Methyl cyclopentylideneacetate

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:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP; minor deviation not affecting the reliability of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Minor deviation: first mutation assay: strain TA98, no bacteria were plated at the mid dose 164 μg/plate. However, since at least a total of five dose levels were used at up to the recommended dose level 5000μg/plate, study reliability is unaffected.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Minor deviation; see above

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

yes

Remarks:

Minor deviation; see above

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Guideline stipulated by the Japanese Ministry of Health, Labour and Welfare, Ministry of Economy, Trade and Industry and Ministry of the Environment (revised March 31st, 2011)

Deviations:

yes

Remarks:

Minor deviation; see above

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected March 2013; signature: May 2013

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl cyclopentylideneacetate

EC Number:

254-837-3

EC Name:

Methyl cyclopentylideneacetate

Cas Number:

40203-73-4

Molecular formula:

C8H12O2

IUPAC Name:

methyl 2-cyclopentylideneacetate

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Physical state: liquid
- Storage condition of test material: In refrigerator (2-8°C) in the dark
- Other: Clear colourless

Method

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

Dose range finding study: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 ug/plate
Experiment 1: 17, 52, 164, 512, 1600, 5000 ug/plate
Experiment 2: 492, 878, 1568, 2800, 5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance formed an emulsion in water and ethanol at a concentration of 50 mg/ml. The test substance was fully soluble in dimethyl sulfoxide (DMSO) at a concentration of 50 mg/ml.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli were counted. The revertant colonies were counted automatically with a Colony Counter.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (bacterial background lawn) and reduction in the number of revertants

OTHER:
Dose range finding test on TA100 and WP2urvA with and without 5% (v/v) S9-mix; First mutation assay on TA1535, TA1537 and TA98 with and without 5% (v/v) S9-mix. To obtain more information about the possible mutagenicity of the test substance, a second mutation experiment was performed on all strains, in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test substance was tested up to the dose level of 5000 μg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Based on the results of the earlier assays.

Evaluation criteria:

See 'Any other information on materials and methods' for details on evaluation of the assay and positive criteria.

Statistics:

No formal hypothesis testing was done. See 'Any other information on materials and methods' for details on the acceptability and evaluation criteria of the assay.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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:

Cytotoxicity was only observed at the top dose of 5000 μg/plate in tester strain TA1535 and TA100 (absence and presence of S9-mix) and TA1537 and TA98 (absence of S9-mix) in experiment 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.

RANGE-FINDING/SCREENING STUDIES:
Test substance was tested in the tester strains TA100 and WP2uvrA with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 17, 52, 164, 512, 1600 and 5000 μg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Dose range finding test: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain
	TA100		WP2uvrA
Without S9-mix
Positive control	1057	± 86	1257	± 41
Solvent control	139	± 26	29	± 5
1.7	146	± 15	35	± 3
5.4	128	± 12	37	± 8
17	130	± 15	35	± 6
52	134	± 6	38	± 3
164	129	± 5	34	± 4
512	126	± 6	36	± 6
1600	121	± 17 n	36	± 3 n
5000	95	± 9 s NP	38	± 15 s NP
With S9-mix #1
Positive control	1420	± 45	275	± 33
Solvent control	132	± 19	38	± 7
1.7	121	± 8	44	± 5
5.4	128	± 9	45	± 6
17	126	± 15	48	± 8
52	123	± 4	44	± 5
164	149	± 38	48	± 4
512	122	± 7	42	± 6
1600	112	± 15 n	37	± 8 n
5000	83	± 12 s NP	28	± 5 s NP

#1          Plate incorporation assay (5% S9)

NP         No precipitate

n            Normal bacterial background lawn

s            Bacterial background lawn slightly reduced

 

Table 2 Experiment 1: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium
	TA1535		TA1537		TA98
Without S9-mix
Positive control	562	± 37	541	± 36	924	± 114
Solvent control	25	± 7	9	± 5	17	± 2
17	29	± 0	9	± 5	19	± 1
52	26	± 5	5	± 2	15	± 5
164	30	± 3	10	± 2	#2
512	30	± 8	9	± 2	16	± 6
1600	27	± 5	4	± 1	19	± 0
5000	21	± 1 n NP	9	± 4 n NP	14	± 1 n NP
With S9-mix #1
Positive control	251	± 27	259	± 41	715	± 48
Solvent control	16	± 3	6	± 1	23	± 1
17	18	± 6	8	± 3	24	± 2
52	18	± 7	13	± 5	20	± 6
164	18	± 4	13	± 10	#2
512	18	± 4	8	± 2	26	± 6
1600	15	± 4	7	± 4	26	± 2
5000	11	± 1 n NP	8	± 1 n NP	19	± 1 n NP

#1          Plate incorporation assay (5% S9)

#2          No bacteria plated

NP         No precipitate

n            Normal bacterial background lawn

 

Table 3 Experiment 2: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1535		TA1537		TA98		TA100		WP2uvrA
Without S9-mix
Positive control	753	± 28	710	± 95	867	± 63	901	± 42	1154	± 106
Solvent control	22	± 5	8	± 4	22	± 6	133	± 8	35	± 11
492	26	± 3	9	± 4	29	± 7	124	± 23	29	± 17
878	26	± 4	5	± 5	21	± 3	131	± 16	33	± 11
1568	22	± 7	7	± 4	25	± 4	123	± 2	32	± 6
2800	24	± 8 n	6	± 2 n	22	± 9 n	107	± 3	26	± 6
5000	17	± 4 s NP	9	± 2 s NP	13	± 6 s NP	93	± 25 s NP	23	± 3 n NP
With S9-mix #1
Positive control	166	± 10	443	± 138	1047	± 68	1275	± 218	153	± 41
Solvent control	13	± 2	14	± 2	39	± 2	134	± 13	42	± 13
492	17	± 6	15	± 3	42	± 2	130	± 16	41	± 8
878	13	± 3	7	± 4	34	± 5	129	± 21	46	± 9
1568	17	± 8	15	± 4	36	± 6	121	± 12	40	± 11
2800	12	± 6 n	4	± 1	33	± 14	103	± 11 n	37	± 5
5000	11	± 4 s NP	13	± 6 n NP	33	± 6 n NP	77	± 2 s NP	21	± 2 n NP

#1          Plate incorporation assay (10% S9)

NP         No precipitate

n            Normal bacterial background lawn

s            Bacterial background lawn slightly reduced

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study the test substance is not considered to be mutagenic. Negative and strain specific positive control values were within laboratory historical control data.

Executive summary:

The study was performed to OECD 471, EU Method B.13/14, EPA OPPTS 870.5100 and the Japan Guidelines for Screening Mutagenicity of Chemicals in accordance with GLP; to evaluate the mutagenic activity of the test substance in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (with independent repeat). The dose range finding test, the test substance was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test substance did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease of the bacterial background lawn, was observed at the top dose of 5000 μg/plate in both tester strains. Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at a concentration range of 17 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In an independent repeat of the assay with additional parameters, the test substance was tested at a concentration range of 492 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Cytotoxicity, as evidenced by a decrease of the bacterial background lawn, was only observed at the top dose of 5000 μg/plate in the tester strains TA1535 and TA100 (absence and presence of S9-mix) and TA1537 and TA98 (absence of S9-mix). The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the conditions of this study it is concluded that that the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.