Registration Dossier

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, OECD 471 Guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

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:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): DIBENZYL TRITHIOCARBONATE
- Physical state: yellow orange-colored solid
- Analytical purity: 96.2
- Purity test date: 2003-10-27
- Lot/batch No.: 1 UG 114
- Expiration date of the lot/batch: November 2004
- Storage condition of test material: at room temperature and protected from light

Method

Target gene:
Histidine operon
Species / strain
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 homogenate was prepared from rats that have been induced with Arochlor 1254
Test concentrations with justification for top dose:
31.3, 62.5, 125, 250 and 500 µg/plate, for both mutagenicity experiments without S9
62.5, 125, 250, 500 and 1000 µg/plate, for the TA 1535, TA 1537 and TA 98 strains in the first experiment with S9.
31.3, 62.5, 125, 250 and 500 µg/plate, for the TA 100 and TA 102 strains in the first experiment with S9 as for all the strains in the second experiment with S9
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9 mix: Sodium azide (NAN3), TA 1535 & TA 100; 9-Aminoacridine (9AA), TA 1537; 2-Nitrofluorene (2NF), TA 98 strain; Mitomycin C (MMC), TA 102 strain. + S9 mix: 2-Anthramine (2AM), TA 1535; TA 1537; TA 98; TA 100 strain and 2AM, TA 102 strains
Details on test system and conditions:
DETERMINATION OF CYTOTOXICITY (preliminary range-finding test)
- Test: in TA 98, TA 100 and TA 102 strains, with or without S9 mix ; 6 dose-levels (one plate/dose level)
- Method: relative total growth (decrease in the number of revertant colonies and/or a thinning of the bacterial lawn);

EXPERIMENTS
Number of independent experiments: 2.

METHOD OF APPLICATION:
* Direct plate incorporation method: for preliminary test and first experiment
* Preincubation: for the second experiment

DURATION
- Preincubation period: 60 min
- Exposure duration: 48-72H

NUMBER OF REPLICATIONS: triplicates
Evaluation criteria:
Reproducible increase in the number of revertant colonies (2-fold for TA98/TA100 and TA102, 3-fold for TA 1535/TA 1537) compared with vehicle controls in any strain at any dose-level and/or evidence of a dose-relationship.
Reference to historical data and consideration to biological relevance may also be taken into account.
Statistics:
Not applicable

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
>= 1000 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
RANGE-FINDING STUDY:
- Results on solubility: freely soluble in the vehicle (DMSO) at 50 mg/mL.
- Results on cytotoxicity:
A moderate to marked toxicity was noted at dose-levels ≥ 1000 µg/plate in the TA 98 and TA 102 strains without S9 mix and in the TA 100 strain with and without S9 mix. A moderate to marked toxicity was observed at dose-levels ≥ 2500 µg/plate in the TA 98 and TA 102 strains with S9 mix.
- Precipitation:
A moderate to marked precipitate was observed in the Petri plates when scoring the revertants generally at dose-levels ≥ 500 µg/plate.

MAIN STUDY:
A moderate to marked precipitate was observed in the Petri plates when scoring the revertants mainly at dose-levels ≥ 250 µg/plate without S9 and ≥ 500 µg/plate with S9

The test item did not induce any noteworthy increase in the number of revertants, with and without S9 mix, in any of the five strains.

Any other information on results incl. tables

First assay

Strain

Compound

Dose level

per plate

S-9

mix

Mean

revertant

colony

counts

SD

Ratio

treated

/

solvent

Individual revertant

colony counts

TA 1535

DMSO

-

10

3

7,13,10

TEST ITEM

31.3 µg

-

5

1

0.5

6, 4, 6

62.5 µg

-

15

3

1.5

12,16,18

125µg

-

9

4

0.9

4,10,12

250µg

-

6

2

0.6

8,7,4

500µg

-

10

2

1.0

12 Sp, 8 Sp, 11 Sp

NAN3

1 µg

-

396

49

39.6

430, 419, 340

DMSO

+

12

6

11, 6,18

TEST ITEM

62.5 µg

+

16

2

1.4

18,14,17

125µg

+

15

4

1.3

19,11,14

250µg

+

18

2

1.5

19, 19, 16

500µg

+

18

10

1.5

8 Mp, 17 Mp, 28 Mp

1000µg

+

11

1

0.9

12 Sp, 11 Sp, 10 Sp

2AM

2 µg

+

204

18

17.5

184, 217, 211

TA 1537

DMSO

-

7

0

7, 7, 7

TEST ITEM

31.3 µg

-

5

1

0.7

5, 4, 6

62.5µg

-

4

3

0.6

7,2,4

125µg

-

5

1

0.7

5,6,4

250µg

-

3

2

0.5

6,2,2

500µg

-

2

2

0.3

1 Sp,4 Sp,2 Sp

9AA

50 µg

-

647

190

92.4

709, 798, 434

DMSO

+

8

3

6, 6,12

TEST ITEM

62.5 µg

+

11

1

1.3

11,

125µg

+

7

2

0.8

4,8,8

250µg

+

12

7

1.5

5,13,18

500 µg

+

10

6

1.2

16 Mp, 7 Mp, 6 Mp

1000µg

+

6

2

0.8

6 Sp,8 Sp,4 Sp

2AM

2µg

+

91

10

11.4

81, 101, 91

TA 98

DMSO

-

28

9

36, 29, 19

TEST ITEM

31.3 µg

-

23

5

0.8

26, 26,17

62.5 µg

-

20

4

0.7

24,19,16

125 µg

-

25

5

0.9

25, 20, 29

250 µg

-

24

5

0.8

28 Mp, 24 Mp, 19 Mp

500µg

-

25

14

0.9

8 Sp,32 Sp,34 Sp

2NF

0.5 µg

-

141

16

5.0

131,160,133

DMSO

+

32

3

32, 29, 35

TEST ITEM

62.5 µg

+

50

5

1.6

48, 47, 56

125 µg

+

59

19

1.8

67, 37, 72

250 µg

+

41

6

1.3

46, 43, 35

500 µg

+

42

13

1.3

30 Mp, 41 Mp, 55 Mp

1000 µg

+

48

30

1.5

79 Sp, 46 Sp, 20 Sp

2AM

2 µg

+

1460

290

45.6

1571,1677,1131

TA 100

DMSO

-

99

25

127, 92, 79

TEST ITEM

31.3µg

-

77

19

0.8

98, 67, 65

62.5µg

-

86

12

0.9

77, 81, 99

125µg

-

95

7

1.0

92, 103,90

250µg

-

71

11

0.7

81 Mp, 60 Mp, 73 Mp

500µg

-

58

30

0.6

24 Sp, 78 Sp, 73 Sp

NAN3

1µg

-

423

48

4.3

442, 458, 368

DMSO

+

95

19

115, 78, 91

TEST ITEM

31.3µg

+

66

3

0.7

67, 63, 69

62.5µg

+

77

4

0.8

77, 73,81

125µg

+

77

15

0.8

61, 90, 80

250µg

+

82

22

0.9

59,85, 102

500µg

+

70

5

0.7

66 Mp, 75 Mp, 68 Mp

2AM

2 µg

+

651

49

6.9

678, 680, 595

TA 102

DMSO

-

370

17

361, 389, 359

TEST ITEM

31.3µg

-

418

25

1.1

398, 410, 446

62.5µg

-

404

19

1.1

387, 425, 399

125µg

-

499

119

1.3

366, 535, 595

250µg

-

432

38

1.2

468 Mp, 436 Mp, 392 Mp

500µg

-

499

131

1.3

635 Sp, 487 Sp, 374 Sp

MMC

0.5µg

-

1599

156

4.3

1422, 1714, 1662

DMSO

+

425

54

363, 459, 454

TEST ITEM

31.3µg

+

508

47

1.2

558, 466, 499

62.5µg

+

550

136

1.3

497, 448, 704

125µg

+

635

137

1.5

792, 569, 543

250µg

+

471

15

1.1

480, 479, 453

500µg

+

720

201

1.7

916 Mp, 729 Mp, 515 Mp

2AM

10µg

+

2162

773

5.1

2635,2581, 1270

SD: Standard deviation

Mp: Moderate precipitate

Sp:Strong precipitate

- : Absence of S-9

+: Presence ofS-9

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

DIBENZYL TRITHIOCARBONATE did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium
Executive summary:

The potential of DIBENZYL TRITHIOCARBONATE to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, TA 100 and TA 102) was evaluated in accordance with the international guidelines (OECD 471, Commission Directive No. B13/14) in compliance with the Principles of Good Laboratory Practice.

DIBENZYL TRITHIOCARBONATE was tested in two independent experiments, with and without a metabolic activation system, both 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). Bacterias were exposed to DIBENZYL TRITHIOCARBONATE at six dose-levels (three plates/dose-level) selected from a preliminary toxicity test: 31.3 to 500 μg/plate without S9 and 62.5 to 1000 µg/plate for the TA 1535, TA 1537 and TA 98 strains in the first experiment with S9 or 31.3 to 500 µg/plate for the TA 100 and TA 102 strains in the first experiment as for all the strains in the second with S9. After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. DIBENZYL TRITHIOCARBONATE 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, DIBENZYL TRITHIOCARBONATE did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.