2-Cyclopentene-1-acetic acid, ethyl ester

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:

2011-05-26 to 2011-07-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study without restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 2-(cyclopent-2-en-1-yl)acetate

EC Number:

605-150-6

Cas Number:

15848-49-4

Molecular formula:

C9H14O2

IUPAC Name:

ethyl 2-(cyclopent-2-en-1-yl)acetate

Test material form:

other: liquid

Details on test material:

- Physical state: liquid
- Molecular formula: C9H14O2
- Molecular weight: 154.21 g/mol

Method

Target gene:

not applicable

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 prepared from phenobarbital /β-naphthoflavone induced male rat liver (age: 8-12 weeks old; induction: 3 consecutive days)

Test concentrations with justification for top dose:

Pre-experiment/Experiment 1 (plate incorporation test):
3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate (with and without metabolic activation)
Experiment 2 (pre-incubation assay):
3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate (without S9-mix)
33, 100, 333, 1000, 2500, and 5000 µg/plate (with S9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (MERCK, 64293 Darmstadt/Germany; purity > 99%)
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria (Maron et al. 1981)*

*Reference:
Maron D.M., J. Katzenellenbogen, and B.N. Ames (1981): Compatibility of organic solvents with the Salmonella/Microsome Test. Mutation Res. 88, 343-350.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Positive control substanbce without metabolic activation; strains TA 1535 (10 µg/plate), TA 100 (10 µg/plate)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine (4-NOPD)

Remarks:

Positive control substance without metabolic activation; strains TA 1537 (50 µg/plate), TA 98 (10 µg/plate)

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Positive control substance without metabolic activation; strain WP2 uvrA (3 µg/plate)

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

Positive control substance with metabolic activation; strains TA 1535 (2.5 µg/plate), TA 1537 (2.5 µg/plate), TA 98 (2.5 µg/plate), TA 100 (2.5 µg/plate), WP2 uvrA (10 µg/plate)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation (experiment 1) and preincubation (experiment 2)

NUMBER OF REPLICATIONS: 3

PRE-EXPERIMENT FOR TOXICITY:
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The pre-experiment was reported as experiment 1.

EXPERIMENTAL PERFORMANCE:
For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µL test solution at each dose level (solvent or reference mutagen solution (positive
control)),
- 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test
without metabolic activation),
- 100 µL bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µL overlay agar
In the pre-incubation assay 100 µL test solution (solvent or reference mutagen solution (positive control)), 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After preincubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark (de Serres and Shelby, 1979)*.

DATA RECORDING:
The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager. Due to reduced background growth and wide spread bacterial colony growth the revertant colonies were partly counted manually.

ACCEPTABILITY OF THE ASSAY:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

*Reference:
de Serres F.J. and M.D. Shelby (1979). Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay. Mutation Res. 64, 159-165

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed (Hollstein, McCann, Angelosanto and Nichols, 1979)*.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration (de Serres and Shelby, 1979)*.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

*References:
- de Serres F.J. and M.D. Shelby (1979). Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay. Mutation Res. 64, 159-165.
- Hollstein,M., J. McCann, F.A. Angelosanto, and W.W. Nichols (1979). Short-term tests for carcinogens and mutagens. Mutation Res. 65, 133-226.

Statistics:

A statistical analysis of the data is not mandatory.

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:

please refer to "Any other information on results incl. tables" (table 1)

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:

cytotoxicity

Remarks:

please refer to "Any other information on results incl. tables" (table 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation of the test item occurred up to the highest investigated dose.

EXPERIMENTS 1 AND 2:
The plates incubated with the test item showed reduced background growth at the following concentrations as shown in Table 1 (please refer to "Any other information on results incl. tables).
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups at the following concentrations as shown in Table 2 (please refer to "Any other information on results incl. tables).
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with PI26133 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1

Strain	Experiment 1		Experiment 2
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	2500 - 5000	/	2500 - 5000	/
TA 1537	5000	/	1000 - 5000	5000
TA 98	5000	/	1000 - 5000	/
TA 100	1000 - 5000	/	1000 - 5000	/
WP2 uvrA	/	/	1000 - 5000	/

Table 2

Strain	Experiment 1		Experiment 2
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	5000	/	2500 - 5000	/
TA 1537	2500 - 5000	/	1000 - 5000	5000
TA 98	5000	/	1000 - 5000	/
TA 100	2500 - 5000	/	2500 - 5000	/
WP2 uvrA	/	/	2500 - 5000	/

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is not mutagenic.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as mutagenic.