2-[(3,5-dimethyl pyrazolyl)carbamoyl]ethyl methacrylate

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:

16 Sep - 21 Oct 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Guidelines for Toxicity Testings of New Chemical Substances

Version / remarks:

Notification No. 1121002 of Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, No. 2 of the Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry, & No. 031121002 of Environmental Policy Bureau, Ministry of the Environment, Japan, November 21, 2003

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[(3,5-dimethyl-1H-pyrazole-1-carbonyl)amino]ethyl 2-methylprop-2-enoate

EC Number:

680-413-6

Cas Number:

217437-44-0

Molecular formula:

C12H17N3O3

IUPAC Name:

2-[(3,5-dimethyl-1H-pyrazole-1-carbonyl)amino]ethyl 2-methylprop-2-enoate

Method

Target gene:

his operon (for S. typhimurium strains) and trp operon (for E. coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Dose-range finding test: 1.2, 4.9, 20, 78, 313, 1250 and 5000 μg/plate
Main test: 39, 78, 156, 313, 625 and 1250 µg/plate

In the dose-range finding test, growth inhibition by the test substance was observed at 1250 μg/plate and above in all strains with and without metabolic activation. Therefore, 1250 μg/plate was selected as top dose for all strains with and without metabolic activation in the main test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was insoluble in water. A solubility test showed that the test substance was soluble at 50 mg/mL in DMSO.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2); 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine 2HCl (ICR-191); 2-Aminoanthracene (2-AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: duplicates each in the dose-range finding study and in the main test


DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition

Evaluation criteria:

The test substance was judged to be positive with regard to mutagenicity, if the number of revertant colonies on the test plates increased significantly in comparison with the control plates (twice as that of the solvent control), and in addition dose-response and reproducibility were observed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA1537, TA1535, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 1250 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 1250 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed either with or without metabolic activation
- Contamination: In a sterility test on the test solution and the S9 mix, no growth of bacteria was observed.

RANGE-FINDING/SCREENING STUDIES: In a preliminary dose-range finding test concentrations of 1.2, 4.9, 20, 78, 313, 1250 and 5000 μg/plate were tested. Growth inhibition by the test substance was observed at 1250 μg/plate and above in all strains with and without metabolic activation. And no precipitation of the test substance on the plates was observed either with or without metabolic activation. Therefore, 1250 μg/plate dose was selected as top dose for all strains both with and without metabolic activation in the main test.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: please refer to table 3
- Negative (solvent/vehicle) historical control data: please refer to table 3

Any other information on results incl. tables

Table 1. Results of dose-range finding test

With or without S9mix	Dose (μg/plate)	Number of revertant colonies/plate
		Base-pair substitution type			Frame-shift type
		TA100	TA1535	WP2uvrA	TA98	TA1537
－S9mix	Solvent Control (DMSO)	136	24	33	23	10
		134 (135)	24 (24)	21 (27)	27 (25)	9 (10)
	1.2	121	19	10	17	12
		116 (119)	19 (19)	21 (16)	18 (18)	13 (13)
	4.9	102	17	20	19	7
		142 (122)	24 (21)	21 (21)	29 (24)	9 (8)
	20	124	14	16	23	5
		103 (114)	17 (16)	24 (20)	21 (22)	9 (7)
	78	135	15	28	13	8
		110 (123)	20 (18)	20 (24)	20 (17)	8 (8)
	313	133	22	23	19	9
		149 (141)	20 (21)	22 (23)	12 (16)	13 (11)
	1250	0*	0*	17*	0*	0*
		0*(0)*	0*(0)*	20*(19)*	0*(0)*	0*(0)*
	5000	0*	0*	11*	0*	0*
		0*(0)*	0*(0)*	23*(17)*	0*(0)*	0*(0)*
＋S9mix	Solvent Control (DMSO)	132	15	28	34	20
		139 (136)	18 (17)	33 (31)	35 (35)	25 (23)
	1.2	146	17	27	32	22
		148 (147)	13 (15)	19 (23)	27 (30)	30 (26)
	4.9	139	14	27	34	19
		121 (130)	12 (13)	28 (28)	38 (36)	23 (21)
	20	121	11	25	29	19
		139 (130)	8 (10)	34 (30)	39 (34)	17 (18)
	78	133	10	29	30	20
		131 (132)	8 (9)	28 (29)	26 (28)	17 (19)
	313	159	13	24	33	20
		130 (145)	17 (15)	20 (22)	22 (28)	20 (20)
	1250	124*	11*	31*	21*	10*
		114*(119)*	14*(13)*	28*(30)*	19*(20)*	16*(13)*
	5000	89*	0*	13*	18*	0*
		101*(95)*	0*(0)*	18*(16)*	17*(18)*	0*(0)*
Positive control not requiring S9mix	Name	AF-2	NaN3	AF-2	AF-2	ICR-191
	Dose (μg/plate)
		0.01	0.5	0.01	0.1	1
	Number of colonies/plate	550	401	125	520	2209
		510 (530)	436 (419)	137 (131)	541 (531)	2186 (2198)
Positive control requiring S9mix	Name	B[a]P	2AA	2AA	B[a]P	B[a]P
	Dose (μg/plate)
		5	2	10	5	5
	Number of colonies/plate	867	369	524	213	83
		859 (863)	331 (350)	484 (504)	219 (216)	90 (87)

AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3: Sodium azide

ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine･2HCl

2AA: 2-Aminoanthracene

B[a]P: Benzo[a]pyrene

*: The growth inhibition of the tested bacterium by the test substance was observed.

The average number of colonies of two plates at each dose is shown in the ( ).

Table 2. Results of main test

With or without S9mix	Dose (μg/plate)	Number of revertant colonies/plate
		Base-pair substitution type			Frame-shift type
		TA100	TA1535	WP2uvrA	TA98	TA1537
－S9mix	Solvent Control (DMSO)	130	30	37	27	16
		151 (141)	28 (29)	31 (34)	16 (22)	17 (17)
	39	152	23	30	21	19
		126 (139)	26 (25)	28 (29)	16 (19)	20 (20)
	78	144	30	26	19	18
		138 (141)	33 (32)	22 (24)	20 (20)	18 (18)
	156	120	26	25	20	26
		140 (130)	32 (29)	31 (28)	15 (18)	21 (24)
	313	149	29	24	21	12
		123 (136)	33 (31)	30 (27)	18 (20)	13 (13)
	625	125	33	29	28	12
		122 (124)	22 (28)	26 (28)	19 (24)	8 (10)
	1250	123*	19*	26*	13*	4*
		90*(107)*	12*(16)*	20*(23)*	9*(11)*	4*(4)*
＋S9mix	Solvent Control (DMSO)	145	19	28	33	23
		151 (148)	25 (22)	29 (29)	31 (32)	25 (24)
	39	142	22	26	23	27
		127 (135)	32 (27)	27 (27)	24 (24)	19 (23)
	78	149	25	27	22	31
		136 (143)	22 (24)	23 (25)	28 (25)	23 (27)
	156	147	38	20	30	27
		118 (133)	24 (31)	30 (25)	27 (29)	31 (29)
	313	131	19	26	22	23
		129 (130)	24 (22)	24 (25)	31 (27)	23 (23)
	625	123	30	33	24	21
		162 (143)	29 (30)	29 (31)	32 (28)	23 (22)
	1250	107*	16*	26*	24*	10*
		110*(109)*	24*(20)*	23*(25)*	24*(24)*	10*(10)*
Positive control not requiring S9mix	Name	AF-2	NaN3	AF-2	AF-2	ICR-191
	Dose (μg/plate)
		0.01	0.5	0.01	0.1	1
	Number of colonies/plate	562	445	147	542	2268
		637 (600)	505 (475)	168 (158)	501 (522)	2061 (2165)
Positive control requiring S9mix	Name	B[a]P	2AA	2AA	B[a]P	B[a]P
	Dose (μg/plate)
		5	2	10	5	5
	Number of colonies/plate	643	346	485	202	72
		681 (662)	390 (368)	436 (461)	204 (203)	92 (82)

AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3: Sodium azide

ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine･2HCl

2AA: 2-Aminoanthracene

B[a]P: Benzo[a]pyrene

*: The growth inhibition of the tested bacterium by the test substance was observed.

The average number of colonies of two plates at each dose is shown in the ( ).

Table 3. Historical background data from Jun to Aug 2004

		without S9mix						with S9mix
strains		mean - 3SD	mean - 2SD	mean	mean + 2SD	mean + 3SD	n	mean - 3SD	mean - 2SD	mean	mean + 2SD	mean + 3SD	n
TA100	solvent control	74	88	115	143	156	349	78	94	126	157	173	324
	positive control	433	477	563	649	693	349	491	578	750	922	1008	324
TA1535	solvent control	9	13	21	29	33	267	4	8	16	23	27	245
	positive control	365	394	451	508	537	267	280	305	354	403	428	245
WP2 uvrA	solvent control	12	16	26	36	40	229	15	20	30	39	44	221
	positive control	105	119	148	176	190	229	364	402	478	553	591	221
TA98	solvent control	7	11	20	29	34	328	13	19	30	42	48	304
	positive control	468	504	577	649	686	328	142	164	207	251	272	304
TA1537	solvent control	1	5	11	18	22	255	6	11	21	30	35	233
	positive control	1683	1820	2093	2367	2503	255	42	52	71	91	101	233
TA1538	solvent control	7	9	14	20	22	20	9	14	24	34	39	20
	positive control	549	589	668	747	787	20	68	85	117	150	166	20

SD: Standard deviation

Applicant's summary and conclusion

Conclusions:

Under the conditions of the Ames test the substance was not mutagenic in any of the five bacterial strains (TA1535, TA1537, TA98, TA100 and WP2 uvrA) tested with and without metabolic activation up to 5000 µg/plate. Cytotoxicity was observed at and above 1250 µg/plate.