2-Propyl-4-methyl-6-(1-methylbenzimidazol-2-yl)benzimidazole

Genetic toxicity: in vitro

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:

2010-09-09 to 2010-10-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to OECD 471 and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,5´-Bi-1H-Benzimidazole,1,7´-dimethyl-2´-propyl

IUPAC Name:

2,5´-Bi-1H-Benzimidazole,1,7´-dimethyl-2´-propyl

Test material form:

not specified

Details on test material:

- Storage condition of test material: Room temperature in the dark (ambient humidity)

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

- Properly maintained: yes- The optical density (OD) at 600 nm of each bacterial culture was determined

Metabolic activation:

with and without

Test concentrations with justification for top dose:

30, 100, 300, 1000, 3000 µg/plate in the plate test and the preincubation test

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, preincubation)DURATION- Preincubation period: 20 min- Exposure duration: 2 days (TA 102: 3 days)NUMBER OF REPLICATIONS: 3 and 6 for negative controlDETERMINATION OF CYTOTOXICITY- Method: background bacterial lawn

Evaluation criteria:

A reproducible, concentration-dependent increase in the number of revertants of at least one tester strain over the vehicle control value and/or outside the historical control data range indicates a genotoxic activity.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

other: historical control data (except for strain TA 102)

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH:- Effects of osmolality:- Evaporation from medium:- Water solubility:- Precipitation: at 300 µg/plate and higher- Other confounding effects:RANGE-FINDING/SCREENING STUDIES: In a previous Ames test, The test substance was soluble in DMSO but showed precipitation on plates at concentrations of 1000, 3000 and 5000 μg/plate. Therefore, 3000 μg/plate was investigated as the highest concentration in this study.COMPARISON WITH HISTORICAL CONTROL DATA: yesADDITIONAL INFORMATION ON CYTOTOXICITY: No bacteriotoxicity was observed up to 3000 μg/plate

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

MUTAGENICITY

The OD600 of the individual overnight bacterial cultures varied between 2.3 and 3.0 (raw data). These values correspond to a bacterial titer of ca 100 million/0.1 mL. The test substance did not induce relevant increases in the number of revertant colonies in different tester strains of S. typhimurium compared to the negative control when tested up to 3000 μg/plate in two different experiments. Metabolic activation by S9 mix did not alter the mutation frequency of these bacterial strains under any test conditions used.

The validity of this study is given since the vehicle control plates showed spontaneous revertants in different tester strains of S.typhimurium at frequencies similar to those described in the literature and within the historical negative control data range (99 % lower

limit – 99 % upper limit) experienced in our laboratory., except for slightly higher number of revertants in strain TA 102 in the negative control using the preincubation method. This minor difference was regarded as incidental without any effect on the validity of the study in

view of the clear positive response of the positive control. The mutagens NaN3, 9-AA, 2-NF, MMC and 2-AA showed the expected strain specific responses in the absence and presence of mammalian liver enzymes, respectively, demonstrating the validity of this study.

Table 1: Mutagenic activity of the test substance in S. typhimurium Plate Test (summary of results) Without metabolic activation

µg/plate	Mean Revertants/Plate
	S. typhimurium
	TA 1535	TA 1537	TA 98	TA 100	TA 102
Negative Control
DMSO	7	3	14	74	438
Test substance
30	6	5	16	69	459
100	5	6	14	71	452
300	4 P	2 P	26 P	64 P	442
1000	7 P	5 P	17 P	67 P	418 P
3000	10 P	5 P	20 P	75 P	322 P
Positive Control
NaN3 (5)	1016	-	-	1124	-
9-AA (50)	-	252	-	-	-
2-NF (10)	-	-	662	-	-
MMC (0.5)	-	-	-	-	1331

Table 2: Mutagenic activity of the test substance in S. typhimurium Plate Test (summary of results)With metabolic activation

µg/plate	Mean Revertants/Plate
	S. typhimurium
	TA 1535	TA 1537	TA 98	TA 100	TA 102
Negative Control
DMSO	13	6	21	91	522
Test substance
30	8	8	17	87	546
100	11	6	15	80	537
300	8 P	4 P	18 P	73 P	516
1000	8 P	4 P	14 P	82 P	488 P
3000	7 P	5 P	14 P	86 P	411 P
Positive Control
2-AA (4)	186	149	1286	1246	-
2-AA (10)	-	-	-	-	1102

Table 3: Mutagenic activity of the test substance in S. typhimurium Preincubation (summary of results) Without metabolic activation

µg/plate	Mean Revertants/Plate
	S. typhimurium
	TA 1535	TA 1537	TA 98	TA 100	TA 102
Negative Control
DMSO	6	5	15	62	427
Test substance
30	8	5	17	60	460
100	6	3	14	59	448
300	7 P	4 P	15 P	56 P	435
1000	4 P	4 P	15 P	49 P	375 P
3000	2 P	4 P	15 P	44 P	355 P
Positive Control
NaN3 (5)	1160	-	-	1314	-
9-AA (50)	-	255	-	-	-
2-NF (10)	-	-	518	-	-
MMC (0.5)	-	-	-	-	1781

Table 4: Mutagenic activity of the test substance in S. typhimurium Preincubation (summary of results) With metabolic activation

µg/plate	Mean Revertants/Plate
	S. typhimurium
	TA 1535	TA 1537	TA 98	TA 100	TA 102
Negative Control
DMSO	10	4	16	84	541
Test substance
30	9	5	18	83	572
100	9	2	19	76	558
300	6 P	4 P	17 P	82 P	562
1000	11 P	3 P	14 P	81 P	523 P
3000	10 P	3 P	17 P	76 P	493 P
Positive Control
2-AA (4)	118	245	1926	1662	-
2-AA (10)	-	-	-	-	1394

P: Preincubation

-: Not tested

Underlined values are regarded as increased

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):negativeThe test substance caused neither base-pair substitutions nor frameshift mutations in different S. typhimurium strains in the presence and absence of metabolic activation when tested up to insoluble concentration. Based on these results it was concluded, that the test substance is "Ames negative".