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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:
2017-07-26 to 2017-08-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Zinc di(benzimidazol-2-yl) disulphide
EC Number:
221-203-2
EC Name:
Zinc di(benzimidazol-2-yl) disulphide
Cas Number:
3030-80-6
Molecular formula:
C7H6N2S.1/2Zn
IUPAC Name:
2-{[(1H-1,3-benzodiazol-2-ylsulfanyl)zincio]sulfanyl}-1H-1,3-benzodiazole
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material: OUCHI SHINKO CHEMICAL INDUSTRIAL CO., LTD.; 608011
- Expiration date of the lot/batch: 2018-08-31

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep to the storeroom with suitable ventilation. Avoid fire, direct sunlight and moisture. Store at room temperature.

Method

Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver)
Test concentrations with justification for top dose:
5, 16, 50, 160, 500, 1600, 5000 µg/plate
Vehicle / solvent:
- Vehicle used: DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
NPD
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine
Remarks:
TA 98 (without S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
SA
Positive control substance:
sodium azide
Remarks:
TA 100, 1535 (without S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
9AA
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 (without S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
MMS
Positive control substance:
methylmethanesulfonate
Remarks:
E.coli (without S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2AA
Positive control substance:
other: 2-aminoanthracene
Remarks:
all Salmonella strains, E.coli (with S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results. A statistical evaluation of the results was not regarded as necessary.

Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups with or without metabolic activation.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 1600 µg/plate onwards
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 1600 µg/plate onwards
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 1600 µg/plate onwards
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 1600 µg/plate onwards
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slight from 1600 µg/plate onwards
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Milky, opalescent plates, in microscope at 40X magnification microdrops (as colloid chemical phenomenon) were observed at the concentration of 5000 μg/plate in the absence and presence of exogenous metabolic activation (±S9 Mix) following the plate incorporation procedure, and at 5000 μg/plate, in the presence of exogenous metabolic activation (+S9 Mix) following the pre-incubation procedure. The obtained microdrops did not disturb the scoring in any case.

RANGE-FINDING/SCREENING STUDIES:
The revertant colony numbers of vehicle control plates in both strains with and without S9 Mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.
In the Informatory Toxicity Test slight inhibitory effect of the test item was observed; that was indicated by lower revertant colony numbers (compared to that of the vehicle and historical control data ranges). A slight inhibitory, cytotoxic effect of the test item was observed in both strains at the concentrations of 5000 and 1600 μg/plate in the presence (+S9 Mix) and furthermore in S. typhimurium TA100 at 5000 μg/plate, in the absence (-S9 Mix) of exogenous metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the performed experiments slight inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was observed in all examined strains. In general the 1600 μg/plate was found to be the lowest cytotoxic concentration, observed in the Initial and Confirmatory Mutation Tests in the case of Salmonella typhimurium TA100 strain, in the presence of exogenous metabolic activation (+S9 Mix).

Any other information on results incl. tables

Table 1 Results

Experiment 1 (SPT)

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and E. coli

 

TA1535

TA1537

TA98

TA100

WP2 uvrA

 

 

 

Results with S9

Untreated control

10.7

7.3

26.3

105

26.7

DMSO control

11

8

25

90.3

23.7

5000

4.7

6.3

17.7

55.7

9.7

1600

7.3

12

23.3

61.7

25.3

500

9

8.3

17

82.3

23.3

160

9.7

7.3

14.7

83

24.3

50

10.3

8.7

20

79.7

28.3

16

13

6.7

26.3

90.7

24

5

10

8

19.7

95.3

23.7

Positive control

219.7

222

1421.3

2406.7

264.3

 

 

 

Results without S9

Untreated control

9.7

9.7

20.7

86.3

16.7

DMSO control

9.3

8.7

20.7

78.7

14.7

5000

3.7

6.7

12

49

15.3

1600

10.3

8.3

17.3

72.7

15.7

500

10.7

9.7

19

73

20.7

160

10.7

10.3

24.7

70.3

24.3

50

11.3

6.7

22

80.7

21.3

16

7

9.3

19.7

81.3

22.3

5

9

7.3

25.3

84.3

22.7

Positive control

589.3

655.3

223

949.3

548.7

 

 

 

 

 

 

Experiment 2 (PIT)

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and E. coli

TA1535

TA1537

TA98

TA100

WP2 uvrA

 

 

 

Results with S9

Untreated control

15

6.3

21

103.7

38

DMSO control

12.7

6

23.3

81

41.7

5000

6.3

2.7

19.3

46.3

28

1600

7.3

9

20

67

32.3

500

8.7

7

25

82

41

160

9

6

23.3

91

47.3

50

13.7

7

25.7

94.3

50.7

16

11

7.7

18.3

96

44.7

5

13

7.7

22

94

49.3

Positive control

154.3

124.3

1226.7

1514.7

181.7

 

 

 

Results without S9

Untreated control

13.3

8.3

23.7

77

30.3

DMSO control

12.7

6.3

18.7

76.3

28.3

5000

6.7

4.7

10.3

43.3

20.7

1600

14.3

7.7

24.3

66.7

27.3

500

12

7.7

24

77.7

42

160

10.3

6.7

21.7

78

25

50

10.7

6

20

85

33.3

16

10.3

5.3

21.3

82

28.7

5

12

10

20

86

25.3

Positive control

1464

1458.7

247.3

1260

816

Applicant's summary and conclusion

Conclusions:
Under the conditions of the above assay, Zinc di(benzimidazol-2-yl) disulphide tested as suspension formulation in a suitable vehicle (DMSO) did not show any mutagenic potential.
Executive summary:

In a reverse mutation assay in bacteria according to OECD guideline 471, strains TA98, TA1537, TA1535 and TA100 of S. typhimurium and Escherichia coli WP2 uvrA were investigated. The test item was dissolved in dimethyl sulfoxide (DMSO) and concentrations of 5000, 1600, 500; 160; 50; 16 and 5 μg/plate were examined.

The five bacterial strains were used to investigate the mutagenic potential of Zinc di(benzimidazol-2-yl) disulphide in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently.

In the performed experiments all of the validity criteria regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled (See: Validity of the Study).

No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with Zinc di(benzimidazol-2-yl) disulphide at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

Sporadic increases in revertant colony numbers compared to the vehicle control values within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

In the performed experiments slight inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was observed in all examined strains. In general the 1600 μg/plate was found to be the lowest cytotoxic concentration, observed in the Initial and Confirmatory Mutation Tests in the case of Salmonella typhimurium TA100 strain, in the presence of exogenous metabolic activation (+S9 Mix).

Milky, opalescent plates, in microscope at 40X magnification microdrops (as colloid chemical phenomenon) were observed at the concentration of 5000 μg/plate in the absence and presence of exogenous metabolic activation (±S9 Mix) following the plate incorporation procedure, and at 5000 μg/plate, in the presence of exogenous metabolic activation (+S9 Mix) following the pre-incubation procedure. The obtained microdrops did not disturb the scoring in any case.

The reported data of this mutagenicity assay show, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, Zinc di(benzimidazol-2-yl) disulphide is considered non-mutagenic in this bacterial reverse mutation assay.