Cyclopentadecanone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Cyclopentadecanone is not genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 August 1997 - 9 September 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 May 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

26 May 1973

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OJ No. L383A, 29-12-1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA Method HG-Gene Muta - S. typhimurium: The Salmonella typhimurium reverse mutation assay

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA HG-Gene Muta - E. coli: The Escherichia coli reverse mutation assay

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

28 January 1985

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble at 50 mg/mL in DMSO

Target gene:

S. typhimurium: his
E.coli: try

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat S9

Test concentrations with justification for top dose:

312.5, 625, 1250, 2500 and 5000 µg/plate (based on maximal solubility of the test article in DMSO)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: based on the solubility of the test substance

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Exposure duration: 3 days

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
relative total growth
- Any supplementary information relevant to cytotoxicity:
Preliminary toxicity study was performed using 6 tester strains. The maximal tested concentration was 5000 µg/plate . Three ten-fold serial dilutions were also tested. A single plate per dose was used.

Evaluation criteria:

If treatment with a substance produces increase in revertant colony numbers of at least twice of the concurrent negative control, with some evidence of dose-response relationship, in two separate experiments, with any bacterial strain either in the presence or the absence of S9, it is considered to show evidence of mutagenic activity in this test system.
If treatment with a test substance does not produce a reproducible increase of at least 1.5 fold of the concurrent solvent control, at any dose level in any strain, it is considered to show no evidence of mutagenic activity in this test system.

Statistics:

No statistical analysis was performed.

Key result

Species / strain:

S. typhimurium TA 98

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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

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:

valid

Positive controls validity:

valid

Key result

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:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test substance was not toxic to tester strains at the highest concentration of 5000 µg/plate

Conclusions:

In the reliable study, performed according to OECD Guidelines 471 and 472, the test substance cyclopentadecanone was not mutagenic in the strains S. typhimurium TA98, TA100, TA1535, TA 1537 and TA1538 and E. coli WP2 uvrA, both with and without metabolic activation, up to the highest tested concentration of 5000 µg/plate.

Executive summary:

In the reliable study performed according to OECD Guidelines 471 and 472, cyclopentadecanone did not increase the number of revertant colonies in the tester strains S. typhimurium TA98, TA100, TA1535, TA1537 and TA1538, and E. coli WP2 uvrA, both in the presence and absence of metabolic activation (rat S9), up to the limit concentration of 5000 µg/plate. No cytotoxicity was observed. DMSO was used as a solvent. The testing was performed in triplicate. Positive controls produced satisfactory mutagenic response. Based on the results of the study, cyclopentadecanone was not mutagenic in bacterial cells.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 September 1997 - 29 October 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro chromosome aberration study

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in DMSO at 50 mg/mL

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: human blood collected aseptically from healthy male donors, pooled and diluted with RPMI 1640 tissue culture medium containing 10% foetal calf serum. Aliquots (0.4 mL of blood : 4.5 mL medium : 0.1 mL phytohemaegglutinin) of the cell suspension were placed in sterile universal containers and incubated at 37 °C in a slanted position for approx. 48 hours. The cultures were gently shaken once daily to resuspend the cells.
- Sex, age and number of blood donors if applicable: not specified
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-stimulated rat liver S9

Test concentrations with justification for top dose:

First test: 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL (3 hours exposure, ±S9)
Second test:
7.8, 15.6, 31.6, 62.5, 90, 125 and 250 µg/mL (continuous treatment, -S9)
7.8, 15.6, 31.6, 62.5, 90, 125, 150 and 250 µg/mL (3 hours exposure, +S9)
The top concentrations were chosen based on the maximal solubility of the substance in DMSO. At a maximal concentration of 500 µg/mL, a precipitate was observed. Further dilutions of 125 and 250 µg/mL also resulted in a precipitate after dosing. After incubation at 37 °C for a few hours, solution containing 100 µg/mL was almost clear. When dosed at a concentration 50 µg/mL, no precipitate was observed. Therefore the highest tested concentration was 500 µg/mL, to ensure that several doses were in insoluble range.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: based on the test substance solubility. The test substance is insoluble in water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 48 hours
- Exposure duration: 3 hours (1st experiment, ±S9, and 2nd experiment, + S9); continuous treatment (2nd experiment, -S9)
- Expression time (cells in growth medium): 18 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid

NUMBER OF REPLICATIONS: two independent experiments, each performed in duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Two hours before the cells were harvested, mitotic activity was arrested by addition of colcemide to each culture at a final concentration of 0.1 µg/mL. After 2 hours incubation, each cell suspenion was transferred to a conical centrifuge tube and centrifuged for 5 minutes at 500 g. The cell pellets were treated with a hypotonic solution of (0.075 M KCl pretreated at 30 °C). After a 10 minutes period of hypotonic incubation at 37 °C, the suspensions were centrifuged at 500 g for 5 minutes and the cell pellets fixed by an addition of freshly prepared cold fixative (methanol : glacial acetic acid 3:1). The fixative was replaced several times.
The pellets were resuspended, then centrifuged at 200 g for 10 minutes and finally resuspended in a small volume of fresh fixative. Two or three drops of the cell suspensions were dropped onto pre-cleaned microscope slides were were then allowed to air-dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and then mounted in DPX.

STAIN (for cytogenetic assays):
10% Giemsa, prepared in buffered water (pH 6.8)

NUMBER OF CELLS EVALUATED: 1000 mitotic cells in each culture, except positive control treated cultures

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): ca. 100 per each culture; only cells with 44-48 chromosomes were analysed

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;

OTHER EXAMINATIONS:
- Determination of polyploidy: yes; at the time of scoring the slides for chromosomal aberrations, slides from all dose elvels chosen for metaphase analysis were also examined for the incidence of polyploid cells. A quantitative analysis was carried out on the solvent control and the highest dose levels chosen for metaphase analysis.
- Determination of endoreplication:yes; at the time of scoring the slides for chromosomal aberrations, slides from all dose elvels chosen for metaphase analysis were also examined for the incidence of endoreduplicated cells. A quantitative analysis was carried out on the solvent control and the highest dose levels chosen for metaphase analysis.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:

Evaluation criteria:

The test substance is considered to cause a positive response if the following conditions are met:
1) Statistically significant increases (p < 0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentrations
2) The increases exceed the negative control range of this laboratory, taken at the 95% confidence limit
3) The increases are reproducible between replicate cultures
4) The increases are not associated with large changes in osmolarity of the treatment medium or extreme toxicity
5) Evidence of a dose-relationship is considered to support the conclusion

A negative response is claimed if no statisctially significant increases in the number of aberrant cells above concurrent control frequencies are observed at any dose level.

Statistics:

Fischer's test

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

METAPHASE ANALYSIS:
First experiment:
In both the absence and the presence of S9 mix, the test substance caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any dose level, when compared with the solvent control. In the absence of S9 mix, no statistically significant increase in the proportion of polyploid cells was seen at the highest dose level chosen for the metaphase analysis (p > 0.01). In the presece of S9 mix, statistically significant increases in the proportion of polyploid cells were seen at the intermediate dose level, 62.5 µg/mL (p < 0.01) and at the highese dose level, 125 µg/mL(p < 0.001) analysed. These increases both lay within the historical control range seen in this laboratory (0-3.0%).

Second experiment:
In both the absence and the presence of S9 mix, the test substance caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any dose level, when compared with the solvent control. No statistically significant increases in the proportion of polyploid cells were seen at the highest dose levels chosen for the metaphase analysis (P > 0.01). Due to the lack of reproducibility of the response seen in the first and second test in the presence of S9 mix, the substance was considered not to show evidence f polyploid-inducing activity.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
First experiment:
In the absence of S9 mix, the test substance reduced the mitotic index to 46% of the solvent control value at 125 µg/mL, and this was selected as the highest dose level for metaphase analysis. At intermediate and low dose levels of 62.5 and 42 µg/mL the mitotic indices were reduced to 69% and 96% of the solvent control value, respectively.
In the presence of S9 mix, the test substance reduced the mitotic index to 28% of the solvent control value at 125 µg/mL, and this was selected as the highest dose level for metaphase analysis. At intermediate and low dose levels of 62.5 and 31.3 µg/mL the mitotic indices were reduced to 64% and 82% of the solvent control value, respectively.

Second experiment:
In the absence of S9 mix, the test substance reduced the mitotic index to 45% of the solvent control value at 90 µg/mL, and this was selected as the highest dose level for metaphase analysis. At intermediate and low dose levels of 62.5 and 42 µg/mL the mitotic indices were reduced to 59% and 76% of the solvent control value, respectively.
In the presence of S9 mix, the test substance reduced the mitotic index to 53% of the solvent control value at 250 µg/mL, and this was selected as the highest dose level for metaphase analysis. At intermediate and low dose levels of 150 and 90 µg/mL the mitotic indices were reduced to 72% and 88% of the solvent control value, respectively.

Conclusions:

The test substance cyclopentadecanone was not clastogenic, when tested in a guideline chromosome aberration assay, both in the presence and absence of metabolic activation, when tested up to cytotoxic doses.

Executive summary:

In the GLP-compliant study performed according to OECD Guideline 473, the test substance cyclopentadecanone did not induce increased frequency of chromosomal aberrations, both with and without metabolic activation, when tested up to cytotoxic doses. The study was performed in two independent experiments, using 3 hours exposure duration, both with and without metabolic activation, in the 1st experiment, and continuous treatment in the absence of metabolic activation and 3 hours exposure duration in the presence of metabolic activation in the 2nd experiment. Cytotoxicity was determined by mitotic indices. Positive controls produced satisfactory response. Based on these results, the test substance cyclopentadecanone was not clastogenic under the conditions of the study.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 August 2016 - 18 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes

Type of assay:

other: mouse lymphoma assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in refregirator (2-8 °C)
- Stability under test conditions: stable for max. 40 minutes at 70 °C
- Solubility and stability of the test substance in the solvent/vehicle: stable in ethanol

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).
Growth medium: casic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium:
For 3 hour exposure: cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure: cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
Selective medium: selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 µg/ml trifluorothymidine (TFT) (Sigma).
Non-selective medium: non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and β-naphthoflavone-induced rat liver S9

Test concentrations with justification for top dose:

First mutagenicity test:
Without S9-mix: 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 35, 40, and 45 μg/ml exposure medium.
With S9-mix: 0.5, 1, 5, 10, 20, 35, 50, 60, 70, 80 and 90 μg/ml exposure medium.

The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.05, 0.1, 0.5, 1, 10, 20, 30 and 40 μg/ml exposure medium.
With S9-mix: 1, 10, 20, 50, 60, 70, 80 and 90 μg/ml exposure medium.

Second mutagenicity test (24 h exposure, -S9):
0.5, 1, 2.5, 5, 10, 20, 30, 35, 40, 45, 50 and 55 µg/ml exposure medium
The dose levels selected to measure mutation frequencies at the TK-locus were: 0.5, 1, 2.5, 5, 10, 20, 30 and 35 µg/ml exposure medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: test substace is insoluble in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Cell density at seeding: 8 x 10e6 cells (10e6/mL) for 3 hours treatment or 6 x 10e6 (1.25 x 10e5 cells/mL) for 24 hours treatment

DURATION
- Exposure duration: 3 hours (+ S9), 3 and 24 hours (-S9)
- Expression time (cells in growth medium): 2 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

STAIN (for cytogenetic assays): 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma), 0.5 mg/mL

NUMBER OF REPLICATIONS: two independent experiments, 5 plates/concentration, solvent controls tested in duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: For determination of the Cloning Efficiency (CE) day2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 10e5 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10e5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

NUMBER OF CELLS EVALUATED: 2000 for the test groups, 1000 for positive controls

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

ACCEPTABILITY CRITERIA:
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10^-6). At least 40% of the IMF should be reflected in the small colony MF. Furthermore, the positive control should have an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150 x 10^-6).

Evaluation criteria:

A test item is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency (MF) of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

Not used.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 12.5 to 200 µg/ml in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3-hour treatment period.
After 3 hours of treatment, in the absence of S9-mix, the relative suspension growth was 59% at the test item concentration of 25 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 50 μg/ml and above.
In the presence of S9-mix, the relative suspension growth was 49% at the test item concentration of 50 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 100 μg/ml and above.
After 24 hours of treatment, the relative suspension growth was 4% at the test item concentration of 50 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at the test item concentration of 100 μg/ml and above.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: 3 hours treatment, -S9: 894 (SD = 247); 24 hours treatment, -S9: 724 (SD = 200); +S9: 1694 (SD 793).
- Negative (solvent/vehicle) historical control data: 3 hours treatment, -S9: 83 (SD 22); 24 hours treatment, -S9: 75 (SD 24); +S9: 84 (SD 27)

EVALUATION OF MUTAGENICITY:
First experiment:
No significant increase in the mutation frequency at the TK locus was observed after treatment with Cyclopentadecanone either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the Cyclopentadecanone treated cultures were comparable to the numbers of small and large colonies of the solvent controls.
An increase above the 95% control limit was observed at the top dose of 30 µg/ml in the absence of S9-mix. Although this increase is above the 95% control limit, no dose-related response is was observed, only at the low Relative Total Growth of 17 %, and the increase in the mutation frequency (167 per 106 survivors) is was below the MF(controls) + 126 (180 per 106 survivors),. tTherefore this increase is considered to be not biologically relevant.

Second experiment:
In the presence of S9-mix, Cyclopentadecanone did not induce an increase in the mutation frequency at the TK locus.

Conclusions:

The test substance cyclopentadecanone was not mutagenic in the mouse lymphoma assay, both in the presence and absence of metabolic activation, when tested up to cytotoxic doses.

Executive summary:

In the GLP-compliant guideline study, the test substance was tested in a mouse lymphoma assay, both in the absence and presence of metabolic activation. Two independent experiments were performed, in which the cells were exposed for 3 hours in the presence and absence of metabolic activation, and for 24 hours in the absence of metabolic activation. The relative total growth of the highest test item concentrations was decreased to 34% and 11% of the concurrent solvent controls following 3 hours exposure in the presence and absence of the S9 mix, respectively, and to 16% following 24 hours exposure. None of the tested concentrations reached a mutation frequency of MF_(controls)+ 126, neither in the absence nor in the presence of S9-mix. The positive controls produced satisfactory response. Based on this, cyclopentadecanone was considered to be not mutagenic in the mouse lymphoma test system under the conditions of the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Cyclopentadecanone is not a genotoxic substance.

Additional information

In a reliable Ames test, performed according to OECD Guidelines 471 and 472, the test substance cyclopentadecanone was not mutagenic in the strains S. typhimurium TA98, TA100, TA1535, TA 1537 and TA1538 and E. coli WP2 uvrA, both with and without metabolic activation, up to the highest tested concentration of 5000 µg/plate. Cyclopentadecanone was not clastogenic, when tested in a OECD guideline 473 chromosome aberration assay, both in the presence and absence of metabolic activation, when tested up to cytotoxic doses. It was was not mutagenic in the mouse lymphoma assay, performed according to OECD guideline 490, both in the presence and absence of metabolic activation, when tested up to cytotoxic doses.

Justification for classification or non-classification

As all in vitro genotoxicity studies with cyclopentadecanone were negative, classification of the substance for genotoxicity is not warranted under Regulation (EC) 1272/2008.