Methyl trans-3-oxo-2-pentylcyclopentanecarboxylate

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:

1 November - 9 December 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Protocol for the study was developed in accordance with the OECD guideline on the bacterial reverse mutation test. Study is performed by a GLP accredited laboratory.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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:

methyl trans-3-oxo-2-pentylcyclopentanecarboxylate

EC Number:

700-527-2

Cas Number:

1271488-66-4

Molecular formula:

C12H20O3

IUPAC Name:

methyl trans-3-oxo-2-pentylcyclopentanecarboxylate

Method

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:

S9-mix

Test concentrations with justification for top dose:

Dose range finding test TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate (in the presence and absence of S9-mix).
Based on the dose range findings, TA1535, TA 1537 and TA98 were exposed to the test system at 100, 333, 1000, 3330, 5000 µg/plate.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

yes

Remarks:

vehicle of test substance

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see details of test system and conditions.

Details on test system and experimental conditions:

A dose range finding test was performed with strains TA100 and WP2uvrA both with an without 5% (v/v) S9-mix. Eight concentrations were tested in triplicate. The results were used for the first experiment.
Five different doses for the TA1535, TA1537 and TA98 were tested, also in triplicate and under the same conditions as part of the first experiment.
In a repeated experiment, the test substance was tested in the presence and absence of 10% (v/v) S9-mix in all tester strains.

A negative control (DMSO) and strain specific positive controls were included. The vehicle of the test substance was DMSO. All positive controls (except for the TA1535 and TA 1537 strains) were dissolved in DMSO.

Positive control without metabolic activation (-S9)
TA 1535: sodium azide dissolved in physiological saline at 5 µg/plate.
TA 1537: 9-aminoacridine dissolved in Milli-Q water at 60 µg/plate.
TA 98: 2-nitrofluorene dissolved in DMSO at 10 µg/plate.
TA100: methylmethanesulfonate dissolved in DMSO at 650 µg/plate.
WP2uvrA: 4-nitroquinoline N-oxide dissolved in DMSO at 10 µg/plate.

Positive control with metabolic activation (+S9) was 2-aminoanthracene in DMSO at the following doses:
TA 1535: 2.5 µg/plate with 5 and 10% DMSO.
TA 1537: 2.5 µg/plate with 5% DMSO and 5µg/plate with 10% DMSO.
TA 98: 1 µg/plate with 5 and 10% DMSO.
TA100: 1 µg/plate with 5% DMSO and 2.5µg/plate with 10% DMSO.
WP2uvrA: 10 µg/plate with 5 and 10% DMSO.

Medium
The agar plates were 9 cm in diameter and contained 25ml glucose-agar medium. The agar for the test with the Salmonella typhimurium strains contained 12.5 µg/plate biotin and 15 µg/plate histidine. The agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan.

Metabolic activation
The metabolic activation system consisted of rat liver microsomal enzymes. Adult male rats were obtained from Charles River, Sulzfeld, Germany. The rats were exposed to phenobarbital (80mg/kg) and β-naphthoflavone (100mg/kg) for three consecutive days prior to the isolation of the enzymes. The S9-mix obtained was characterised with the mutagens benzo-(a)-pyrene and 2-aminoanthracene which require metabolic activation in tester strain TA98 at a dose level of 5 µg/plate and 1 µg/plate resp. It was stored at -196°C for a maximum of 1 year.

Mutation assay
0.1ml of the bacterial culture of a tester strain, 0.1ml of the test substance in DMSO and either 0.5ml S9-mix (for metabolic activation) or 0.5ml 0.1M phosphate buffer (in case of non-metabolic activation) were added to a 3ml solution of 0.6% (w/w) bacteriological agar and 0.5% (w/v) sodium chloride in Milli-Q water. After mixing the ingredients, the mixture was put on a selective agar plate and allowed to solidify.
The plates were subsequently inverted and incubated in the dark at 37±1.0°C for 48±4h. After the incubation period, the colonies were counted either manually (<40 colonies) or automatically using a Biocount 4000 Pro-S-colony counter.

Toxicity controls
To determine the toxicity of the test substance, the reduction of the bacterial background lawn, the increase in the size of microcolonies and the reduction of the revertant colonies were examined. The condition of the bacterial background lawn is evaluated both macroscopically and microscopically by using a dissecting microscope. The thinning of the microcolony and the extend of precipitation has been scored. The reduction in the number of revertant colonies compared to the solvent control has been scored as well. Any mean plate count equal to the minimal count of the historical control range was considered non-toxic. The toxicity control is based on expert judgement.

Evaluation criteria:

The Samonella typhimurium and the Escherichia coli reverse mutation assays are considered acceptable if:
1) the vehicle control is within historical range in each strain (Table 4).
2) the responses of the positive controls are in historical range and the mean plate count should be at least three times the concurrent vehicle control group mean (Table 4 and 5).
3) the selected dose range should include a clearly toxic concentration or shows limited solubility as demonstrated by the preliminary toxicity range finding test, or it should extend to 5000 µg/plate.

Statistics:

The test substance was considered not mutagenic if:
1) the total number of revertants in tester strain TA100 is not greater than two times the concurrent control and the total number of revertants in tester strains TA1535, TA1537, TA98, WP2uvrA is not greater than three times the concurrent control.
2) the negative response should be reproducible in at least one independently repeated experiment (Table 2 and 3).

The test substance is considered mutagenic if:
1) the total number of revertants in tester strain TA100 is greater than two times the concurrent control and the total number of revertants in tester strains TA1535, TA1537, TA98, WP2uvrA is greater than three times the concurrent control.
2) a positive response in one of the tester strains is 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:

cytotoxicity

Remarks:

see table 1

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, but tested up to precipitating concentrations

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

A dose range finding test was performed at a concentration range of 3-5000 µg/plate on the strains (Tables 2a and 2b). On the basis of this test a range of 100-5000 µg/plate was selected.
Precipitation of the test substance was only observed at the end of the incubation period at a concentration of 5000 µg/plate.
The results on the toxicity of the test substance can be found in Table 1. In the strain WP2uvrA, no biologically relevant reduction of the bacterial background lawn and no decrease in the number of revertants were observed.
Details of the results of experiment 1 and 2 can be found in Tables 2 and 3 resp. In both experiments, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Minimal concentration of observed toxicity of the test substance in the tester strains (details on bacterial background lawn and revertant colonies are not shown).

		dose
Experiment	Strain	µg/plate
		-S9	+S9
1	TA100	3330	3330
1	TA1537	3330
1	TA98	5000	5000
2	TA1535	3330	5000
2	TA1537	3330
2	TA98	3330
2	TA100	3330	5000

Table 2a Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli strain. Mean number of revertant colonies per three replicate plate (± standard deviation) without metabolic activation - Experiment 1.

	TA1535				TA1537				TA98				TA100				WP2uvrA
positive control	882	±	32		251	±	27		1021	±	65		956	±	12		1264	±	63
solvent control	11	±	0		4	±	3		15	±	5		84	±	17		15	±	3
Dose (µg/plate)
3													81	±	7		16	±	4
10													84	±	13		22	±	2
33													87	±	11		20	±	3
100	12	±	2		7	±	1		15	±	5		87	±	7		19	±	4
333	10	±	1		6	±	3		14	±	4		92	±	7		19	±	3
1000	8	±	3		5	±	3		15	±	5		82	±	2		18	±	3
3330	6	±	3		4	±	1 m		14	±	3		56	±	3 s		14	±	4
5000	4	±	1		MC	±	e		11	±	2 s		37	±	5 m		15	±	3

Table 2b Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli strain. Mean number of revertant colonies per three replicate plate (± standard deviation) with metabolic activation (5% S9 v/v) - Experiment 1.

	TA1535				TA1537				TA98				TA100				WP2uvrA
positive control	314	±	16		336	±	70		983	±	32		1174	±	6		318	±	9
solvent control	8	±	2		6	±	1		25	±	2		70	±	10		17	±	5
3													79	±	10		26	±	2
10													79	±	7		19	±	5
33													75	±	11		22	±	2
100	8	±	2		5	±	2		21	±	1		76	±	8		20	±	3
333	7	±	3		6	±	1		22	±	6		74	±	2		24	±	3
1000	5	±	3		9	±	5		26	±	5		62	±	7		17	±	4
3330	4	±	2		3	±	1		20	±	5		62	±	8 s		20	±	6
5000	9	±	6		5	±	2		18	±	7 m		56	±	7 m		13	±	3

Table 3a Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli strain. Mean number of revertant colonies per three replicate plate (± standard deviation) without metabolic activation - Experiment 2.

	TA1535				TA1537				TA98				TA100				WP2uvrA
positive control	921	±	36		203	±	40		995	±	58		902	±	48		1100	±	71
solvent control	7	±	3		4	±	1		16	±	1		88	±	7		18	±	6
100	8	±	2		5	±	1		15	±	2		85	±	4		15	±	1
333	7	±	2		4	±	2		19	±	8		76	±	7		14	±	3
1000	7	±	1		4	±	2		15	±	2		91	±	2		17	±	5
3330	3	±	1 s		1	±	1 s		6	±	3 s		59	±	12 s		12	±	5
5000	3	±	2 m		MC		e		4	±	3 m		MC		e		13	±	4

Table 3b Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli strain. Mean number of revertant colonies per three replicate plate (± standard deviation) with metabolic activation (10% S9 v/v) - Experiment 2.

	TA1535				TA1537				TA98				TA100				WP2uvrA
positive control	169	±	5		452	±	28		381	±	38		913	±	264		316	±	37
solvent control	9	±	2		4	±	2		26	±	8		67	±	8		19	±	1
100	6	±	3		9	±	2		28	±	8		78	±	11		21	±	3
333	6	±	5		5	±	2		26	±	4		79	±	13		17	±	5
1000	9	±	2		5	±	2		27	±	3		66	±	7		19	±	5
3330	8	±	3		2	±	1		23	±	8		56	±	2		17	±	1
5000	5	±	0 s		4	±	2		21	±	4		72	±	6 s		14	±	2

s     Bacterial background lawn slightly reduced

m    Bacterial background lawn moderately reduced

e     Bacterial background lawn extremely reduced

SP    Slight Precipitate

MC   Microcolonies

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

All bacterial strains showed negative responses over the entire dose range in the two independent experiments.
The negative and strain specific controls were within the historical range indicating that the test conditions were adequate and the metabolic system worked properly.
Based on the results, it is concluded that the test substance is not mutagenic in the Salmonella typhimurium and the Escherichia coli reverse mutation assay.

Executive summary:

The mutagenicty of the test substance was evaluated by a Salmonella typhimurium reverse mutation assay with four histidine-requiring strains (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain (WP2uvrA). The test was performed in two independent experiments in the presence and absence of metabolic activation (S9-mix) at a concentration range of 100 - 5000 µg/plate. In most of the Salmonella typhimurium strains, toxicity of the test substance was observed at a dose level of 3330µg/plate without S9 and in some strains at 5000 µg/plate (except TA100: 3330 µg/plate). No toxicity was observed in the Escherichia coli strain. Slight precipitation was observed at 5000 µg/plate. The test substance did not induce a significant dose-related increase in the number of revertants colonies in all strains both in the presence and absence of metabolic activation (S9). All negative and positive controls were within historical ranges. It is therefore concluded that the test substance is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assays.