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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication

Data source

Reference
Reference Type:
publication
Title:
Salmonella Mutagenicity Tests: III. Results From the Testing of 255 Chemicals
Author:
Errol Zeiger
Year:
1987
Bibliographic source:
Environmental Mutagenesis

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Preincubation Assay
The preincubation assay was performed as described previously [Haworth et al,1983]. The test chemical, Salmonella culture, and S-9 mix or buffer were incubated at 37"C, without shaking, for 20 min. Chemicals known or suspected to be volatile were incubated in capped tubes. The top agar was added, and the contents of the tubes were mixed and poured onto the surface of petri dishes that contained Vogel- Bonner medium [Vogel and Bonner, 19561. The histidine-revertant (his') colonies arising on these plates were counted following 2 days incubation at 37°C. The plates were hand-counted when a precipitate was present; otherwise automatic colony counters were used.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzimidazole-2-thiol
EC Number:
209-502-6
EC Name:
Benzimidazole-2-thiol
Cas Number:
583-39-1
Molecular formula:
C7H6N2S
IUPAC Name:
1H-benzimidazole-2-thiol
Details on test material:
SOURCE OF TEST MATERIAL
- Test substance: 2-mercaptobenzimidazole (benzimidazole-2-thiol)
- Source and lot/batch No.of test material: Aldrich
- Expiration date of the lot/batch: No data
- Purity: 98%
- Substance type: Organic
- Physical state: Solid
- Molecular formula: C7H6N2S
- Molecular weight: 150.204 g/mol

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Arocolor 1254-induced rat and Syrian hamster livers.
Test concentrations with justification for top dose:
0, 3.3, 10.0, 33.0, 100.0, 200.0, 333.0, 667.0, 1000.0, 3333.0, 6666.0, 6667.0, 10000.0 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO
Controlsopen allclose all
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
For strains TA1535 and TA 100 in the absence of metabolic activation.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
For strain TA 1537 in the absence of metabolic activation.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamin
Remarks:
For strain TA98 in the absence of metabolic activation.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
In the presence of metabolic activation for all strains.
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other:

OTHER: No data available
Rationale for test conditions:
No data
Evaluation criteria:
An individual trial was judged mutagenic (+) if a dose-related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic C+W) if a low-level dose response was seen. A trial was considered questionable (?) if a dose related increase was judged insufficiently high to justify a call of " + W," if only a single dose was elevated over the control, or if a non-dose-related increase was seen. The distinctions between a weak mutagenic response and a mutagenic response, or between a weak mutagenic response and a questionable mutagenic response are highly subjective.

A chemical was judged to be mutagenic (+), or weakly mutagenic (+W), if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials. A chemical was considered to be questionable (?) if a reproducible increase of hist revertants did not meet the criteria for either a " + " or " + W," or if only single doses produced an increase in his+ revertants in repeat trials. The chemicals were decoded by the chemical repository only after a determination had been made regarding their mutagenicity or nonmutagenicity .
Statistics:
Mean and SEM

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: The chemical was tested initially in a toxicity assay to determine the appropriate dose range. The toxicity assay was performed by using TA100 or the system developed by Waleh et al. Toxic concentrations were those at which a decrease in the number of his+ colonies was seen or at which there was a clearing in the density of the background lawn

COMPARISON WITH HISTORICAL CONTROL DATA: No data available

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available
Remarks on result:
other: No mutagenic potential was observed

Any other information on results incl. tables

Table: Mutagenicity of 2-Mercaptobenzimidazole (benzimidazole-2-thiol)

Dose (µg/plate)

TA100

-S9

10% HLI

30% HLI

10% RLI

30%HLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

98

5.6

68

3.2

143

3.3

81

0.9

125

2.8

3

 

 

 

 

 

 

 

 

 

 

10

 

 

 

 

 

 

 

 

 

 

33.3

93

2.5

 

 

123

5.5

 

 

117

3.8

100.0

96

2.2

59

1.8

131

22.1

76

1.2

118

3.1

200

 

 

 

 

 

 

 

 

 

 

333.0

77

5.3

64

2.9

115

11.2

74

5.4

94

10.4

667.0

 

 

 

 

 

 

 

 

 

 

1000

57

0.7

63

2.0

122

16.7

69

3.5

92

6.2

3333

34s

2.3

62

5.2

84

20.2

39

2.3

69s

5.0

6666

 

 

14

2.0

 

 

14

2.2

 

 

6667

 

 

 

 

38

7.0

 

 

49s

15.6

10000

 

 

 

 

 

 

 

 

 

 

Positive control

1060

27.8

901

4.7

871

37.4

1415

31.7

598

19.5

 

Dose (µg/plate)

TA1535

-S9

10% HLI

30% HLI

10% RLI

30%HLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

28

2.9

7

1.7

12

2.6

7

0.6

12

2.0

3

 

 

 

 

 

 

 

 

 

 

10

 

 

 

 

 

 

 

 

 

 

33.3

24

4.5

 

 

 

 

 

 

 

 

100.0

28

3.3

8

2.0

10

2.6

6

1.2

9

2.0

200

 

 

 

 

 

 

 

 

 

 

333.0

29

1.8

7

1.3

10

0.9

8

2.4

9

1.3

667.0

 

 

 

 

 

 

 

 

 

 

1000

40

0.9

8

2.0

7

2.3

7

0.7

5

1.2

3333

35

4.3

6

0.9

5

0.7

4

0.6

5

2.0

6666

 

 

5

0.9

3

0.3

3

0.3

3

0.7

6667

 

 

 

 

 

 

 

 

 

 

10000

 

 

 

 

 

 

 

 

 

 

Positive control

1009

33.1

119

10.6

119

10.8

114

7.7

96

4.3

 

Dose (µg/plate)

TA97

-S9

10% HLI

30% HLI

10% RLI

30%HLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

100

6.9

86

1.8

194

6.9

84

3.5

167

3.2

3

106

7.7

 

 

 

 

 

 

 

 

10

102

6.5

 

 

182

11.1

 

 

174

4.4

33.3

94

8.8

 

 

189

8.3

 

 

149

17.9

100.0

86

2.3

80

2.0

188

6.7

83

1.9

167

4.4

200

75

1.8

 

 

 

 

 

 

 

 

333.0

 

 

73

4.3

152

9.7

72

1.7

110

3.8

667.0

 

 

 

 

58

8.0

 

 

96

7.2

1000

 

 

16

6.1

 

 

8

1.5

 

 

3333

 

 

20

3.4

 

 

8

1.5

 

 

6666

 

 

2

1.2

 

 

1

0.6

 

 

6667

 

 

 

 

 

 

 

 

 

 

10000

 

 

 

 

 

 

 

 

 

 

Positive control

656

7.0

910

11.9

839

38.1

1402

4204

557

2.3

 

Dose (µg/plate)

TA98

-S9

10% HLI

30% HLI

10% RLI

30%HLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

24

3.9

21

2.2

34

0.9

71

3.7

21

3.2

3

 

 

 

 

 

 

 

 

 

 

10

 

 

 

 

 

 

 

 

 

 

33.3

20

3.2

 

 

 

 

 

 

 

 

100.0

12

1.2

19

2.8

32

0.9

26

3.3

 

 

200

 

 

 

 

 

 

 

 

 

 

333.0

14

3.8

25

4.8

37

0.7

24

1.0

24

0.0

667.0

 

 

 

 

 

 

 

 

 

 

1000

17

1.2

20

3.2

33

4.6

22

4.4

23

3.8

3333

14

1.0

23

1.5

50

4.8

19

3.1

20

5.5

6666

 

 

25

5.0

 

 

23

2.3

 

 

6667

 

 

 

 

44

4.7

 

 

27

5.8

10000

 

 

 

 

32

1.5

 

 

24

2.6

Positive control

2029

18.0

823

20.7

90

0.0

1328

40.0

474

0.6

 

s: slight clearing of background material

Applicant's summary and conclusion

Conclusions:
Test substance was tested for its genotoxic potetial in the Salmonella/microsome assay with preincubation using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activation (S9 mix from Arocolor 1254-induced rat and Syrian hamster livers.). The substance was not likly to exibit gene mutations.

Executive summary:

Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters was performed to evaluate the mutagenic nature of the test substance using S. typhimurium tester strains TA1535, TA97, TA98 and TA100. The study was performed as per the preincubation assay and the preincubation time was 20 mins and the plates were incubated for 48 hrs.  The test compound was used at a dosage level of 0, 3.3, 10.0, 33.0, 100.0, 200.0, 333.0, 667.0, 1000.0, 3333.0, 6666.0, 6667.0, 10000.0 µg/plate in the preincubation assay of 48 hrs. Concurrent solvent and positive control chemicals were included in the study. Test substance did not induce a reproducible, dose-related increase in his+revertants over the corresponding solvent in the S. typhimurium tester strains TA1535, TA1537, TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence is negative for mutation in vitro.

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