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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-10-21 to 2004-11-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate coherence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
• Other name : MTSC
• batch number: 0404001019
• Sponsor's filing number: GRL 0035/04
• description:
- at receipt: white powder
- on the Study plan and on the test article description: white solid
• container: one glass flask
• date of receipt: 25 August 2004
• storage conditions: at room temperature, protected from light and humidity
• purity: 99.7%
• composition: see analytical certificate
• expiry date: 30 April 2006.

Method

Target gene:
Histidine operon.
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiments without S9 mix

Experiments with S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
The test item was tested in a preliminary test and two mutagenicity experiments.
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix was performed according to the direct plate
incorporation method. The second experiment with S9 mix was performed according to the preincubation method.

DURATION
- Preincubation period: 60 minutes, 37°C
- Exposure duration: 48 to 72 hours, 37°C in growth medium

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies and/or thinning of the bacterial lawn
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a
positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
Not applicable.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The test item was freely soluble in the vehicle (DMSO) at 100 mg/mL.
Consequently, with a treatment volume of 50 μL/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 μg/plate.
No precipitate was observed in the Petri plates when scoring the revertants at any dose-level.
No noteworthy toxicity was noted towards the three strains used, with and without S9 mix.
The individual results of the preliminary toxicity test are provided in table 1.

TOXICITY AND MUTATIONS :
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.
Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level for the main test was 5000 μg/plate, according to the criteria specified in the international guidelines.
The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 μg/plate, for both mutagenicity experiments with and without S9 mix.
No toxicity was noted towards all the strains used, both with and without S9 mix.
The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.
The findings of the individual tests are provided in tables 2 (first experiment) and 3 (second experiment).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Tables of results: Ames test with MTSC

 

1/ First experiment / Direct plate incorporation method

 

strain

compound

Dose level (µg/ plate)

S9-mix

Mean revertant colony counts

SD

Ration treated/

solvent

Individual revertant colony counts

 

 

TA 1535

DMSO

 

-

11

2

 

14

10

10

Test item

312.5

-

9

3

0.8

8

13

7

625

-

13

4

1.1

14

8

16

1250

-

14

4

1.2

10

13

18

2500

-

11

1

1.0

10

11

12

5000

-

13

2

1.2

12

12

16

NAN3

1

-

583

26

51.5

588

555

607

DMSO

 

+

9

4

 

10

5

12

Test item

312.5

+

12

1

1.4

13

11

13

625

+

12

2

1.3

12

13

10

1250

+

13

2

1.4

13

14

11

2500

+

16

3

1.8

16

19

14

5000

+

14

1

1.5

14

13

14

2AM

2

+

219

22

24.4

201

214

243

 

 

TA 1537

DMSO

 

-

5

3

 

5

2

8

Test item

312.5

-

7

2

1.5

10

6

6

625

-

6

2

1.1

8

5

4

1250

-

3

4

0.5

7

0

1

2500

-

5

2

0.9

6

6

2

5000

-

5

3

0.9

7

6

1

9AA

50

-

657

332

131.3

999

335

636

DMSO

 

+

6

3

 

2

8

8

Test item

312.5

+

9

2

1.4

6

10

10

625

+

9

2

1.6

7

11

10

1250

+

10

2

1.7

12

8

11

2500

+

9

3

1.4

11

10

5

5000

+

7

3

1.2

7

5

10

2AM

2

+

62

11

10.4

74

60

53

 

 

TA 98

DMSO

 

-

22

5

 

24

17

36

Test item

312.5

-

15

7

0.7

8

22

14

625

-

17

9

0.8

18

8

25

1250

-

20

6

0.9

26

14

20

2500

-

33

3

1.5

30

32

36

5000

-

27

8

1.2

20

36

26

2NF

0.5

-

274

17

12.3

260

269

292

DMSO

 

+

27

4

 

24

32

25

Test item

312.5

+

27

6

1.0

34

24

24

625

+

27

4

1.0

22

30

29

1250

+

26

2

1.0

28

24

26

2500

+

22

8

0.8

18

17

31

5000

+

17

7

0.6

11

24

16

2AM

2

+

775

383

28.7

525

583

1216

 

 

TA 100

DMSO

 

-

167

25

 

176

176

139

Test item

312.5

-

147

16

0.9

166

138

137

625

-

139

25

0.8

155

110

152

1250

-

130

13

0.8

145

119

126

2500

-

112

9

0.7

104

110

121

5000

-

103

7

0.6

104

109

96

NAN3

1

-

828

54

5.0

881

773

928

DMSO

 

+

129

14

 

120

122

146

Test item

312.5

+

132

1

1.0

132

133

132

625

+

131

9

1.0

126

126

141

1250

+

122

6

0.9

127

125

115

2500

+

90

17

0.7

81

80

110

5000

+

119

39

0.9

92

163

101

2AM

2

+

1312

983

10.1

773

716

2447

 

 

TA 102

DMSO

 

-

316

7

 

319

322

308

Test item

312.5

-

321

21

1.0

298

338

328

625

-

342

19

1.1

340

362

325

1250

-

350

10

1.1

343

346

361

2500

-

298

16

0.9

283

296

314

5000

-

281

27

0.9

311

272

260

MMC

0.5

-

2370

62

7.5

2298

2405

2406

DMSO

 

+

342

12

 

333

355

337

Test item

312.5

+

383

69

1.1

454

378

316

625

+

393

27

1.2

365

419

396

1250

+

429

7

1.3

423

437

428

2500

+

377

55

1.1

398

419

315

5000

+

342

32

1.0

362

359

305

2AM

10

+

1809

206

5.3

2025

1788

1614

SD: standard deviation

- : absence S9

+: presence S9

 

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under these experimental conditions, the test item MTSC did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.
Executive summary:

The potential of 4 -methylthiosemicarbazide to induce reverse mutation was evaluated in Salmonella typhimurium. The study was performed according to the international guidelines (OECD 471 and Commission Directive No. B13/14) and in compliance with the principles of Good Laboratory Practice.

A preliminary toxicity test was performed to define the dose-levels of MTSC to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The test item 4-METHYLTHIOSEMICARBAZIDE was dissolved in dimethylsulfoxide (DMSO). The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level for the main test was 5000 μg/plate, according to the criteria specified in the international guidelines. The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 μg/plate, for both mutagenicity experiments with and without S9 mix. No toxicity was noted towards all the strains used, both with and without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains. Under these experimental conditions, the test item MTSC did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.