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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CR SB37 was not mutagenic in the reverse mutation analysis ofSalmonellatyphimuriumup 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 16, 2017 to December 22, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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:
Rat liver S9 mix and hamster liver 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 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
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

R plasmida

TA98

+

+

+

+

TA100

+

+

+

+

TA102

+

ϴ

+

+

TA1535

+

+

+

ϴ

TA1537

+

+

+

ϴ

a R plasmid: Ampicillin resistance were used for R plasmid test.

+ 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)

Average of coloniesa

1

2

3

Without S9 activation

Negative control group

182

185

199

189 ± 9

Positive control groupb

854

980

845

893±75

Test group

5

180

201

183

188 ± 11

2.5

173

184

192

183 ± 10

1.25

199

180

172

184 ± 14

0.625

185

195

184

188 ± 6

0.3125

192

204

183

193 ± 11

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

b Positive control group: 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

Rat liver S9 activation

Negative control group

37

31

33

34 ± 3

Positive control groupb

256

199

218

224 ± 29*

Test group

5

38

39

39

39 ± 1

2.5

34

38

39

37 ± 3

1.25

31

39

37

36 ± 4

0.625

43

36

30

36 ± 7

0.3125

43

48

33

41 ± 8

Hamster liver S9 activation

Negative control group

36

32

38

35 ± 3

Positive control group

201

270

235

235 ± 35*

Test group

5

31

34

35

33 ± 2

2.5

31

26

36

31 ± 5

1.25

41

30

41

37 ± 6

0.625

40

42

43

42 ± 2

0.3125

41

48

45

45 ± 4

Without S9 activation

Negative control group

31

33

39

34 ± 4

Positive control group

205

249

240

231 ± 23*

Test group

5

31

36

34

 34 ± 3

2.5

30

36

42

 36 ± 6

1.25

30

35

36

 34 ± 3

0.625

33

34

36

 34 ± 2

0.3125

35

36

35

 35 ± 1

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

b Positive control group:

Rat liver S9 activation and Hamster liver S9 activation group: Benzo [a] pyrene (4.0μg/plate).

Without S9 activation group: 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

Rat liver S9 activation

Negative control group

185

165

182

177 ± 11

Positive control groupb

662

683

608

651 ± 39*

Test group

5

173

175

177

175 ± 2

2.5

173

179

172

175 ± 4

1.25

178

182

183

181 ± 3

0.625

171

193

182

182 ± 11

0.3125

198

184

182

188 ± 9

Hamster liver S9 activation

Negative control group

175

167

173

172 ± 4

Positive control group

684

614

699

666 ± 45*

Test group

5

182

183

186

184 ± 2

2.5

184

188

176

183 ± 6

1.25

176

171

180

176 ± 5

0.625

170

174

196

180 ± 14

0.3125

177

173

178

176 ± 3

Without S9 activation

Negative control group

163

168

173

168 ± 5

Positive control group

675

579

606

620 ± 50*

Test group

5

164

162

177

168 ± 8

2.5

178

168

158

168 ± 10

1.25

178

176

167

174 ± 6

0.625

167

187

182

179 ± 10

0.3125

180

165

160

168 ± 10

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

b Positive control group:

Rat liver S9 activation and Hamster liver S9 activation group:2-Aminoanthracene (4.0μg/plate).

Without S9 activation group: 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

Rat liver S9 activation

Negative control group

438

436

447

440 ± 6

Positive control groupb

871

939

901

904 ± 34*

Test group

5

441

445

466

451 ± 13

2.5

472

465

464

467 ± 4*

1.25

429

432

419

427 ± 7

0.625

425

431

424

427 ± 4

0.3125

433

442

438

438 ± 5

Hamster liver S9 activation

Negative control group

405

412

441

419 ± 19

Positive control group

896

870

830

865 ± 33*

Test group

5

448

419

441

436 ± 15

2.5

425

446

452

441 ± 14

1.25

455

448

439

447 ± 8

0.625

425

442

442

436 ± 10

0.3125

462

452

442

452 ± 10

Without S9 activation

Negative control group

399

383

387

390 ± 8

Positive control group

840

972

870

894 ± 69*

Test group

5

423

433

422

426 ± 6

2.5

454

457

435

449 ± 12*

1.25

420

449

433

434 ± 15

0.625

413

428

417

419 ± 8

0.3125

429

462

448

446 ± 17*

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

b Positive control group:

Rat liver S9 activation and Hamster liver S9 activation group: 2-Aminoanthracene (10.0μg/plate).

Without S9 activation group: 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

Rat liver S9 activation

Negative control group

17

17

15

16 ± 1

Positive control groupb

356

482

286

375 ± 99*

Test group

5

9

13

10

11 ± 2

2.5

7

13

12

11 ± 3

1.25

18

17

12

16 ± 3

0.625

16

17

13

15 ± 2

0.3125

15

19

16

17 ± 2

Hamster liver S9 activation

Negative control group

20

29

27

25 ± 5

Positive control group

250

240

232

241 ± 9*

Test group

5

21

18

22

20 ± 2

2.5

17

32

22

24 ± 8

1.25

22

21

23

22 ± 1

0.625

23

15

22

20 ± 4

0.3125

17

23

22

21 ± 3

Without S9 activation

Negative control group

15

15

18

16 ± 2

Positive control group

204

199

271

225 ± 40*

Test group

5

15

19

18

17 ± 2

2.5

12

11

8

10 ± 2

1.25

13

18

10

14 ± 4

0.625

8

10

10

9 ± 1

0.3125

14

14

12

13 ± 1

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

b Positive control group:

Rat liver S9 activation and Hamster liver S9 activation group:2-Aminoanthracene (4.0μg/plate).

Without S9 activation group: 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

Rat liver S9 activation

Negative control group

10

10

9

10 ± 1

Positive control groupb

425

538

344

436 ± 97*

Test group

5

7

8

9

8 ± 1

2.5

8

6

13

9 ± 4

1.25

7

6

10

8 ± 2

0.625

9

8

13

10 ± 3

0.3125

7

8

13

9 ± 3

Hamster liver S9 activation

Negative control group

15

14

10

13 ± 3

Positive control group

431

441

455

442 ± 12*

Test group

5

10

10

9

10 ± 1

2.5

8

10

13

10 ± 3

1.25

8

9

10

9 ± 1

0.625

10

9

12

10 ± 2

0.3125

10

9

11

10 ± 1

Without S9 activation

Negative control group

10

7

10

9 ± 2

Positive control group

312

336

358

335 ± 23*

Test group

5

10

6

6

7 ± 2

2.5

11

12

8

10 ± 2

1.25

9

12

9

10 ± 2

0.625

10

8

9

9 ± 1

0.3125

8

4

8

7 ± 2

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

b Positive control group:

Rat liver S9 activation and Hamster liver S9 activation group: 2-Aminoanthracene (4.0μg/plate).

Without S9 activation group: 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, CR SB37 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 M62-170900011001EN 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 SB37 show that test validity criteria was met.

Based on the preliminary assay results, 5 mg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CR SB37 at 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate, negative control and positive controls were tested in tester strains TA98, TA100, TA102, TA1535 and TA1537 in triplicate with or without S9 mix activation. The metabolite activations were rat liver S9 mix activation and hamster liver S9 mix activation. Results showed that CR SB37 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5 mg/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 SB37 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5 mg/plate in the absence and presence of S9 metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Based on the preliminary assay results, 5 mg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CR SB37 at 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate, negative control and positive controls were tested in tester strains TA98, TA100, TA102, TA1535 and TA1537 in triplicate with or without S9 mix activation. The metabolite activations were rat liver S9 mix activation and hamster liver S9 mix activation. Results showed that CR SB37 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5 mg/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 SB37 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5 mg/plate in the absence and presence of S9 metabolic activation.

Justification for classification or non-classification

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