Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 13, 2016 to August 23, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
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:
S9 Mix
Controls
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

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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
cytotoxicity
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

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

130

Positive control groupa

1329

Test group

 

5

153

2.5

146

1.25

148

0.625

202

0.3125

176

Without S9 Mix

Negative control group

121

Positive control group

850

Test group

 

5

146

2.5

127

1.25

141

0.625

135

0.3125

134

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

53

43

36

44 ± 9

Positive control groupb

248

233

213

231 ± 18*

Test group

 

 

 

 

5

32

36

31

33 ± 3

2.5

34

32

35

34 ± 2

1.25

26

38

37

34 ± 7

0.625

23

38

28

30 ± 8

0.3125

33

32

34

33 ± 1

Without S9 Mix

Negative control group

40

23

23

29 ± 10

Positive control group

359

312

326

332 ± 24*

Test group

 

 

 

 

5

21

24

26

24 ± 3

2.5

28

26

30

28 ± 2

1.25

29

24

26

26 ± 3

0.625

21

24

21

22 ± 2

0.3125

24

35

26

28 ± 6

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

264

230

273

256 ± 23

Positive control groupb

1152

1264

1215

1210 ± 56*

Test group

 

 

 

 

5

236

216

255

236 ± 20

2.5

237

207

187

210 ± 25

1.25

207

221

210

213 ± 7

0.625

260

229

213

234 ± 24

0.3125

216

266

283

255 ± 35

Without S9 Mix

Negative control group

271

228

252

250 ± 22

Positive control group

741

726

802

756 ± 40*

Test group

 

 

 

 

5

201

223

197

207 ± 14

2.5

231

222

192

215 ± 20

1.25

217

250

217

228 ± 19

0.625

249

241

208

233 ± 22

0.3125

228

190

229

216 ± 22

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

356

340

328

341 ± 14

Positive control groupb

716

730

764

737 ± 25*

Test group

 

 

 

 

5

344

332

318

331 ± 14

2.5

375

287

307

323 ± 46

1.25

336

308

318

321 ± 14

0.625

327

305

286

306 ± 21

0.3125

343

321

371

345 ± 25

Without S9 Mix

Negative control group

335

370

355

353 ± 18

Positive control group

782

831

903

839 ± 61*

Test group

 

 

 

 

5

302

328

334

321 ± 17

2.5

356

307

370

344 ± 33

1.25

341

284

302

309 ± 29

0.625

368

326

307

334 ± 31

0.3125

320

354

381

352 ± 31

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

31

18

21

23 ± 7

Positive control groupb

281

266

300

282 ± 17*

Test group

 

 

 

 

5

12

11

17

13 ± 3

2.5

9

25

20

18 ± 8

1.25

21

19

10

17 ± 6

0.625

25

24

23

24 ± 1

0.3125

22

15

17

18 ± 4

Without S9 Mix

Negative control group

33

31

23

29 ± 5

Positive control group

263

277

244

261 ± 17*

Test group

 

 

 

 

5

28

22

33

28 ± 6

2.5

18

21

25

21 ± 4

1.25

19

26

27

24 ± 4

0.625

28

19

24

24 ± 5

0.3125

31

25

25

27 ± 3

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

16

15

19

17 ± 2

Positive control groupb

345

343

379

356 ± 20*

Test group

 

 

 

 

5

15

17

20

17 ± 3

2.5

24

14

13

17 ± 6

1.25

31

19

21

24 ± 6

0.625

25

14

21

20 ± 6

0.3125

17

19

22

19 ± 3

Without S9 Mix

Negative control group

17

8

16

14 ± 5

Positive control group

618

709

775

701 ± 79*

Test group

 

 

 

 

5

16

15

18

16 ± 2

2.5

18

16

21

18 ± 3

1.25

22

27

22

24 ± 3

0.625

20

18

22

20 ± 2

0.3125

22

15

15

17 ± 4

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).

Applicant's summary and conclusion

Conclusions:
According to OECD 471 test method, CR SB08 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 M62-151100072001EN 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 CR SB08 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 CR SB08 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 CR SB08 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, CR SB08 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5mg/plate in the absence and presence of S9 metabolic activation.