2-(4-tert-butylphenyl)-6-cyano-5-[bis(ethoxycarbonylmethyl)carbamoyloxy]-1H-pyrrolo[1,2-b][1,2,4] triazole-7-carboxylic acid 2,6-di-tert-butyl-4-methylcyclohexylester

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:

25 Jul - 08 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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

448-050-6

EC Name:

-

Cas Number:

444065-11-6

Molecular formula:

C41 H57 N5 O8

IUPAC Name:

2,6-di-tert-butyl-4-methylcyclohexyl 5-{[bis(2-ethoxy-2-oxoethyl)carbamoyl]oxy}-2-(4-tert-butylphenyl)-6-cyano-1H-pyrrolo[1,2-b][1,2,4]triazole-7-carboxylate

Details on test material:

- Name of test material (as cited in study report): UC-141
- Physical state: White powder
- Analytical purity: 99.7% (area%), 98.2% (mass%)
- Lot/batch No.: CE-203
- 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 (Salmonella strains)
trp operon (Escherichia 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 Aroclor 1254

Test concentrations with justification for top dose:

Range-finding study (results included in first experiment): 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate; TA 100 and WP2 uvr A; with and without metabolic activation
First experiment: 3, 10, 33, 100, 333 and 1000 μg/plate; TA 1535, TA 1537 and TA 98; with and without metabolic activation
Second experiment: 10, 33, 100, 333 and 1000 μg/plate; TA 1535, TA 1537, TA 100 and TA 98, and WP2 uvr A; with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Test substance concentrations were prepared directly prior to use and used within 4 hours after preparation.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

vehicle control with and without metabolic activation

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium azide, 9-aminoacridine, daunomycin, methylmethanesulfonate, 4-nitroquinoline N-oxide, 2-aminoanthracene (see 'Any other information on results incl. tables' for details).

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 replications each, in 2 independent experiments

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

OTHER: In a range-finding study with strain TA 100 and the WP2 uvr A strain, with and without S9-mix, 8 concentrations (3-5000 μg/plate) were tested in triplicate. The dose range finding test results were reported as a part of the first experiment of the mutation assay. The highest concentration of the test substance used in the subsequent mutation assay was the level at which the test substance exhibited limited solubility.

Evaluation criteria:

a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

A test substance is considered negative (not mutagenic) in the test if:
a) 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.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 μg/pIate and above (see Table 1 and 2). The precipitate does not influence automated counting of the plate.

RANGE-FINDING/SCREENING STUDIES: The results of the range-finding study was used as the first of two independent experiments. Precipitation of UC-141 on the plates was observed at the start and at the end of the incubation period at 333 μg/plate and above with and without metabolic activation in all strains. No cytotoxicity was observed at any dose level in any strain, with or without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes (data not shown). The results of the negative and positive control data fell within the historical range,

ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed up to and including 5000 μg/plate with and without metabolic activation in TA 100 and WP2 uvr A; and up to and including 1000 μg/plate with and without metabolic activation in TA 1535, TA 1537 and TA 98.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Test results of experiment 1, including range-finding study (plate incorporation)

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2 uvr A	TA98	TA1537
–	Solvent control	138 ± 13	9 ± 2	13 ± 3	17 ± 1	7 ± 3
–	3	143 ± 21	6 ± 2	16 ± 4	14 ± 2	5 ± 2
–	10	147 ± 2	7 ± 1	14 ± 4	13 ± 4	6 ± 1
–	33	148 ± 15	4 ± 1	19 ± 3	15 ± 2	6 ± 1
–	100	147 ± 3	6 ± 2	20 ± 5	12 ± 3	4 ± 1
-	333	150 ± 17	6 ± 1	17 ± 7	16 ± 1	3 ±1
-	1000P	161 ± 8	-	12 ± 4	-	-
-	3330P	167 ± 13	-	19 ± 6	-	-
–	5000P	152 ± 23	-	13 ± 3	-	-
Positive controls, –S9	Name	MMS	SA	4NQO	DM	9AA
	Concentrations (μg/plate)	650	1	10	4	60
	Mean No. of colonies/plate (average of 3 ± SD)	1025 ± 27	207 ±8	712 ± 6	317 ± 69	265 ± 24
+	Solvent control	136 ± 2	9 ± 2	18 ± 1	23 ± 2	6 ± 1
+	3	130 ± 18	7 ± 1	19 ± 2	20 ± 2	5 ± 2
+	10	135± 9	8 ± 2	27 ± 5	23 ± 3	4 ± 1
+	33	149 ± 9	7 ± 1	20 ± 6	24 ± 3	5 ± 2
+	100	155 ± 3	5 ± 1	15 ± 3	22 ± 2	4 ± 2
+	333	142 ± 15	8 ± 2	21 ± 2	20 ± 3	6 ± 1
+	1000P	142 ± 11	-	20 ± 3	-	-
+	3330P	142 ± 27	-	15 ± 1	-	-
+	5000P	141 ± 17	-	15 ± 3	-	-
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1	1	5	1	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	825 ± 35	153 ± 14	90 ± 2	529 ± 46	279 ± 14

SA = sodium azide

DM = daunomycine

MMS = methylmethanesulfonate

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2-Aminoanthracene

P = Slight precipitate

 

Table 2: Test results of experiment 2 (plate incorporation)

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2 uvr A	TA98	TA1537
–	Solvent control	146 ± 5	8 ± 4	9 ± 5	13 ± 2	4 ± 3
–	10	131 ± 9	9 ± 4	8 ± 2	13 ± 5	7 ± 4
–	33	147 ± 13	6 ± 2	7 ± 2	19 ± 4	6 ± 3
–	100	143 ±13	4 ± 1	6 ± 2	13 ± 3	6 ± 2
-	333	138 ± 18	8 ± 4	8 ± 4	12 ± 2	6 ± 5
-	1000P	147 ± 2	8 ± 3	10 ± 1	17 ± 3	6 ± 2
Positive controls, –S9	Name	MMS	SA	4NQO	DM	9AA
	Concentrations (μg/plate)	650	1	10	4	60
	Mean No. of colonies/plate (average of 3 ± SD)	808 ± 18	794 ± 14	751 ± 14	661 ± 112	454 ± 32
+	Solvent control	110 ± 9	7 ± 3	12 ± 5	26 ± 9	4 ± 1
+	10	115 ± 11	10 ± 3	9 ± 3	15 ± 1	7 ± 3
+	33	122 ± 5	6 ± 1	8 ± 2	19 ± 3	6 ± 4
+	100	105 ± 13	4 ± 3	7 ± 1	19 ± 5	8 ± 3
+	333	116 ± 5	10 ± 3	8 ± 5	18 ± 3	6 ± 2
	1000P	106 ± 14	10 ± 3	7 ± 1	16 ± 4	4 ± 2
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1	1	5	1	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	719 ± 34	117 ± 11	142 ± 21	302 ± 14	257 ± 18

SA = sodium azide

DM = daunomycine

MMS = methylmethanesulfonate

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2-Aminoanthracene

P = Slight precipitate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

CLP: not classified
DSD: not classified