2-(piperidin-4-yl)-1H-1,3-benzodiazole

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 22, 2007 - December 21, 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(piperidin-4-yl)-1H-1,3-benzodiazole

EC Number:

609-547-5

Cas Number:

38385-95-4

Molecular formula:

C12H15N3

IUPAC Name:

2-(piperidin-4-yl)-1H-1,3-benzodiazole

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: sponsor
- Lot: 0604X10

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in darkness.
- Solubility and stability of the test substance in the solvent/vehicle: Sodium phosphate buffer, 200mM, pH=7.4, was used as the vehicle to prepare the item concentrations. A stock concentration of 100 mg/ml was prepared in DMSO from which 1:5 dilutions were made.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The top concentration of the test item was toxic for Salmonella typhimurium so, the following concentrations were tested: 20; 4; 0.8; 0.16; and 0.032 mg/ml

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Solvent is compatible with the survival of the bacteria and the S9 activity.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cumene hydroperoxide

other: 2-aminoantracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation
Each point of the two series of tubes (with and without S9) was tested in duplicate and with the following compposition: phosphate buffer (or S9 micture), 2E9 cell/ml bacterial culture and the solvent (negative control), the test item (each of the five concentrations) or the reference item (positive controls). The tubes were placed in a water bath at 37ºC for 45 minutes. Then 2ml of surface agar supplemented with histidine/biotin 0,5 mM was added to each tube and poured onto a minimum agar plate. The plates were left to set for 1 hour and they were then placed in the incubator at 37 ºC for 48-72 hours.


DURATION
- Preincubation period: 45 minutes
- Exposure duration:48 -72 hours

SELECTION AGENT (mutation assays): The lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize an essential amino acid.

NUMBER OF REPLICATIONS: 2.

DETERMINATION OF CYTOTOXICITY
- Method: Visual observation of the colonies.

OTHER EXAMINATIONS:
Phenotype and sterility controls were also performed.

- OTHER:
Solutions preparation: Sodium phosphate buffer, 200mM, pH=7.4, was used as the vehicle to prepare the item concentrations. In all cases, these concentrations were prepared on the day they were used. A stock concentration of 100mg/ml was prepared in DMSO from which 1:5 dilutions were carried out.

Test system: Prior to the study, the master plates of each strain were prepared. The strains were plated out in minimum agar plates enriched with Biotin 0.5 mM and Histidine 0.1 M. In the case of strains TA98 and TA100 the plates also contained ampicillin 8 mg/ml and in the case of strain TA102 they contained Tetracycline 8 mg/ml, in addition to Histidine, Biotin and Ampicillin. The plates were cultivated for 48 hours at 37ºC.

Rationale for test conditions:

The top concentration of the test item, 100 mg/ml, was toxic for Salmonella typhimurium so, the following concentrations were tested: 20; 4; 0.8; 0.16 and 0.032 mg/ml.

Evaluation criteria:

Criteria conclusion: the result of the test is considered as positive if the test item induce an increase of colonies with respect to non-treated plates, dependent on the concentration of one, or several of the 5 strains, without and/or with metabolic activation.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

Any other information on results incl. tables

The conditions listed below indicate that the tests are acceptable:

1. The plates show a firm, uniform lawn, which demonstrates that there is no toxicity in the concentrations that were taken as a reference to evaluate the mutagenic power.

2. The number of colonies in the spontaneous mutation plates is within the normal range for each strain.

3. The positive controls induce a clear increase in the number of revertants in all cases.

4. The phenotype control plates show the expected results for each strain.

From the results expressed on the tables below it can be deduced that the test item does not induce an increase in colonies in any of the strains used in this study, neither in the presence of S9 nor in its absence.

Calculation of the mutation index (MI)

MI = nº. of mut. in a dose / nº. of mut. in the control

Strain TA98
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	13/15	14.0	--	19/20	19.5	--
0mg/ml	16/19	17.5	--	20/22	21.0	--
32mg/ml	22/15	18.5	1.057	22/17	19.5	0.929
160mg/ml	14/18	16.0	0.914	16/23	19.5	0.929
800mg/ml	20/14	17.0	0.971	14/22	18.0	0.857
4000mg/ml	14/18	16.0	0.914	16/18	17.0	0.810
20000mg/ml	18/15	16.5	0.943	27/12	19.5	0.929
Control +	>2000/>2000	>2000	>114.286	>2000/>2000	>2000	>95.238

 

Strain TA100
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	176/183	169.5	--	192/197	194.5	--
0mg/ml	180/164	172.0	--	215/207	211.5	--
32mg/ml	173/169	171.0	0.994	173/162	167.5	0.794
160mg/ml	168/159	163.5	0.951	168/172	170.0	0.806
800mg/ml	163/170	166.5	0.968	132/196	164.0	0.777
4000mg/ml	181/176	178.5	1.038	160/188	174.0	0.825
20000mg/ml	186/190	188.0	1.093	200/213	206.5	0.979
Control +	>2000/>2000	>2000	>11.628	>2000/>2000	>2000	>9.479

 

Strain TA102
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	312/321	316.5	--	412/420	416.0	--
0mg/ml	326/325	325.5	--	400/410	405.0	--
32mg/ml	317/329	323.0	0.992	438/422	430.0	1.062
160mg/ml	330/322	326.0	1.002	407/410	408.5	1.009
800mg/ml	327/333	330.0	1.014	428/430	429.0	1.059
4000mg/ml	315/320	317.0	0.974	410/412	411.0	1.015
20000mg/ml	317/321	319.0	0.980	426/410	418.0	1.032
Control +	>2000/>2000	>2000	>6.144	920/880	900.0	2.222

 

Strain TA1535
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	6/6	6.0	--	17/16	16.5	--
0mg/ml	7/4	5.5	--	19/17	18.0	--
32mg/ml	8/5	6.5	1.182	20/22	21.0	1.167
160mg/ml	7/5	6.0	1.091	21/19	20.0	1.111
800mg/ml	6/5	5.5	1.000	17/23	20.0	1.111
4000mg/ml	5/5	5.0	0.909	26/18	22.0	1.222
20000mg/ml	5/6	5.5	1.000	22/23	22.5	1.250
Control +	>1500/>1500	>1500	>272.727	253/249	251.0	13.944

  

Strain TA1537
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	4/5	4.5	--	8/10	9.0	--
0mg/ml	7/6	6.5	--	9/11	10.0	--
32mg/ml	6/6	6.0	0.923	4/7	5.5	0.550
160mg/ml	8/6	7.0	1.077	10/7	8.5	0.850
800mg/ml	5/7	6.0	0.923	6/1	3.5	0.350
4000mg/ml	6/7	6.5	1.000	3/5	4.0	0.400
20000mg/ml	4/6	5.0	0.769	13/4	8.5	0.850
Control +	165/179	172.0	26.462	192/184	188.0	18.800

--: It was not possible to count colonies

 

Results of the phenotype control

	TA98	TA100	TA1535	TA1537	TA102
Ampicilyne	Resistant	Resistant	Sensitive	Sensitive	Resistant
Violet Crystal	Sensitive	Sensitive	Sensitive	Sensitive	Sensitive
UV light	Sensitive	Sensitive	Sensitive	Sensitive	Sensitive
Tetracycline	n.t	n.t	n.t	n.t	Resistant

n.t.: not tested

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:

The test item does not induce a dose-dependent increase in Salmonella typhimurium strains: TA98, TA100, TA102, TA1535 and TA1537. Therefore, it was not considered as mutagenic under test conditions.

Executive summary:

A Bacterial reverse mutation test was performed according OECD guideline 471 with GLP. Bases on previous toxicity test, 1 -2E9 cell/mL os Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102 were exposed to 0.032, 0.16, 0.8, 4 and 20 mg/mL test item, solvent and positive controls with and without metabolic activation (two replicates each). The incubation mixtures were pre-incubated at 37 ºC for 45 minutes and incubated at 37 ºC for 48 -72 hours. Then, the revertant colonies were counted. Phenotype and sterility controls were also performed. The plates showed a firm, uniform lawn, which demostrates that there was no toxicity. The number of colonies in the spontaneous mutation plates was within the normal range for each strain. The positive controls induced a clear inclease in the number of revertants in all cases and the phenotype control plates show the expected results for each strain. The test item do not induce a dose-dependent increase in Salmonella typhimurium strains: TA98, TA100, TA102, TA1535 and TA1537. Therefore, it was not considered as mutagenic under test conditions.