1-(allyloxy)-2-methyl-1-oxopropan-2-yl 2-chloro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]benzoate

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:

1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to guideline; under GLP conditions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: THE MINISTRY OF HEALTH AND WELFARE (MHW), Japan (1986), Information in the Guidelines of Toxicity Studies Required for Applications for Approval to Manufacture (Import) Drugs (Part 1), Notification No. 1039 of the Pharmaceutical Affairs Bureau.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-(allyloxy)-2-methyl-1-oxopropan-2-yl 2-chloro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]benzoate

EC Number:

603-837-5

Cas Number:

134605-64-4

Molecular formula:

C20H18ClF3N2O6

IUPAC Name:

1-(allyloxy)-2-methyl-1-oxopropan-2-yl 2-chloro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]benzoate

Test material form:

not specified

Details on test material:

- Physical state: Solid
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature

Method

Target gene:

Histidine and tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

Range finding test: 20.58, 61.73, 185.19, 555.56, 166.67 and 5000.00 μg/plate
Main test: 61.73, 185.19, 555.56, 166.67 and 5000.00 μg/plate
Confirmatory test: 312.50, 625.00, 1250.00, 2500.00 and 5000.00 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Not reported

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: with metabolic activation: 2-aminoanthracene, cyclophosphamide; without metabolic activation: sodium azide, 4-nitroquinoline, mitomycin-C, 2-nitrofluorene, 9-aminoacridine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Expression time (cells in growth medium): 48h at 37 degrees C

NUMBER OF REPLICATIONS: 3

Preliminary test: A range finding test was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA with and without metabolic activation at six concentrations of the test substance and one negative control according to a Standard Operating Procedure of Genetic Toxicology. The highest
concentration applied was 5000.0 ug/plate. The five lower concentrations decreased by a factor of three. The plates were inverted and incubated for about 48 hours at 37±1.5°C in darkness.

Main test: The mutagenicity test was performed with the Salmonella typhimurium strains T A 98, TA 100, TA 102, TA 1535, TA 1537 and with the Escherichia coli strain WP2 uvrA with and without metabolic activation according to Standard Operating Procedures of Genetic Toxicology. Each of the five concentrations of the test substance, a negative and a positive control were tested, using three plates per test substance concentration and controls. The highest concentration applied was determined in the preliminary range finding test and the four lower concentrations. The plates were inverted and incubated for about 48 hours at 37±1.5°C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.

Evaluation criteria:

A test is considered acceptable if the mean colony counts of the negative control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response.

Criteria for a positive response: The test substance will be considered to be positive in the test system if one or both of the
following conditions are met:
At least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: TA 98, TA 1535, TA 1537, E. coli WP2 uvrA.
A reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1. 5 for strains T A 100 or T A 102.
Generally a concentration-related effect should be demonstrable.

Statistics:

A statistical analysis was not performed.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Preliminary test: Six concentrations of the test material ranging from 20.6 to 5000.0 μg/plate were tested with strain Salmonella typhimurium TA 100 and strain Escherichia coli WP2 uvr A to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without metabolic activation. Normal background growth was observed with both strains. The numbers of revertant colonies were not reduced. Precipitates of the test compound were visible on the plates at concentrations of 555.6 μg/plate and higher. From the results obtained the highest concentration suitable for the mutagenicity test was selected to be 5000.0 ug/plate with and 5000.0 μg/plate without metabolic activation.

- Main test: In the original experiments performed with and without metabolic activation, treatment of strains TA 98, TA 100, TA 102, TA 1535, TA 1537 and WP2 uvrA with the test material did not lead to an increase in the incidence of either histidine- or tryptophan-prototrophic mutants in comparison with the negative control.

- Confirmatory test: these experiments performed with and without metabolic activation, again after treatment of strains TA 98, TA 100, TA 102, TA 1535, TA 1537 and WP2 uvrA with the test material, no increase in the incidence of either histidine- or tryptophan-prototrophic mutants was observed in comparison with the negative control. In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were slightly reduced with certain strains at higher concentrations in the presence of metabolic activation. Therefore, the test substance exerted no toxic effect on the growth of the bacteria in the absence of metabolic activation but a slight toxicity on certain strains in the presence of metabolic activation. Precipitates of the test compound were visible on the plates at concentrations of 1666.7 μg/plate and higher in the absence of S9 and at 5000 μg/plate in the presence of S9.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Main test - revertants

+/- S9 Mix	Test material concentration (ug/plate)	Number of revertants (colonies/plate)
		TA 100		TA 1535		WP2 uvrA		TA 102		TA 98		TA 1537
		Replicate	Mean	Replicate	Mean	Replicate	Mean	Replicate	Mean	Replicate	Mean	Replicate	Mean
+ S9 Mix	Solvent control	137	152	8	11	38	37	291	315	39	39	10	10
		158		12		37		329		49		12
		160		14		36		326		29		8
	61.73	160	145	9	10	36	38	320	332	32	37	10	12
		135		12		36		326		41		13
		140		9		41		350		38		14
	185.19	124	134	12	11	42	36	328	336	40	41	12	11
		140		13		41		351		38		13
		137		8		25		328		45		9
	555.56	114	136	8	10	29	29	389	392	40	40	8	7
		146		12		25		376		31		6
		148		9		33		411		48		7
	1666.67	110	126	5	5	25	27	348	365	29	26	4	6
		120		2		25		384		26		4
		149		7		30		364		24		9
	5000.00	128	129	11	6	21	19	422	408	20	27	7	7
		137		2		24		415		31		7
		122		5		13		386		29		6
- S9 Mix	Solvent control	122	125	9	11	25	22	374	352	23	23	11	13
		105		12		21		338		25		12
		149		13		19		345		21		17
	61.73	153	139	21	18	24	21	358	363	15	18	8	8
		141		20		20		388		16		10
		124		14		20		343		24		7
	185.19	128	145	19	16	25	28	325	336	27	22	9	10
		138		15		17		324		21		10
		168		13		42		360		19		11
	555.56	127	142	10	10	27	24	378	400	19	22	11	9
		137		10		26		382		21		9
		162		11		18		440		25		8
	1666.67	163	165	14	15	21	22	399	431	20	24	7	10
		160		15		17		442		29		15
		173		15		27		453		23		9
	5000.00	165	164	22	16	17	19	394	404	23	23	9	8
		158		12		22		383		25		9
		168		13		19		434		20		7
Positive control +S9	Name	2-Aminoanthracene		Cyclophosphamide		2-Aminoanthracene		2-Aminoanthracene		2-Aminoanthracene		2-Aminoanthracene
	Concentration (ug/plate)	1.5		200		20		5		1.5		1.5
	Replicates	2101		291		1170		2036		1777		377
		2120		233		1128		2196		1891		352
		1866		276		1147		2190		1849		292
	Mean	2029		267		1148		2141		1839		340
Positive control -S9	Name	Sodium azide		Sodium azide		4-NQO		Mitomycin-C		2-nitrofluorene		9-aminoacridine
	Concentration (ug/plate)	2		2		2		0.5		5		80
	Replicates	761		782		468		890		1118		1022
		801		823		531		853		1356		1108
		784		710		573		884		1308		1056
	Mean	782		772		524		876		1261		1062

Confirmatory test - revertants

+/- S9 Mix	Test material concentration (ug/plate)	Number of revertants (colonies/plate)
		TA 100		TA 1535		WP2 uvrA		TA 102		TA 98		TA 1537
		Replicate	Mean	Replicate	Mean	Replicate	Mean	Replicate	Mean	Replicate	Mean	Replicate	Mean
+ S9 Mix	Solvent control	133	150	9	10	37	39	253	264	33	41	15	16
		134		10		42		271		38		12
		182		12		39		268		51		21
	312.50	134	134	9	11	32	34	277	287	32	29	13	14
		151		14		37		326		32		14
		116		10		33		257		24		15
	625	141	129	8	6	31	28	319	322	38	31	8	7
		123		5		24		327		37		9
		123		6		30		321		18		5
	1250	112	123	5	5	27	25	325	332	30	33	10	8
		121		4		21		324		32		11
		136		6		28		348		36		3
	2500	122	124	3	4	29	24	230	271	15	17	4	5
		111		4		26		300		17		5
		138		4		18		282		19		6
	5000.00	144	119	3	3	31	25	241	278	18	22	7	6
		114		3		22		314		19		8
		100		4		22		280		28		2
- S9 Mix	Solvent control	150	134	8	11	29	28	328	327	17	20	7	7
		126		11		19		330		20		4
		127		13		36		324		23		9
	61.73	149	133	13	12	21	22	329	333	21	24	6	8
		144		12		17		332		24		8
		105		12		27		339		27		10
	185.19	163	150	11	12	31	25	379	362	31	25	10	10
		147		14		17		356		17		11
		139		10		26		352		26		8
	555.56	151	149	9	12	31	25	310	314	29	26	8	9
		114		15		27		294		29		9
		182		12		18		338		21		11
	1666.67	144	144	16	14	19	20	369	343	24	23	13	8
		144		11		14		364		20		5
		145		15		27		297		26		5
	5000.00	151	149	7	9	18	19	329	332	17	18	8	9
		153		8		17		314		21		9
		144		11		22		352		17		10
Positive control with S9	Name	2-Aminoanthracene		Cyclophosphamide		2-Aminoanthracene		2-Aminoanthracene		2-Aminoanthracene		2-Aminoanthracene
	Concentration (ug/plate)	1.5		200		20		5		1.5		1.5
	Replicates	2009		253		484		833		1913		328
		1985		249		482		860		2199		283
		1790		257		493		843		1908		232
	Mean	1928		253		486		845		2007		281
Positive control without S9	Name	Sodium azide		Sodium azide		4-NQO		Mitomycin-C		2-nitrofluorene		9-aminoacridine
	Concentration (ug/plate)	2		2		2		0.5		5		80
	Replicates	843		752		458		2144		1122		1033
		907		819		500		1873		1179		1113
		914		829		517		1902		1172		1122
	Mean	888		800		492		1973		1158		1089

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study, the test material and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.

Executive summary:

The study was performed to the requirements of OECD Guideline 471, EU Method B.14, MHW (Japan) and U SEPA OTS 798.5265 under GLP conditions, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA102, TA1535, TA1537 and E. coli strains WP2 uvrA, in both the presence and absence of S-9 mix. A preliminary test was performed to determine the test concentrations for main study. In the main study the test substance was evaluated at a concentration of up to 5000 µg/plate. Positive controls appropriate for each strain, in the presence and absence of S9-mix, were included. A separate confirmatory study was performed at a concentration up to 5000 µg/plate. The test substance did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the strains tested, either in the presence or absence of S9-mix. The sensitivity of the test system and the metabolic activity of the S9-mix were clearly demonstrated by the increases in the numbers of revertant colonies induced by the positive control substances. It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 and E. coli strains WP2 uvrA in both the presence and absence of S-9 mix.