2-[1-(2-{4-[2-(4,4-dimethyl-4,5-dihydro-1,3-oxazol-2-yl)propan-2-yl]phenyl}ethyl)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-[1-(2-{4-[2-(5,5-dimethyl-4,5-dihydro-1,3-oxazol-2-yl)propan-2-yl]phenyl}ethyl)piperidin-4-yl]-1H-1,3-benzodiazole

EC Number:

813-966-0

Cas Number:

202189-81-9

Molecular formula:

C28H36N4O

IUPAC Name:

2-[1-(2-{4-[2-(5,5-dimethyl-4,5-dihydro-1,3-oxazol-2-yl)propan-2-yl]phenyl}ethyl)piperidin-4-yl]-1H-1,3-benzodiazole

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: sponsor
- Lot: 5BM

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 composition: phosphate buffer (or S9 mixture), 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 37 ºC for 45 minutes. Then 2 mL of surface agar supplemented with histidine/biotin 0.5 mM was added to each tube and poured out 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 contaided tetracycline 8 mg/ml, in addition to Histidine, Biotin and Ampicillin. The plates were cultivated for 48 hous at 37 ºC.

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.	22/20	21.0	--	20/17	18.5	--
0mg/ml	18/20	19.0	--	15/16	15.5	--
32mg/ml	17/17	17.0	0.895	18/14	16.0	1.032
160mg/ml	27/25	26.0	1.368	20/13	16.5	1.065
800mg/ml	16/22	19.0	1.000	24/15	19.5	1.258
4000mg/ml	16/19	17.5	0.921	18/24	21.0	1.355
2000mg/ml	26/24	25.0	1.316	27/15	21.0	1.355
Control +	>2000/>2000	>2000	>105.263	>2000/>2000	>2000	129.032

 

Strain TA100
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	173/179	176.0	--	200/184	192.0	--
0mg/ml	180/176	178.0	--	191/197	194.0	--
32mg/ml	172/174	173.0	0.972	182/175	178.5	0.920
160mg/ml	181/173	177.0	0.994	190/194	192.0	0.990
800mg/ml	168/174	171.0	0.961	181/176	178.5	0.920
400mg/ml	190/176	183.0	1.028	198/179	188.5	0.972
20000mg/ml	184/191	187.5	1.053	207/210	208.5	1.075
Control +	>2000/>2000	>2000	>11.236	>2000/>2000	>2000	>10.309

 

Strain TA102
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	330/324	327.0	--	420/416	418.0	--
0mg/ml	321/324	322.5	--	380/410	395.0	--
32mg/ml	330/326	328.0	1.017	426/430	428.0	1.084
160mg/ml	321/337	329.0	1.020	412/400	406.0	1.028
800mg/ml	320/326	323.0	1.002	430/422	426.0	1.078
4000mg/ml	329/334	331.5	1.028	416/420	418.0	1.058
20000mg/ml	323/329	326.0	1.011	410/420	415.0	1.021
Control +	>2000/>2000	>2000	>6.202	880/900	890.0	2.253

 

Strain TA1535
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	6/7	6.5	--	13/15	14.0	--
0mg/ml	8/6	7.0	--	16/17	16.5	--
32mg/ml	4/5	4.5	0.643	18/18	18.0	1.091
160mg/ml	7/8	7.5	1.071	16/18	17.0	1.030
800mg/ml	7/5	6.0	0.857	22/15	18.5	1.121
4000mg/ml	6/5	5.5	0.786	24/17	20.5	1.242
20000mg/ml	5/7	6.0	0.857	21/20	20.5	1.242
Control +	>1500/>1500	>1500	>214.286	245/257	251.0	15.212

  

Strain TA1537
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	4/4	4.0	--	10/7	8.5	--
0mg/ml	7/6	6.5	--	6/8	7.0	--
32mg/ml	7/5	6.0	0.923	6/8	7.0	1.000
160mg/ml	5/4	4.5	0.692	4/8	6.0	0.857
800mg/ml	2/5	3.5	0.538	13/10	11.5	1.643
4000mg/ml	5/4	4.5	0.692	12/7	9.5	1.357
20000mg/ml	7/5	6.0	0.923	9/5	7.0	1.000
Control +	164/171	167.5	25.769	180/191	185.5	26.500

--: 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. Therefore, it was considered as non-mutagenic under test conditions.

Executive summary:

A Bacterial reverse mutation test was performed according OECD guideline 471 with GLP. Based on a previous toxicity test, 1-2E9 cell/mL of 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 demonstrates 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 increase in the number of revertants in all cases and the phenotype control plates show the expected results for each strain. The test item does not induce a dose-dependent increase in any of the Salmonella typhimurium. Therefore, the test item was determined to be non-mutagenic under test conditions.

.