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
Study period:
18 February 2014 - 27 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD 471. GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
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:
-
EC Number:
440-240-7
EC Name:
-
Molecular formula:
C19H20N4.H2O
IUPAC Name:
1,7'-dimethyl-2'-propyl-1H,1'H-2,5'-bibenzimidazole hydrate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: 2-N-propyl-4-methyl-6-(1-methylbenzimidazol-2-yl)-benzimidazole monohydrate
- Physical state: white powder
- Analytical purity: 100.5% (Potentiometric assay)
- Lot/batch No.: M09532R
- Storage condition: Controlled room temperature

Method

Target gene:
Histidine-requiring gene in Salmonella typhimurium and Tryptophan-requiring gene in the strain of Escherichia coli.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver
Test concentrations with justification for top dose:
5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO) and Acetone. The test item was insoluble in Distilled water. The formulation at 100 mg/mL concentration using Acetone as vehicle was inhomogeneous suspension; however, the formulations at 100 mg/mL concentration using DMSO as vehicle was suitable for the test. Therefore, DMSO was selected as vehicle (solvent) of the study. Purity conversion was applied in the main experiment (ie. concentration of the test item in the dosing formulation was adjusted according to the water content of the provided material). For the purity conversion, the 94.4% purity data of the test item was taken into consideration, thus a correction factor of 1.06 was used at dose formulation preparation.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO and distilled water
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100, TA1535, -S9; 2 µg/plate
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine
Remarks:
TA98, -S9; 4 µg/plate
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537, -S9; 50 µg/plate
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA, -S9; 2 µg/plate
Positive controls:
yes
Positive control substance:
other: 2-aminoantracene
Remarks:
all Salmonella strains, +S9; 2 µg/plate
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
WP2 uvrA, +S9; 50 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Plate incorporation (initial mutation test):
Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item (or controls) and other components (top agar, overnight culture of test strain, phosphate buffer (pH 7.4) or S9 mix) were prepared freshly and added to the overlay (45°C). This solution was mixed and poured on the surface of minimal agar plates. After preparation, the plates were incubated at 37°C for 48 hours.

Pre-incubation (confirmatory mutation test):
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control or concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes; the content was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. After preparation, the plates were incubated at 37°C for 48 hours.

NUMBER OF REPLICATIONS: 3 replicates per dose and controls.

EVALUATION OF EXPERIMENTAL DATA:
The colony numbers were determined by manual counting.
Visual examination of the plates was performed, precipitation or signs of growth inhibition (if any) were recorded.
The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated.
Evaluation criteria:
Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
Criteria for a Negative Response:
A test article was considered non-mutagenic if it produced 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.
Statistics:
The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system). No signs of inhibitory, cytotoxic effects were observed in the preliminary experiment in any examined bacterial strain at any concentration with or without metabolic activation. Precipitate / slight precipitate was detected on the plates in the both tester strains at 5000, 2500, 1000 and 316 μg/plate concentrations with and without metabolic activation.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was detected on the plates in the main tests in all examined strains at 5000 and 1581 μg/plate concentrations with and without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean values of revertant colonies of the vehicle (solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the vehicle (solvent) control and were within the normal biological variability of the test system.

Summary Table of the Initial Mutation Test (test item):

Concentration (µg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

5000

Mean

19.7

32.3

70.7

72.0

4.7

5.7

6.0

4.7

27.0

26.0

MF

0.95

1.02

0.85

0.67

1.00

0.46

0.86

0.61

0.80

0.83

1581

Mean

19.3

20.7

72.3

87.7

7.7

9.0

4.7

4.7

23.7

31.3

MF

0.94

0.65

0.87

0.81

1.64

0.73

0.67

0.61

0.70

1.00

500

Mean

25.3

31.3

70.3

97.3

9.7

8.7

5.3

4.7

33.3

38.7

MF

1.23

0.99

0.84

0.90

2.07

0.70

0.76

0.61

0.99

1.23

158.1

Mean

21.3

29.3

78.3

90.0

8.3

8.3

8.7

8.0

27.0

32.7

MF

1.03

0.93

0.94

0.84

1.79

0.68

1.24

1.04

0.80

1.04

50

Mean

21.7

28.7

89.7

100.3

10.0

9.7

8.7

7.0

21.7

31.0

MF

1.05

0.91

1.08

0.93

2.14

0.78

1.24

0.91

0.64

0.99

15.81

Mean

21.7

25.7

67.3

107.0

10.7

11.3

4.3

10.7

27.7

33.3

MF

1.05

0.81

0.81

0.99

2.29

0.92

0.62

1.39

0.82

1.06

5

Mean

27.7

36.0

63.0

103.0

7.3

11.3

6.0

6.3

29.7

30.3

MF

1.34

1.14

0.76

0.96

1.57

0.92

0.86

0.83

0.88

0.97

Summary Table of the Confirmatory Mutation Test (test item):

Concentrations (µg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

5000

Mean

20.7

24.7

78.3

88.7

6.3

13.0

3.7

10.3

20.0

32.7

MF

1.24

0.78

0.80

0.83

0.54

1.22

0.55

0.94

0.95

1.07

1581

Mean

22.3

32.0

77.7

110.0

8.7

11.7

4.3

8.3

20.3

29.7

MF

1.34

1.01

0.79

1.03

0.74

1.09

0.65

0.76

0.97

0.97

500

Mean

15.3

29.3

85.0

106.3

10.0

13.0

6.3

5.7

23.7

28.7

MF

0.92

0.93

0.87

1.00

0.86

1.22

0.95

0.52

1.13

0.93

158.1

Mean

24.0

30.0

81.7

100.7

10.0

11.0

11.3

8.3

22.7

31.3

MF

1.44

0.95

0.83

0.95

0.86

1.03

1.70

0.76

1.08

1.02

50

Mean

20.3

26.7

91.3

109.7

11.7

14.0

5.7

8.7

20.7

33.0

MF

1.22

0.84

0.93

1.03

1.00

1.31

0.85

0.79

0.98

1.08

15.81

Mean

22.0

29.3

89.3

109.7

10.3

9.0

8.0

8.3

22.3

32.0

MF

1.32

0.93

0.91

1.03

0.89

0.84

1.20

0.76

1.06

1.04

5

Mean

17.7

26.7

88.0

102.0

11.7

14.7

6.0

13.3

23.3

30.0

MF

1.06

0.84

0.90

0.96

1.00

1.38

0.90

1.21

1.11

0.98

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative (with and without metabolic activation)

The test item had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.
Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD Guideline 471 (GLP study). The experiments were carried out using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA in the presence and absence of metabolic activation (S9 fraction prepared from the livers of rats). Based on the results of the Solubility Test, the test item was dissolved in DMSO. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the main tests were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate. In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the vehicle (solvent) control and were within the normal biological variability of the test system. Precipitation was detected on the plates in the main tests in all examined strains at 5000 and 1581 μg/plate concentrations with and without metabolic activation. The mean values of revertant colonies of the vehicle (solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main test, the examined concentration range was considered to be adequate. The study was considered to be valid. In conclusion, the test item had no mutagenic activity in the examined bacterial strains under the test conditions of this study.