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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 August 22, 2016 to December 09, 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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Pyridinium, 1,1'-[(6,13-dichloro-4,11-disulfo-3,10-triphenodioxazinediyl)bis[imino-2,1-ethanediylimino[6-[(2,5-disulfophenyl)amino]-1,3,5-triazine-4,2-diyl]]]bis[3-carboxy-, dihydroxide, bis(inner salt), hexasodium salt
EC Number:
279-256-2
EC Name:
Pyridinium, 1,1'-[(6,13-dichloro-4,11-disulfo-3,10-triphenodioxazinediyl)bis[imino-2,1-ethanediylimino[6-[(2,5-disulfophenyl)amino]-1,3,5-triazine-4,2-diyl]]]bis[3-carboxy-, dihydroxide, bis(inner salt), hexasodium salt
Cas Number:
79771-28-1
Molecular formula:
C52H38Cl2N16O24S6.6Na
IUPAC Name:
Pyridinium, 1,1'-[(6,13-dichloro-4,11-disulfo-3,10-triphenodioxazinediyl)bis[imino-2,1-ethanediylimino[6-[(2,5-disulfophenyl)amino]-1,3,5-triazine-4,2-diyl]]]bis[3-carboxy-, dihydroxide, bis(inner salt), hexasodium salt
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Everzol SB26
- Substance type: Powder
- Composition of test material, percentage of components: 78.12%
- Lot/batch No.: 3540
- Storage condition of test material: Ambient

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:
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

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

200

Positive control groupa

1234

Test group

 

5

177

2.5

164

1.25

212

0.625

191

0.3125

205

Without S9 Mix

Negative control group

171

Positive control group

770

Test group

 

5

142

2.5

137

1.25

131

0.625

138

0.3125

171

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

29

38

40 ± 12

Positive control groupb

234

226

218

226 ± 8*

Test group

 

 

 

 

5

31

28

35

31 ± 4

2.5

28

30

34

31 ± 3

1.25

35

31

31

32 ± 2

0.625

40

35

41

39 ± 3

0.3125

57

46

42

48 ± 8

Without S9 Mix

Negative control group

31

40

23

31 ± 9

Positive control group

283

257

228

256 ± 28*

Test group

 

 

 

 

5

27

25

28

27 ± 2

2.5

34

22

35

30 ± 7

1.25

31

27

24

27 ± 4

0.625

31

29

35

32 ± 3

0.3125

27

36

29

31 ± 5

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

210

193

200

201 ± 9

Positive control groupb

1332

1415

1372

1373 ± 42*

Test group

 

 

 

 

5

176

171

178

175 ± 4

2.5

204

222

178

201 ± 22

1.25

206

199

189

198 ± 9

0.625

211

203

186

200 ± 13

0.3125

219

204

192

205 ± 14

Without S9 Mix

Negative control group

214

204

185

201 ± 15

Positive control group

842

819

827

829 ± 12*

Test group

 

 

 

 

5

209

235

195

213 ± 20

2.5

205

195

209

203 ± 7

1.25

187

223

212

207 ± 18

0.625

209

204

219

211 ± 8

0.3125

216

177

233

209 ± 29

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

486

435

461

461 ± 26

Positive control groupb

1065

1136

1073

1091 ± 39*

Test group

 

 

 

 

5

387

409

393

396 ± 11

2.5

442

454

470

455 ± 14

1.25

407

383

428

406 ± 23

0.625

426

409

461

432 ± 27

0.3125

489

466

446

467 ± 22

Without S9 Mix

Negative control group

400

339

372

370 ± 31

Positive control group

839

834

870

848 ± 20*

Test group

 

 

 

 

5

371

363

357

364 ± 7

2.5

353

375

336

355 ± 20

1.25

372

390

361

374 ± 15

0.625

418

357

404

393 ± 32

0.3125

390

315

375

360 ± 40

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

20

15

24

20 ± 5

Positive control groupb

278

301

285

288 ± 12*

Test group

 

 

 

 

5

23

17

16

19 ± 4

2.5

14

25

18

19 ± 6

1.25

27

20

22

23 ± 4

0.625

16

17

29

21 ± 7

0.3125

29

27

23

26 ± 3

Without S9 Mix

Negative control group

22

21

20

21 ± 1

Positive control group

259

244

246

250 ± 8*

Test group

 

 

 

 

5

18

26

22

22 ± 4

2.5

20

24

25

23 ± 3

1.25

28

30

19

26 ± 6

0.625

25

18

31

25 ± 7

0.3125

30

27

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

20

18

17

18 ± 2

Positive control groupb

477

445

451

458 ± 17*

Test group

 

 

 

 

5

10

7

13

10 ± 3

2.5

12

17

11

13 ± 3

1.25

15

22

23

20 ± 4

0.625

16

15

10

14 ± 3

0.3125

11

13

19

14 ± 4

Without S9 Mix

Negative control group

18

16

16

17 ± 1

Positive control group

606

768

713

696 ± 82*

Test group

 

 

 

 

5

13

10

20

14 ± 5

2.5

17

11

19

16 ± 4

1.25

10

9

11

10 ± 1

0.625

14

13

12

13 ± 1

0.3125

18

15

23

19 ± 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, Everzol SB26 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 M62-151100132001EN 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 Everzol SB26 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 Everzol SB26 at 0.3125, 0.625, 1.25, 2.5 and 5 mg/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.

In the range-finding test of the Everzol SB26, no toxicity was observed in all five tester strains up to 5mg/plate in the absence and presence of metabolite activations. Results showed that Everzol SB26 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, Everzol SB26 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5mg/plate in the absence and presence of S9 metabolic activation.

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