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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Evercion SR61 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).

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 03, 2016 to December 22, 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 determined
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 determined
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 determined
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 determined
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 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

226

Positive control groupa

1125

Test group

 

5

118

2.5

171

1.25

194

0.625

200

0.3125

207

Without S9 Mix

Negative control group

185

Positive control group

657

Test group

 

5

160

2.5

133

1.25

179

0.625

214

0.3125

255

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

42

32

49

41 ± 9

Positive control groupb

284

314

344

314 ± 30*

Test group

 

 

 

 

5

55

44

53

51 ± 6

2.5

44

65

42

50 ± 13

1.25

42

42

33

39 ± 5

0.625

54

49

53

52 ± 3

0.3125

43

56

71

57 ± 14

Without S9 Mix

Negative control group

41

33

30

35 ± 6

Positive control group

233

208

225

222 ± 13*

Test group

 

 

 

 

5

24

17

23

21 ± 4

2.5

22

25

39

29 ± 9

1.25

23

26

21

23 ± 3

0.625

32

16

23

24 ± 8

0.3125

30

40

26

32 ± 7

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

219

295

205

240 ± 48

Positive control groupb

1208

1429

1475

1371 ± 143*

Test group

 

 

 

 

5

203

195

218

205 ± 12

2.5

237

247

197

227 ± 26

1.25

194

200

196

197 ± 3

0.625

159

257

235

217 ± 51

0.3125

212

256

285

251 ± 37

Without S9 Mix

Negative control group

140

185

180

168 ± 25

Positive control group

789

720

771

760 ± 36*

Test group

 

 

 

 

5

130

175

171

159 ± 25

2.5

152

194

166

171 ± 21

1.25

166

139

208

171 ± 35

0.625

171

178

205

185 ± 18

0.3125

186

154

223

188 ± 35

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

434

392

422

416 ± 22

Positive control groupb

897

881

911

896 ± 15*

Test group

 

 

 

 

5

443

520

460

474 ± 40

2.5

425

437

443

435 ± 9

1.25

461

465

439

455 ± 14

0.625

460

379

425

421 ± 41

0.3125

422

463

388

424 ± 38

Without S9 Mix

Negative control group

411

381

412

401 ± 18

Positive control group

866

840

859

855 ± 13*

Test group

 

 

 

 

5

315

396

414

375 ± 53

2.5

381

394

433

403 ± 27

1.25

389

446

401

412 ± 30

0.625

364

422

369

385 ± 32

0.3125

435

397

402

411 ± 21

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

19

25

27

24 ± 4

Positive control groupb

276

307

300

294 ± 16*

Test group

 

 

 

 

5

29

25

19

24 ± 5

2.5

23

20

16

20 ± 4

1.25

14

19

24

19 ± 5

0.625

28

18

16

21 ± 6

0.3125

15

19

18

17 ± 2

Without S9 Mix

Negative control group

15

17

19

17 ± 2

Positive control group

240

299

276

272 ± 30*

Test group

 

 

 

 

5

11

13

16

13 ± 3

2.5

19

23

21

21 ± 2

1.25

22

18

24

21 ± 3

0.625

13

21

20

18 ± 4

0.3125

21

19

18

19 ± 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: 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

9

12

13

11 ± 2

Positive control groupb

417

451

409

426 ± 22*

Test group

 

 

 

 

5

10

8

15

11 ± 4

2.5

9

15

14

13 ± 3

1.25

14

17

13

15 ± 2

0.625

17

11

14

14 ± 3

0.3125

21

16

18

18 ± 3

Without S9 Mix

Negative control group

14

12

10

12 ± 2

Positive control group

1019

947

1207

1058 ± 134*

Test group

 

 

 

 

5

10

12

10

11 ± 1

2.5

8

8

15

10 ± 4

1.25

7

9

10

9 ± 2

0.625

14

9

9

11 ± 3

0.3125

16

8

7

10 ± 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: 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, Evercion SR61 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 M62-151100096001EN 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 Evercion SR61 show 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 Evercion SR61 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 genotoxicity was observed in all five tester strains up to 5mg/plate in the absence and presence of metabolite activations.Results showed that Evercion SR61 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, Evercion SR61 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 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, 5mg/plate was set as the highest dose in this study. Inthe mutagenicity assay, five doses of Evercion SR61 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 cytotoxicity was observed in all five tester strains up to 5mg/plate in the absence and presence of metabolite activations.Results showed that Evercion SR61 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, Evercion SR61 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5mg/plate in the absence and presence of S9 metabolic activation.

Justification for classification or non-classification