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

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:

May 16, 2002 - July 5, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2007

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

EC Number:

606-467-2

Cas Number:

202189-76-2

Molecular formula:

C23H29NO4S

IUPAC Name:

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

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: sponsor
- Code: W-02-7046-A1

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: DMSO was used as a vehicle to prepare the test item concentrations. A stock concentration of 100 mg/ml was prepared from which 1:5 dilutions were carried out. The test item precipitates in contact with the phospate tampon in concentrations of 10 and 20mg/ml.
- 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:

Due to the fact that during the initial tests the concentrations of 10 and 20 mg/ml, precipitated in contact with the phosphate tampon, the following concentrations were used in successive tests: 4; 0.8; 0.16; 0.032 and 0.0064 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 two serioes 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 one of the five concentrations) or the reference item (positive control). The tubes were place in a water bath at 37ºC for 45 minutes. Then 2ml of surface agar supplemented with histidine/biotine 0.5mM 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 37ºC 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. In cases where a positive may exist the assay has been duplicated which results in 4 replicates for some concentrations.

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

OTHER EXAMINATIONS:
Phenotype and sterility controls were also performed.

- OTHER:
Solutions preparation: DMSO was used as a vehicle to prepare the test item concentrations. In all cases, these concentrations were prepared on the day they were used. A stock concentration of 100mg/ml was prepared from which 1:5 dilutions were carried out.

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

Rationale for test conditions:

Due to the fact that during the initial tests the concentrations of 10 and 20 mg/ml, precipitated in contact with the phosphate tampon, the following concentrations were used in successive tests: 4; 0.8; 0.16; 0.032 and 0.0064 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 1535

Metabolic activation:

with and without

Genotoxicity:

positive

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 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 3

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 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

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 100

Metabolic activation:

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: The concentrations of 10 and 20 mg/ml, precipitated in contact with the phosphate tampon

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 there is no toxicity on the concentration taken as a reference to evaluate the mutagenic power.

2. The number of colonies on the spontaneous mutation colonies 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 induce an increase of colonies with respect to non-treated plates, dependent on the concentration of strains TA1535 (with and without S9) and TA100 (with S9).

- The test item does not induce an increase in colonies in the rest of the strains used for this assay.

- The mutation index (MI) calculated from the average os the number of colonies counted on each plate, show values of 1.74 and 3.11 for the strain TA1535 (without and with S9) and 1.46 on the TA100 (with S9) strain.

The results described indicate that:

- The test item is not mutagenic for the strains TA98, TA102 and TA1537

- The test item is mutagenic for the strains TA1535 and TA100

Calculation of the mutation index (MI)

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

Strain TA1535
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	20/28	24.00		19/25/18/25	21.75
0.00 (mg/ml)	17/20/23/27	21.75		23/23/17/20	20.75
0.0064 (mg/ml)	16/22	19.00	0.874	23/20/22	21.67	1.044
0.032 (mg/ml)	16/13	14.50	0.667	22/23/13/19	19.25	0.928
0.16 (mg/ml)	17/19	18.00	0.828	25/14/18/24	20.25	0.976
0.8 (mg/ml)	28/22/25/32	26.75	1.230	47/35/30/32	36.00	1.735
4 (mg/ml)	28/39/45/42	38.50	1.770	66/65/62/65	64.50	3.108
Control +	480/470	475.00	21.839	134/172	153.00	7.373

 

Strain TA1000
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	110/116	113.00		95/108	101.50
0.00 (mg/ml)	89/90	89.50		100/138/146/80	116.00
0.0064 (mg/ml)	108/99	103.50	1.156	132/135/134/129	132.50	1.142
0.032 (mg/ml)	88/106	97.00	1.084	138/111/134/117	125.00	1.078
0.16 (mg/ml)	102/95	98.50	1.101	127/124/149/132	133.00	1.147
0.8 (mg/ml)	112/106	109.00	1.218	138/125/161/160	146.00	1.259
4 (mg/ml)	124/100	112.00	1.251	168/144/183/182	169.25	1.459
Control +	505/487	496.00	5.542	952/946/940/1108	986.50	8.504

 

Strain TA98
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	28/28	28.00		36/38	37.00
0.00 (mg/ml)	20/29	24.50		33/33	33.00
0.0064 (mg/ml)	21/23	22.00	0.898	34/26	30.00	0.909
0.032 (mg/ml)	20/27	23.50	0.959	33/34	33.50	1.015
0.16 (mg/ml)	22/30	26.00	1.061	28/29	28.50	0.864
0.8 (mg/ml)	28/30	29.00	1.184	32/29	30.50	0.924
4 (mg/ml)	30/23	26.50	1082	38/26	32.00	0.970
Control +	>1000/>1000	>1000	>40.816	973/965	969.00	29.364

 

Strain TA1537
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	23/23	23.00		28/40	34.00
0.00 (mg/ml)	21/18	19.50		26/24	25.00
0.0064 (mg/ml)	20/22	21.00	1.077	28/24	26.00	1.040
0.032 (mg/ml)	21/21	21.00	1.077	29/29	29.00	1.160
0.16 (mg/ml)	21/21	21.00	1.077	30/25	27.50	1.100
0.8 (mg/ml)	23/21	22.00	1.128	28/26	27.00	1.080
4 (mg/ml)	25/19	22.00	1.128	28/30	29.00	1.160
Control +	941/786	863.50	44.282	112/92	102.00	4.080

 

 

Strain TA102
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	270/--	270.00		355/316	335.50
0.00 (mg/ml)	261/260	260.50		315/295	305.00
0.0064 (mg/ml)	228/239	233.50	0.896	250/240	245.00	0.803
0.032 (mg/ml)	256/244	250.00	0.960	222/227	224.50	0.736
0.16 (mg/ml)	200/231	215.50	0.827	282/286	284.00	0.931
0.8 (mg/ml)	229/268	248.50	0.954	320/284	302.00	0.990
4 (mg/ml)	250/254	252.00	0.967	321/282	301.50	0.989
Control +	512/568	540.00	2.069	>1000/>1000	>1000	>3.279

--: 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	-	-	-	-	Resistant

 

Applicant's summary and conclusion

Conclusions:

The test item induce a dose-dependent increase in Salmonella typhimurium strain TA1535 (with and without S9) and TA100 (with S9). Therefore, it was considered as 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 0.0064, 0.032, 0.16, 0.8 and 4 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 colonies 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 induce a dose-dependent increase in TA1535 (with and without S9) and TA100 (with S9). Therefore, the test item was determined to be mutagenic under test conditions.