Cyclopentanone

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:

from 2014-11-28 to 2015-04-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD 471 guideline study in compliance with the GLP. No deviation from the protocol of the study.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cyclopentanone

EC Number:

204-435-9

EC Name:

Cyclopentanone

Cas Number:

120-92-3

Molecular formula:

C5H8O

IUPAC Name:

cyclopentanone

Test material form:

other: liquid stored at controlled room temperature

Details on test material:

- Name of test material (as cited in study report): Cyclopentanone
- Physical state: colourless liquid
- Storage condition of test material: room temperature

Method

Target gene:

- Histidine: TA 1535, TA 100, TA 1537 and TA 98
-Tryptophan: Escherichia coli WP2 uvr A

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: see Table 7.6.1/1

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: see Table 7.6.1/1

Metabolic activation:

with and without

Metabolic activation system:

S9 from induced phenobarbital/Beta-naphthoflavone rat liver.

Test concentrations with justification for top dose:

- Range finding test: 10; 100; 500; 1000; 2500 and 5000 µg/plate
- First experiment (Direct plate incorporation method, with and without metabolic activation): 312.5, 625, 1250, 2500 and 5000 µg/plate
- Second experiment (Direct plate incorporation method, without metabolic activation) (Pre-incubation method, with metabolic activation): 312.5, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water for injections, batch No. 4F1081 (CDM Lavoisier)
- Justification for choice of solvent/vehicle: the substance was soluble in water (solubility: 301 g/l at 20°C)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

methylmethanesulfonate

other: 4-nitroquinoline 1-oxide (4NQO); 2-anthramine (2AM)

Remarks:

see Table 7.6.1/2

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate
incorporation method in agar. The second experiment with S9 mix was performed according to the pre-incubation method.

DURATION
- Incubation period: at 37°C for 48 to 72 hours.
- Preincubation period: 60 min at 37ºC.

NUMBER OF REPLICATIONS: 3 plates/dose/strain. Two independent experiments were performed.

DETERMINATION OF CYTOTOXICITY
- Method: The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a
thinning of the bacterial lawn.

OTHER: SCORING METHOD: the number of revertants per plate was scored for each strain and for each experimental point using an automatic counter (Sorcerer Automatic Colony Counter for the scoring of colonies and Ames Study Manager for the data management, Perceptive Instruments Ltd, Bury St Edmunds IP33 3TA, UK).

Evaluation criteria:

Acceptance criteria :
Each main experiment was considered valid if the following criteria are fully met:
- the mean number of revertants in the vehicle controls is consistent with our historical data, in each strain and test condition (Appendix 2),
- at least five analyzable dose-levels (i.e. including at least three non-cytotoxic dose-levels) are obtained for each strain and test condition,
- the mean number of revertants in the positive controls is higher than that of the vehicle controls (at least 2-fold increase (for the TA 98, TA 100 and WP2 uvrA strains) or at least 3-fold increase (for the TA 1535 and TA 1537 strains)).

When these criteria were not met for one strain or test condition, the corresponding results were invalidated and the experiment was repeated.

Evaluation criteria :
In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.

The test item is considered to have shown mutagenic activity in this study if:
- a reproducible 2-fold increase (for the TA 98, TA 100 and WP2 uvrA strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose-level,
- and/or a reproducible dose-response relationship is evidenced.

The test item is considered to have shown no mutagenic activity in this study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and WP2 uvrA strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose-levels,
- nor any evidence of a dose-response relationship is noted.

Statistics:

No data

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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: no

RANGE-FINDING/SCREENING STUDIES:
To assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100 and WP2 uvrA strains, both with and without S9 mix.
Using a test item concentration at 50 mg/mL in the vehicle and a treatment volume of 100 µL/plate, the highest recommended dose-level of 5000 µg/plate was achievable. Thus, the dose-levels selected for the preliminary test were 10, 100, 500, 1000, 2500 and 5000 µg/plate. The evaluation of the
toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.
No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose levels. No noteworthy toxicity was noted towards the three strains used, either with or without S9 mix.


COMPARISON WITH HISTORICAL CONTROL DATA: The control data reported in these report are in the range of the historical control data observed in the laboratory. The study was therefore considered valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxicity was noted towards all the strains used.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

 See attached document    

 

Applicant's summary and conclusion

Conclusions:

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

Under the experimental conditions of this study, the test item Cyclopentanone did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli strains, either in the presence or in the absence of a rat liver metabolizing system.

Executive summary:

This study was performed to investigate the potential of the test item, Cyclopentanone, to induce reverse mutations in Salmonella typhimurium and Escherichia coli. The study was performed according to OECD guideline no. 471 and EC guideline n° B13/14 and in compliance with the Principles of Good Laboratory Practice. 

 

A preliminary toxicity test was performed to define the dose-levels of Cyclopentanone to be used for the mutagenicity study. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).

Four strains of bacteria Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100) and one strain of Escherichia coli (WP2 uvrA) were used. Each strain was exposed to five dose-levels of the test item (three plates/dose‑level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test item was dissolved in water for injections.

 

The test item was freely soluble in the vehicle at 50 mg/mL. Consequently, with a maximum dose-volume of 100 µL/plate, the dose-levels for the preliminary toxicity test were 10, 100, 500, 1000, 2500 and 5000 µg/plate.

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose‑levels, and no noteworthy toxicity was noted towards the three strains used, either with or without S9 mix.

Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

The selected treatment-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate, for both mutagenicity experiments with and without S9 mix.

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose‑levels, and no toxicity was noted towards all the strains used.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, either with or without S9 mix. These results met thus the criteria of a negative response.

Under the experimental conditions of this study, the test item Cyclopentanone did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli strains, either in the presence or in the absence of a rat liver metabolizing system.