2-hexyldecanoic acid [4-(6-tert-butyl-7-chloro-1H-pyrazolo[1,5-b][1,2,4]triazol-2-yl)phenylcarbamoyl]methylester

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:

17 Jul - 04 Aug 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

published June 2000

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): UM-235
- Physical state: white powder
- Analytical purity: 98.8 %
- Purity test date: 22 Jul 2002
- Lot/batch No.: #SI-1
- Expiration date of the lot/batch: 01 Jan 2005
- Storage condition of test material: at room temperature in the dark

Method

Target gene:

his operon (S. typhimurium), trp operon (E. coli)

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 Aroclor 1254

Test concentrations with justification for top dose:

first and second experiment: 3, 10, 33, 100 and 333 µg/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

methylmethanesulfonate

other: daunomycin, 2-aminoanthracene

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction of bacterial background lawn, increase in size of microcolonies, reduction of revertant colonies

Evaluation criteria:

A test is considered negative (not mutagenic) if
- the total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation
- the negative response should be reproducible in at least one independently repeated experiment

A test substance is considered positive (mutagenic) if
- it induces a number of revertant colonies, dose related, greater than 2-times the number of revertants induced by solvent control in any of the tester strains, either with or without metabolic activation
- the positive response should be reproducible in at least one independently repeated experiment
For more criteria see tables under "Any other information on material and methods including tables"

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation was observed at concentrations of 100 µg/plate and above in the top agar and on the plates at 333 µg/plate at the start and the end of the experiment.

RANGE-FINDING/SCREENING STUDIES:
A dose-range finding test was performed with S. typhimurium TA100 and E. coli WP2 strains with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate. The highest concentration chosen for the main assay was the level at which the test substance showed limited solubility (333 µg/plate).

COMPARISON WITH HISTORICAL CONTROL DATA:
yes, see acceptability of the assay under any other information on materials and methods including tables

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results of Experiment I

with or without S9 mix (5%)	test substance concentration	Mean number of revertant colonies per plate (average of 3 plates ± SD)
	[µg/plate]	Base-pair substitution type		Frameshift type
		TA100	TA1535	TA98	TA1537	WP2 uvrA
-	DMSO	138 ± 6	8 ± 2	11 ± 2	4 ± 1	9 ± 2
-	3	144 ± 4	7 ± 4	13 ± 2	5 ± 2	13 ± 5
-	10	154 ± 20	11 ± 3	12 ± 4	4 ± 1	9 ± 1
-	33	135 ± 11	9 ± 3	14 ± 2	3 ± 2	10 ± 3
-	100	142 ± 13	11 ± 2	13 ± 4	4 ± 1	10 ± 2
-	333^SP	142 ± 9	8 ± 3	13 ± 1	5 ± 2	10 ± 4
positive control, -S9	Name	SA	9AC	DM	MMS	4 -NQO
	Concentrations [µg/plate]	650	5	4	60	10
	Mean No. of colonies/plate (average of 3 ± SD)	962 ± 22	244 ± 16	516 ± 71	280 ± 46	812 ± 73
+	DMSO	140 ± 21	10 ± 1	17 ± 1	4 ± 4	10 ± 1
+	3	136 ± 15	7 ± 2	21 ± 2	5 ± 0	10 ± 2
+	10	151 ± 1	6 ± 1	18 ± 2	5 ± 3	8 ± 1
+	33	155 ± 7	7 ± 2	20 ± 2	5 ± 1	11 ± 1
+	100	138 ± 10	8 ± 3	19 ± 3	6 ± 2	10 ± 2
+	333^SP	137 ± 14	6 ± 3	18 ± 3	6 ± 4	9 ± 2
positive control, +S9	Name	2 -AA	2 -AA	2 -AA	2 -AA	2 -AA
	Concentration [µg/plate]	1	2.5	2.5	2.5	5
	Mean No. of colonies/plate (average of 3 ± SD)	717 ± 50	100 ± 4	520 ± 7	75 ± 16	73 ± 11

SP = Slight Precipitate

Table 2: Results of Experiment II

with or without S9 mix (10%)	test substance concentration	Mean number of revertant colonies per plate (average of 3 plates ± SD)
	[µg/plate]	Base-pair substitution type		Frameshift type
		TA100	TA1535	TA98	TA1537	WP2 uvrA
-	DMSO	132 ± 8	9 ± 2	12 ± 3	8 ± 2	7 ± 2
-	3	149 ± 22	16 ± 3	14 ± 3	7 ± 4	11 ± 4
-	10	146 ± 17	10 ± 2	13 ± 1	8 ± 2	12 ± 0
-	33	144 ± 5	9 ± 2	12 ± 4	7 ± 3	9 ± 4
-	100	134 ± 11	10 ± 2	16 ± 2	9 ± 1	9 ± 1
-	333^SP	148 ± 19	11 ± 1	11 ± 4	5 ± 1	8 ± 3
positive control, -S9	Name	SA	9AC	DM	MMS	4 -NQO
	Concentrations [µg/plate]	650	5	4	60	10
	Mean No. of colonies/plate (average of 3 ± SD)	965 ± 32	355 ± 75	326 ± 57	297 ± 6	1323 ± 72
+	DMSO	135 ± 9	11 ± 4	19 ± 6	10 ± 4	8 ± 3
+	3	123 ± 13	9 ± 2	19 ± 1	10 ± 4	9 ± 4
+	10	131 ± 21	11 ± 1	22 ± 4	6 ± 2	11 ± 3
+	33	131 ± 10	8 ± 1	17 ± 5	11 ± 3	7 ± 1
+	100	117 ± 26	13 ± 3	20 ± 2	14 ± 2	9 ± 4
+	333^SP	133 ± 3	8 ± 3	19 ± 1	9 ± 3	10 ± 3
positive control, +S9	Name	2 -AA	2 -AA	2 -AA	2 -AA	2 -AA
	Concentration [µg/plate]	2.5	5	5	5	10
	Mean No. of colonies/plate (average of 3 ± SD)	829 ± 14	136 ± 6	196 ± 35	218 ± 31	120 ± 13

SP = Slight Precipitate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative