± 62*

2.5

765

667

765

732 ± 57*

1.25

691

619

561

624 ± 65*

0.625

413

425

461

433 ± 25*

0.3125

329

378

381

363 ± 29*

Without S9 Mix

Negative control group

201

147

164

171 ± 28

Positive control group

738

797

759

765 ± 30*

Test group

 

 

 

 

5

498

422

560

493 ± 69*

2.5

449

410

478

446 ± 34*

1.25

488

479

555

507 ± 42*

0.625

459

408

450

439 ± 27*

0.3125

366

379

358

368 ± 11*

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

482

478

410

457 ± 40

Positive control groupb

1025

939

996

987 ± 44*

Test group

 

 

 

 

5

528

596

487

537 ± 55

2.5

636

596

653

628 ± 29

1.25

583

646

576

602 ± 39

0.625

484

480

540

501 ± 34

0.3125

621

530

491

547 ± 67

Without S9 Mix

Negative control group

401

365

446

404 ± 41

Positive control group

925

920

844

896 ± 45*

Test group

 

 

 

 

5

533

534

383

483 ± 87

2.5

528

462

505

498 ± 34

1.25

589

461

490

513 ± 67

0.625

485

515

467

489 ± 24

0.3125

486

594

499

526 ± 59

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

17

25

26

23 ± 5

Positive control groupb

347

388

379

371 ± 22*

Test group

 

 

 

 

5

111

120

118

116 ± 5*

2.5

164

139

131

145 ± 17*

1.25

76

74

81

77 ± 4*

0.625

59

54

74

62 ± 10*

0.3125

37

45

39

40 ± 4

Without S9 Mix

Negative control group

22

23

24

23 ± 1

Positive control group

228

235

213

225 ± 11*

Test group

 

 

 

 

5

40

38

44

41 ± 3

2.5

113

138

92

114 ± 23*

1.25

88

83

87

86 ± 3*

0.625

54

53

55

54 ± 1*

0.3125

49

42

39

43 ± 5

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

14

9

12

12 ± 3

Positive control groupb

450

476

438

455 ± 19*

Test group

 

 

 

 

5

89

96

74

86 ± 11*

2.5

95

125

115

112 ± 15*

1.25

63

94

102

86 ± 21*

0.625

55

57

53

55 ± 2*

0.3125

33

30

33

32 ± 2

Without S9 Mix

Negative control group

10

15

12

12 ± 3

Positive control group

899

863

1020

927 ± 82*

Test group

 

 

 

 

5

78

85

66

76 ± 10*

2.5

40

64

62

55 ± 13*

1.25

59

86

37

61 ± 25*

0.625

72

60

54

62 ± 9*

0.3125

44

40

41

42 ± 2*

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, Everlan SL65 was mutagenic in the reverse mutation analysis of Salmonella typhimurium.
Executive summary:

This test using the procedures outlined in the SuperLub Study Plan for M62-151100105001EN which is based on the SOP for the OECD 471 (SOPF-203) and OECD 471 (OECD, 1997). The results of this OECD 471 test forEverlan SL65show that test validity criteria was met.

Based on the preliminary assay results, 5mg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of Everlan SL65 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. At least one dose in four tester strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 of test groups, the results showed that the number of revertants increase twice more than negative controls group. Based on the data obtained from this study, it was concluded that under the test condition, Everlan SL65 was mutagenic in the reverse mutation analysis of Salmonella typhimurium.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 25, 2016 - Dec 30, 2016
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
OECD 476:2015
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro gene mutation study in mammalian cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1
Details on mammalian cell type (if applicable):
- Source of cells: Food Industry Research and Development Institute
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
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1
Metabolic activation:
with and without
Genotoxicity:
positive
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

167.4 ± 2.1

100.0

Positive controld

153.2 ± 4.7

91.5 ± 2.8

Test groups (mg/mL)

 

 

2.0

76.6 ± 3.8

45.8 ± 2.3

1.0

184.2 ± 2.9

110.0 ± 1.7

0.5

187.4 ± 5.8

111.9 ± 3.4

0.25

184.4 ± 4.6

110.2 ± 2.7

Without S9 Mix

Negative controlc

195.8 ± 2.9

100.0

Positive controld

111.8 ± 11.3

57.1 ± 5.8

Test groups (mg/mL)

 

 

2.0

105.0 ± 6.0

53.6 ± 3.1

1.0

158.8 ± 6.8

81.1 ± 3.5

0.5

154.0 ± 1.6

78.7 ± 0.8

0.25

183.4 ± 7.1

93.7 ± 3.6

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

7.4 ± 0.9

18.9

Positive controld

61.0 ± 9.9

187.7*

Test groups (mg/mL)

 

 

2.0

45.0 ± 11.0

98.3*

1.0

25.4 ± 4.7

49.9*

0.5

13.2 ± 3.1

58.4*

0.25

15.6 ± 1.7

27.3

Without S9 Mix

Negative controlc

9.6 ± 2.3

16.0

Positive controld

142.0 ± 20.3

272.0*

Test groups (mg/mL)

 

 

2.0

18.6 ± 3.9

35.0

1.0

13.6 ± 3.8

20.7

0.5

11.0 ± 2.0

26.7

0.25

12.8 ± 3.3

27.0

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).

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.

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

Conclusions:
According to OECD 476 test method, Everlan SL65 was positive 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 M62-151100112001EN 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 Everlan SL65 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 Everlan SL65 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 significantly different from the negative control group for 0.5-2.0 mg/mL of the test groups in the presence of S9 Mix. Based on the data obtained from this study, it was concluded that under the test condition, Everlan SL65 was positive effect in mammalian cell gene mutation test (in vitro).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec 02,2015 - Dec 14, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
OECD 473:2014
Deviations:
no
GLP compliance:
yes
Type of assay:
other: In vitro Mammalian Chromosome Aberration Test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1
Details on mammalian cell type (if applicable):
- Source of cells: Food Industry Research and Development Institute
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
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

1.009 ± 0.135

100.0

Positive controld

1.038 ± 0.080

102.8 ± 7.9

Test group (mg/mL)

 

 

2.0

0.479 ± 0.010

47.5 ± 1.0

1.0

1.199 ± 0.020

118.8 ± 2.0

0.5

1.045 ± 0.024

103.6 ± 2.4

0.25

1.103 ± 0.114

109.3 ± 11.2

0.125

1.236 ± 0.063

122.5 ± 6.2

Processed for 3 hours

(without S9 Mix)

Negative control

1.933 ± 0.183

100.0

Positive control

1.209 ± 0.218

62.5 ± 11.2

Test group (mg/mL)

 

 

2.0

0.553 ± 0.065

28.6 ± 3.4

1.0

1.201 ± 0.197

62.1 ± 10.2

0.5

1.175 ± 0.215

60.8 ± 11.1

0.25

1.288 ± 0.162

66.6 ± 8.4

0.125

1.309 ± 0.242

67.7 ± 12.5

Processed for 20 hours

(without S9 Mix)

Negative control

1.670 ± 0.130

100.0

Positive control

1.192 ± 0.105

71.4 ± 6.3

Test group (mg/mL)

 

 

2.0

0.092 ± 0.006

5.5 ± 0.4

1.0

0.329 ± 0.037

19.7 ± 2.2

0.5

1.168 ± 0.215

69.9 ± 12.9

0.25

1.102 ± 0.100

66.0 ± 5.9

0.125

1.241 ± 0.063

74.3 ± 3.8

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

13.3

10.3

19.3

Test group (mg/mL)

 

 

 

2.0

4.0

1.3

1.0

7.0

2.0

7.3

0.5

4.0

0.3

1.3

0.25

0.7

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, Everlan SL65 was positive 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 M62-151100106002EN 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 Everlan SL65 show that test validity criteria was met.

Based on the results of the cell viability test, 2.0 mg/mL as the highest dosage was used to perform the test in 3-hours treatment with or without S9 Mix and 1.0 mg/mL as the highest dosage was used to perform the test in 20-hours treatment without S9 Mix. In the chromosome aberration test, three doses of Everlan SL65, negative and positive controls were tested in triplicate with or without S9 Mix. In test group, the cell proportions with abnormal chromosome in all dosage with S9 Mix for 3 hours and in highest dosage without S9 Mix for 20 hours gave the similar reaction as the positive controls. Based on the data obtained from this study, it was concluded that under the test condition, Everlan SL65 was positive effect in mammalian chromosome aberration test (in vitro).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

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 Everlan SL65 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. At least one dose in four tester strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 of test groups, the results showed that the number of revertants increase twice more than negative controls group. Based on the data obtained from this study, it was concluded that under the test condition, Everlan SL65 was mutagenic in the reverse mutation analysis of Salmonella typhimurium.

 

Mammalian chromosomal aberration test (OECD TG473)

Based on the results of the cell viability test, 2.0 mg/mL as the highest dosage was used to perform the test in 3-hours treatment with or without S9 Mix and 1.0 mg/mL as the highest dosage was used to perform the test in 20-hours treatment without S9 Mix.In the chromosome aberration test, three doses of Everlan SL65, negative and positive controls were tested in triplicate with or without S9 Mix. In test group, the cell proportions with abnormal chromosome in all dosage with S9 Mix for 3 hours and in highest dosage without S9 Mix for 20 hours gave the similar reaction as the positive controls.

Based on the data obtained from this study, it was concluded that under the test condition, Everlan SL65 was positive effect in mammalian chromosome aberration test (in vitro).

 

Mammalian cell gene mutation test (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 Everlan SL65 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 significantly different from the negative control group for 0.5-2.0 mg/mL of the test groups in the presence of S9 Mix. Based on the data obtained from this study, it was concluded that under the test condition, Everlan SL65 was positive effect in mammalian cell gene mutation test (in vitro).

Justification for classification or non-classification