Oxacycloheptadecan-2-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Oxacycloheptadecan-2-one 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 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 December 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA, Method: HG-GEne Muta - S. typhimurium: The Salmonella typhimurium revere mutation assay

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA, Method: HG-Gene Muta - E. coli: The Escherichia coli reverse utation assay, 1984

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

28 January 1985, 11 September 1989, 31 March 1987, 16 June 1987

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium: his
E. coli: trp

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity test: 5, 50, 500 and 5000 µg/plate
Mutation test 1: 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate
Mutation test 2: 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate
The top dose corresponded to the maximal solubility of the test substance in the chosen solvent.

Vehicle / solvent:

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

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: two independent experiments, each dose in each experiment tested in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in revertant colony counts or absence of a complete background bacterial lawn

Evaluation criteria:

If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S09 mix, it is considered to show evidence of mutagenic activity in this test system.
If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, the following approach is taken in order to resolve the issue of the substance's mutagenic activity in this test system:
1) Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that alrady tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies the first paragraph, the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
2) IF no clear "positive" response is obtained, the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al. (1989) and will usually be analysis of variance followed by Dunnett's test.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

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

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

S. typhimurium TA 1538

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

Key result

Species / strain:

E. coli WP2 uvr A

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:
Four concentrations of test substance were assessed for toxiciyt using the six tester strains. The highest concentration was 50 mg/mL of test substance in the chosen solvent, which provided a final concentration of 5000 µg/plate. Three 10-fold serial diluions of the highest concentration were also tested. The chosen solvent, dimethyl sulphoxide, was used as the negative control. Any toxic effects of the test substance can be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data, mean (minimum-maximum):
TA 1535:
ENNG, -S9: 351.9 (68-2849)
AA, +S9: 151.3 (44-2171)
TA1537:
9-AC, -S9: > 3500 (> 3500- > 3500)
AA, +S9: 82.4 (29-714)
TA1538:
NF, -S9: 403.2 (79-767)
AA, +S9: 174.3 (40-1725)
TA98:
NF, -S9: 277.7 (68-782)
AA, +S9: 175.3 (57-1617)
TA 100:
ENNG, -S9:
472.3 (178-2564)
AA, +S9: 443.0 (159-2498)
WP2 uvrA:
ENNG, - S9: 853.4 (373-2331)
AA, +S9: 394.5 (143-2345)

- Negative (solvent/vehicle) historical control data:
TA1535: -S9: 15.8 (8-20), +S9: 16.3 (7-21)
TA1537: -S9: 10.0 (4-16), +S9: 11.1 (2-19)
TA1538: -S9: 13.0 (7-21), +S9: 14.5 (8-23)
TA98: -S9: 24.5 (16-34), +S9: 26.9 (17-37)
TA100: -S9: 125.4 (81-168), +S9: 129.7 (80-165)
WP2 uvrA: -S9: 68.1 (43-88); +S9: 67.9 (46-88)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test substance was toxic towards all the tester strains at 5000 µg/plate. 5000 µg/plate was chosen as the top dose elvel in the first mutation test, but a total of eight dose levels were included to ensure that sufficient non-toxic concentrations were assessed.
No other toxicity except at the highest dose level was observed in the first mutation test, therefore a total of only six dose levels were chosen for the second test.

Conclusions:

The test substance oxacycloheptadecan-2-one was not mutagenic in the Ames test performed according to OECD guidelines 471 and 472, both with and without metabolic activation, when tested up to cytotoxic concentrations.

Executive summary:

In a GLP-compliant OECD Guideline 472 and 472 study, oxacycloheptadecan-2 -one was tested in S. typhimurium TA98, TA100, TA1535, TA1537 and TA1538 strains and E. coli WP2 uvrA strain both in the absence and presence of metabolic activation, up to the maximal concentration of 5000 µg/plate. Cytotoxicity was observed at the highest concentration. Two independent mutation tests were performed, and each concentration was tested in triplicate. No evidence of mutagenic activity was seen at any dose level in either mutation test. The concurrent positive controls produced satisfactory results. Therefore oxacycloheptadecan-2 -one was concluded to be non-mutagenic in bacterial cells under the conditions of the study.

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 in mammalian cells

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: human blood was collected aseptically from healthy male donors, pooled and diluted with RPMI 1640 tissue culture medium (Imperial) containing 10% foetl calf serum. Aliquots (0.4 mL blood: 4.5 mL medium : 0.1 mL phytohaemagglutinin) of the cell suspension were placed in steril universal containers and incubated at 37 °C in a slanted position for ca. 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-induced rat liver S9

Test concentrations with justification for top dose:

First test: 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL
Second test:
-S9: 15.6, 31.3, 62.5, 125, 187.5, 250 and 375 µg/mL
+S9: 31.3, 62.5, 125, 250, 375, 500 and 750 µg/mL
The top doses were chosen based on the maximal solubility of the test substance. The highest dose level of 2000 µg/mL was chosen to ensure that several dose levels were in the insoluble range.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: based on the solubility of the active substance (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 medium
- Cell density at seeding (if applicable):

DURATION
- Exposure duration: first test: 3 hours (± S9); second test: 3 hours (+S9), continuous treatment (-S9)
- Expression time (cells in growth medium): first test and second test in the absence of S9: 18 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid

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

NUMBER OF REPLICATIONS: two independent experiments, each concentration tested in duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid to each culture at a final concentration of 0.1 µg/mL. After 2 hours incubation, each cell suspension was transferred to a conical centrifuge tube and centrifuged for 5 minutes at 500 g. The cell pellets were treated with a hypotonic solution (0.075 M KCl prewarmed at 37 °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 addition of freshly prepared cold fixative (3 parts methanol :1 part glacial acetic acid). 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.

NUMBER OF CELLS EVALUATED: ca. 100 metaphase per culture

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): ca. 100

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, at the time of soring the slides for chromosomal aberrations, slides from all dose levels chosen for metaphase analysis were also examined for the incidence of polyploid and endoreduplicated cells. A quantitative analysis for polyploide was carried out on the solvent control and the highest dose level chosen for metaphase analysis.
- Determination of endoreplication: yes, at the time of soring the slides for chromosomal aberrations, slides from all dose levels chosen for metaphase analysis were also examined for the incidence of polyploid and endoreduplicated cells.

Evaluation criteria:

The test substance is considered to cause a positive resposne if the following conditions are met:
- Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentrations
- The increases exceed the negative control range of this laboratory, taken at the 95% confidence limit
- The increases are reproducible between replicate cultures
- The increases are not associated with large changes in osmolality or the treatment medium or extreme toxicity
- Evidence of a dose-relationship is considered to support the conclusion
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed at any dose levels.

Statistics:

The number of aberrant metaphase figures in each treatment group was compared with the solvent control value using Fisher's test. The number of polyploid cells in the highest dose level analysed was compared with the solvent control value using Fisher'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:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Aberrant cells, % mean (range of means):
Without gaps: -S9: 18.64 (4.92-62.50), +S9: 21.33 (4.50-61.50)
With gaps: -S9: 19.25 (5.50-62.50); +S9: 21.92 (4.50, 63.50)

- Negative (solvent/vehicle) historical control data:
Aberrant cells, % mean (range of means):
Without gaps: -S9: 1.02 (0-6.5), +S9: 0.99 (0-6.5)
With gaps: -S9: 1.17 (0-7.75); +S9: 1.19 (0; 6.75)
Polyploid cells:
-S9: 0.18 (0, 1.0)
+S9: 0.48 (0, 3.0)


ADDITIONAL INFORMATION ON CYTOTOXICITY:
First test:
In the absence of S9 mix, the test substance reduced the mitotic index to 48% of the solvent control value at 250 µg/mL and was excessively toxic at higher dose levels. This concentration was selscted as the highest dose for metaphase analysis. The intermediate and low dose levels chosen for metaphase analysis were 125 and 31.3 µg/mL, which reduced the mitotic index to 56% and 80% of the solvent control value, respectively.
In the presence of S9 mix, the test substance reduced the mitotic index to 43% of the solvent control value at 500 µg/mL and was excessively toxic at higher dose levels. This concentration was selscted as the highest dose for metaphase analysis. The intermediate and low dose levels chosen for metaphase analysis were 250 and 62.5 µg/mL, which reduced the mitotic index to 59% and 66% of the solvent control value, respectively.

Second test:
In the absence of S9 mix, the test substance reduced the mitotic index to 36% of the solvent control value at 250 µg/mL. This concentration was selscted as the highest dose for metaphase analysis. The intermediate and low dose levels chosen for metaphase analysis were 187.5 and 31.3 µg/mL, which reduced the mitotic index to 54% 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 750 µg/mL. This concentration was selscted as the highest dose for metaphase analysis. The intermediate and low dose levels chosen for metaphase analysis were 500 and 250 µg/mL, which reduced the mitotic index to 47% and 81% of the solvent control value, respectively.

METAPHASE ANALYSIS:
In both tests, in both the absence and the presence of S9 mix, the test substance cause 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 atthe highest dose levels chosen for the metaphase analysis (P > 0.01)

Conclusions:

The test substance oxacycloheptadecan-2-one did not induce increase in the number of chromosomal aberrations in a guideline study with human lymphocytes, both with and without metabolic activation, when tested up to cytotoxic concentrations.

Executive summary:

In a GLP-compliant OECD guideline 473 study, the test substance oxacycloheptadecan-2 -one was tested in human lymphocytes, both in the presence and absence of metabolic activation, in two independent experiments. In the first experiments, cells were exposed for three hours both in the presence and absence of metabolic activation; in the second experiment, continuous treatment was used in the absence of S9 mix, while three hour exposure was used in the presence of S9 mix. Approximately 100 metaphase figures were examined. Three dose levels were chosen for metaphase analysis. Cytotoxicity was observed at all selected dose levels. In both the absence and the presence of S9 mix, oxacycloheptadecan-2 -one 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 fo polyploid cells were seen at the highest dose levels chosen for the metaphase analysis (p> 0.01). Positive controls produced satisfactory response. Based on these results, oxacycloheptadecan-2 -one was considered to be not clastogenic in human lymphocytes 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 - 21 November 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:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
In refrigerator (2-8°C)
- Stability at higher temperatures: yes, maximum temperature: 40°C, maximum duration: 20 minutes

Target gene:

Thymidine kinase 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
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital-induced rat liver microsomal enzymes (S9)

Test concentrations with justification for top dose:

The test concentrations were selected based on the results of the dose-range finding study. In the dose range finding study, concentration range of 12.5-200 μg/ml was used.
1st mutagenicity test:
Without S9-mix: 0.125, 0.25, 0.5, 1, 5, 10, 20, 30, 35, 40 and 45 μg/ml exposure medium. The dose levels selected to measure mutation frequencies at the TK-locus were 0.25, 0.5, 1.5, 10, 20, 30 and 35 μg/ml.
With S9-mix: 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 μg/ml exposure medium. The dose levels selected to measure mutation frequencies at the TK-locus were 0.78, 1.56, 3,13, 6.25, 12.5, 25, 50 and 100 μg/ml.

2nd mutagenicity test:
1st experiment:
Without S9: 0.625, 1.25, 2.5, 5, 10, 15, 17.5, 20, 22.5, 25, 27.5 and 30 μg/ml. As no dose level with a cell survival below 45% was reached, the repeat experiment was performed with the following dose selection:
2nd experiment:
Without S9: 5, 10, 15, 20, 25, 27.5, 30, 32.5, 37.5, 40 and 45 μg/ml exposure medium. The dose levels selected to measure mutation frequencies at the TK-locus were 5, 10, 15, 20, 25, 27.5, 30 and 37.5 μg/ml.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: based on the results of the solubility test, the test item was insoluble in DMSO.

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: 10^6 cells/ml for 3 hour treatment, 1.25 x 10^5 cells/ml for 24 hour treatment

DURATION
- Preincubation period:
- Exposure duration: 3 hours with and without metabolic activation (experiment 1), 24 hours without metabolic activation (experiment 2)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): trifluorothymidine (TFT) medium

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 control tested in duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 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 10^5 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/well for the test groups, 1000/well for positive controls

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test item is considered positive (mutagenic) in the mutation assay if it induces a 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.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

1st experiment, 3 hours exposure

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

1st experiment, 3 hours exposure

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

2nd experiment, 24 hours exposure

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
In the solubility test, upon mixing with exposure medium, the test item precipitated at concentrations of 31.3 μg/ml and above directly after adding to the exposure medium and after 5 minutes, and precipitation was still present at concentrations of 125 μg/ml and above after 3 hour treatment.
In the dose range finding test, it showed that upon mixing with exposure medium, the test item precipitated at concentrations of 100 μg/ml and above directly after adding to the exposure medium and after 3 hours, and at concentrations of 50 μg/ml and above after 24 hours.
1st mutagenicity experiment: the test item precipitated in the exposure medium at the test item concentration of 100 μg/ml.


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. In the absence of S9-mix with 3 hours exposure, 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 with 3 hours exposure, no toxicity in the suspension growth was observed up to and including the highest test item concentration of 200 μg/ml, compared to the suspension growth of the solvent control. In the absence of S9 mix, with 24 hours exposure, the relative suspension growth was 13% 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.


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 CYTOTOXICITY:
1st mutagenicity experiment:
In the absence of S9-mix, the dose levels of 0.125 to 10 μg/ml showed no cytotoxicity. Therefore, the dose level of 0.125 μg/ml was not regarded relevant for mutation frequency measurement. The dose levels of 40 and 45 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
In the presence of S9-mix, no severe toxicity was observed and all dose levels were evaluated.
In the absence of S9-mix, the relative total growth of the highest test item concentration was 7% compared to the total growth of the solvent controls.
In the presence of S9-mix, no toxicity was observed up to and including the highest precipitating dose level.

2nd mutagenicity experiment:
The dose levels of 30 and 32.5 μg/ml showed similar cell growth delay. Therefore, the dose level of 32.5 μg/ml was not regarded relevant for mutation frequency measurement.
The dose levels of 40 and 45 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
The effect of the test item was evaluated up to the dose levels of 30 μg/ml giving a RTG value of 22%. Above this dose level the RTG was below the acceptable limit of 10%.

EVALUATION OF MUTAGENICITY:
1st mutagenicity experiment:
No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.
2nd mutagenicity experiment:
No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Conclusions:

In a GLP-compliant OECD guideline 490 study, the test substance oxacycloheptadecan-2-one 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 OECD guideline 490 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. In the first experiemtn the test item was tested up to concentrations of 35 and 100 μg/ml in the absence and presence of S9 mix, respectively. The relative total growth of the highest test item concentrations was decreased to 7% of the concurrent solvent controls following 3 hours exposure in the absence of the S9 mix. No toxicity was observed at this dose level in the presence of S9 mix. The test item precipitated in the culture medium at the dose level of 100 μg/ml. In the second experiment, the effect of the test item was evaluated up to the dose level of 30 μg/ml. At this concentration, relative total growth was 22% of the control value. Above this dose level the RTG was below the acceptable limit of 10%. No precipitation was observed up to the concentration of 30 μg/ml.

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, oxacycloheptadecanone 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

Oxacycloheptadecan-2-one is not a genotoxic substance.

Additional information

In a reliable Ames test, performed according to OECD Guidelines 471 and 472, the test substance oxacycloheptadecan-2-one 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. Oxacycloheptadecan-2-one 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 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.