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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:
weight of evidence
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:
Gene mutation toxicity study of the test chemical
Author:
Zeiger et al
Year:
1992
Bibliographic source:
Environmental and Molecular 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:
Ames Salmonella typhimurium test was conducted on Salmonella typhimurium strains TA100,TA1535,TA1537,TA97,TA98 to evaluate the mutagenic effect of the test chemical
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzimidazole
EC Number:
200-081-4
EC Name:
Benzimidazole
Cas Number:
51-17-2
Molecular formula:
C7H6N2
IUPAC Name:
1H-1,3-benzodiazole
Details on test material:
- Name of test material: Benzimidazole
- IUPAC name: 1H-benzimidazole
- Molecular formula: C7H6N2
- Molecular weight: 118.1384 g/mol
- Substance type: Organic
- Physical state: Solid
- Purity: Analyzed purity 99%+, Vendor's purity: 98%
- Impurities (identity and concentrations): No data

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA100,TA1535,TA1537,TA97,TA98
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:
The S-9 (9,000 x g supernatant) fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers were used
Test concentrations with justification for top dose:
0, 33, 100, 333, 1000, 3333 or 6666 µg/plate
Vehicle / solvent:
DMSO
Controls
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
sodium azide
other: 4-nitro-o-phenylenediamine (TA98 and TA1538; Without S9); 2-aminoanthracene (All strains; with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period:20 min
- Exposure duration: 2 days (48 hrs)
- Expression time (cells in growth medium): 2 days (48 hrs)
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells):no data

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

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

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

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

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
Magnitude of the dose dependent increase in his+ revertants, and the shape of the dose response.

Evaluations were made at both the individual trial and chemical levels. Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?),
or nonmutagenic (-), depending on the magnitude of the increase in his+ revertants, and the shape of the dose response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “+ W”, if only a single dose was elevated over the control, or if a weak increase was not dose-related.

A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible, dose-related response over the solvent control, under a single metabolic activation condition, in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in hisf revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response.
Statistics:
No data

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA100, TA1535, TA1537, TA97, TA98
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
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Benzimmidazole was run initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay. The toxicity assay was performed using TA100or the system developed by Waleh et al. [1982]. Toxic concentrations were defined as those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data
Remarks on result:
other: No mutagenic potential

Any other information on results incl. tables

For strain TA100:

Dose

NA

10%HLI

30% HLI

10%RLI

30%RLI

µG/plate

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

95

8.4

136

24.4

133

3.8

90

5.8

123

11.5

33

90

4.6

 

 

 

 

 

 

 

 

100

90

5.2

142

5.7

124

1.2

84

0.3

123

9.0

333

102

4.7

153

3.5

127

13.6

101

6.2

127

2.2

1000

110

13.9

146

4.4

125

9.2

96

4.7

114

12

3333

101

1.7

130

3.3

110

6.9

101

5.5

110

18.6

6666

 

 

104

15.7

92

2.9

87

3.7

73

3.7

POS

497

38.9

458

84.4

380

14.6

339

69.0

413

11.3

 

For strain TA1535:-

Dose

NA

10%HLI

30% HLI

10%RLI

30%RLI

µG/plate

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

12

1.9

12

0.3

10

2.7

7

1.8

12

2.5

33

19

5.0

 

 

 

 

 

 

 

 

100

19

2.8

12

2

12

1.5

10

0

10

1.9

333

19

0.6

7

2

9

2.0

13

2.6

11

1.3

1000

17

1.5

9

2.7

8

0.7

9

0.7

11

2.1

3333

11

2.5

11

2.1

5

0.9

9

1.0

8

1.2

6666

 

 

5

0.9

5

1.3

7

1.8

3

1.5

POS

332

14.2

168

20.4

310

24.2

198

10.7

100

0.9

 

For strain TA1537:-

Dose

NA

10%HLI

30% HLI

10%RLI

30%RLI

µG/plate

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

5

1.2

9

2.3

8

2.3

8

1.7

8

0.9

33

8

2.0

 

 

 

 

 

 

 

 

100

6

0.7

6

1.0

9

1.2

9

0.9

7

0.6

333

6

0.9

6

0.9

6

1.5

6

1.3

8

1.7

1000

8

1.5

10

2.1

6

1.9

7

2.1

9

1.7

3333

7

1.5

6

1.5

7

0.9

10

1.5

7

2.0

6666

 

 

5

2

6

0.3

5

1.7

5

2.0

POS

376

62.4

50

2.9

49

2.6

43

2.8

56

4.2

 

For strain TA97:-

Dose

NA

10%HLI

30% HLI

10%RLI

30%RLI

µG/plate

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

164

12.2

166

6.1

152

7.5

177

10.7

183

9.2

33

165

6.1

 

12.8

 

 

 

 

 

 

100

180

4.4

174

9.8

179

5.5

191

5.2

95

6.4

333

183

9

184

2.8

178

8.2

180

15.7

190

11.7

1000

156

31.4

184

10.6

159

17.6

201

15.5

206

1.5

3333

103

3.3

171

4.0

118

2

167

16.4

99

27.0

6666

 

 

142

 

57

8.3

87

3.6

139

13.7

POS

986

88.5

613

20.5

396

6.8

498

3.9

440

6.9

 

 

 

 

 

 

 

 

 

For strain TA98:-

Dose

NA

10%HLI

30% HLI

10%RLI

30%RLI

µG/plate

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

17

2.9

31

2.7

50

2.1

17

1.2

46

3.6

33

17

1.7

 

 

 

 

 

 

 

 

100

19

1.9

28

1.2

49

4.4

22

2.2

52

2.0

333

18

2.1

30

1.5

50

3.2

24

0.6

47

4.4

1000

17

1.9

25

1.5

49

3.3

24

4.5

53

2.3

3333

8

1.9

26

2.4

42

0.9

20

1.3

46

3.1

6666

 

 

28

1.2

32

9.1

17

3.3

31

11.7

POS

675

40.9

384

45.2

211

7.1

318

33.8

311

6.4

Applicant's summary and conclusion

Conclusions:
Non-mutagenic effects were known in Ames assay by using Salmonella typhimurium of strain TA100, TA1535, TA1537, TA97, TA98 when exposed with the test chemical in preincubation method and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. Ames assay was performed by the preincubation protocol using Salmonella typhimurium strains  TA100, TA1535, TA1537, TA97, TA98 both in the presence and absence of S9 metabolic activation system. The study was performed at dose levels of 0, 33, 100, 333, 1000, 3333 or 6666 µg/plate following a preincubation time of 20 mins. Concurrent solvent and positive controls were included in the study. The evaluation was done considering the magnitude of the dose dependent increase in his+ revertants, and the shape of the dose response. The test chemical did not cause a dose dependent increase in the number of histidine revertants in Salmonella typhimurium strains  TA100, TA1535, TA1537, TA97, TA98 both with and witthout S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.