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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From to July 25, 2016 October 07, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
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

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:
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 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

266

Positive control groupa

1173

Test group

5

259

2.5

302

1.25

270

0.625

258

0.3125

243

Without S9 Mix

Negative control group

223

Positive control group

796

Test group

5

275

2.5

243

1.25

280

0.625

282

0.3125

274

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

34

30

38

34 ± 4

Positive control groupb

262

243

236

247 ± 13*

Test group

5

25

35

26

29 ± 6

2.5

24

25

29

26 ± 3

1.25

29

28

34

30 ± 3

0.625

32

35

31

33 ± 2

0.3125

29

53

34

39 ± 13

Without S9 Mix

Negative control group

20

31

25

25 ± 6

Positive control group

338

358

373

356 ± 18*

Test group

5

21

31

32

28 ± 6

2.5

33

29

30

31 ± 2

1.25

27

26

24

26 ± 2

0.625

20

26

27

24 ± 4

0.3125

26

24

32

27 ± 4

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

255

240

237

244 ± 10

Positive control groupb

1471

1308

1355

1378 ± 84*

Test group

5

339

211

293

281 ± 65

2.5

278

295

255

276 ± 20

1.25

230

275

228

244 ± 27

0.625

219

279

240

246 ± 30

0.3125

248

249

261

253 ± 7

Without S9 Mix

Negative control group

178

159

186

174 ± 14

Positive control group

857

884

809

850 ± 38*

Test group

5

184

176

207

189 ± 16

2.5

185

178

183

182 ± 4

1.25

194

167

169

177 ± 15

0.625

179

150

158

162 ± 15

0.3125

188

185

166

180 ± 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 (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

407

397

436

413 ± 20

Positive control groupb

1133

964

1054

1050 ± 85*

Test group

5

386

397

367

383 ± 15

2.5

379

397

411

396 ± 16

1.25

413

396

409

406 ± 9

0.625

298

373

394

388 ± 12

0.3125

468

376

409

418 ± 47

Without S9 Mix

Negative control group

369

419

385

391 ± 26

Positive control group

1015

1071

912

999 ± 81*

Test group

5

343

341

356

347 ± 8

2.5

407

392

368

389 ± 20

1.25

384

371

341

365 ± 22

0.625

320

371

374

355 ± 30

0.3125

385

353

371

370 ± 16

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

24

17

21

21 ± 4

Positive control groupb

584

497

538

540 ± 44*

Test group

5

22

23

21

22 ± 1

2.5

20

21

13

18 ± 4

1.25

26

23

20

23 ± 3

0.625

28

14

21

21 ± 7

0.3125

19

18

21

19 ± 2

Without S9 Mix

Negative control group

17

18

20

18 ± 2

Positive control group

238

261

237

245 ± 14*

Test group

5

18

16

12

15 ± 3

2.5

23

18

14

18 ± 5

1.25

19

27

19

22 ± 5

0.625

27

20

19

22 ± 4

0.3125

20

25

25

23 ± 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

12

15

14

14 ± 2

Positive control groupb

296

289

295

293 ± 4*

Test group

5

7

10

19

12 ± 6

2.5

10

19

13

14 ± 5

1.25

12

7

15

11 ± 4

0.625

13

10

16

13 ± 3

0.3125

11

14

18

14 ± 4

Without S9 Mix

Negative control group

12

15

11

13 ± 2

Positive control group

993

912

860

922 ± 67*

Test group

5

17

10

9

12 ± 4

2.5

12

10

8

10 ± 2

1.25

15

13

12

13 ± 2

0.625

9

21

6

12 ± 8

0.3125

12

16

16

15 ± 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).

Applicant's summary and conclusion

Conclusions:
According to OECD 471 test method, CR SB87 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 M62-151100120001EN 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 SB87 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 CR SB87 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 toxicity was observed in all five tester strains up to 5mg/plate in the absence and presence of metabolite activations. Results showed that CR SB87 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 SB87 was not mutagenic in the reverse mutation analysis ofSalmonellatyphimuriumup to 5mg/plate in the absence and presence of S9 metabolic activation.