4-Cyclopentadecen-1-one, (4Z)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to 5000 µg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.

Link to relevant study records

Reference

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 September 09 to 29, 2015

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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Program (inspected on June 17, 2015 / Signed on September 24, 2015)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Storage conditions of test material: 11-25 °C, dry, keep container closed.

Target gene:

Histidine and tryptophan gene for Salmonella typhimurium and Escherichia coli, respectively.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: other: TA 100, TA1535 and E. Coli WP2 sensitive to agents inducing base pair mutation. TA1537, TA1538 and TA98 sensitive to agents inducing frame-shift mutations.

Metabolic activation:

with and without

Metabolic activation system:

10% S9-mix: S9 from the livers of male rats treated with phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day by oral route, for 3 days prior to preparation on day 4).

Test concentrations with justification for top dose:

Experiment 1 (Plate Incorporation Method):
1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix. The maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 2 (Pre-Incubation Method):
- Salmonella strains TA100 and TA98 and Escherichia coli strain WP2uvrA (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate. The maximum concentration was 5000 μg/plate (the maximum recommended dose level).
- Salmonella strains TA1535 and TA1537 (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate. The test item was cytotoxic in Salmonella strains TA1535 and TA1537 at and above 1500 μg/plate in Experiment 1.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.
- Preparation of test materials: The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (8.8%) of the test item. All formulations were used within four hours of preparation and were assumed to be stable for this period. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.

Untreated negative controls:

yes

Remarks:

untreated: spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9-mix

Untreated negative controls:

yes

Remarks:

untreated: spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: The bacteria used in the test were obtained from the University of California, Berkeley, and from the British Industrial Biological Research Association.

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes with shaking
- Exposure duration: approximately 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

NUMBER OF REPLICATIONS: Triplicate plates per dose level in experiment 1 and experiment 2.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning.

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using an automated colony counting system. Several manual counts were required due to bubbles in the base agar slightly distorting the actual plate count.

Rationale for test conditions:

Experiment 1 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 2 - test item dose levels were selected in Experiment 2 (main test) in order to achieve both four non-toxic dose levels and the toxic limit of the test item.
- Salmonella strains TA100 and TA98 and Escherichia coli strain WP2uvrA (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate. The maximum concentration was 5000 μg/plate (the maximum recommended dose level).
- Salmonella strains TA1535 and TA1537 (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate. The test item was cytotoxic in Salmonella strains TA1535 and TA1537 at and above 1500 μg/plate in Experiment 1.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- Fold increases greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

Key result

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:

cytotoxicity

Remarks:

Toxicity to strains TA1535 and TA1537 at and above 1500 μg/plate and from 500 μg/plate in Experiments 1 and 2, respectively, with and without S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration.
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
In both experiments 1 and 2, there were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).
In Experiment 2, small, statistically significant increases in revertant colony frequency were observed in the second mutation test at 5000 μg/plate (TA98) and 0.5 μg/plate (TA1537) in the absence of S9-mix only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility.

COMPARISON WITH HISTORICAL CONTROL DATA:
The individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.9 times the concurrent vehicle controls.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test item was cytotoxic in Salmonella strains TA1535 and TA1537 at and above 1500 μg/plate and from 500 μg/plate in Experiments 1 and 2, respectively, in both the presence and absence of metabolic activation (S9-mix). No toxicity was noted to Salmonella strains TA100 and TA98 and Escherichia coli strain WP2uvrA.

OTHERS:
- The test material formulation, amino acid supplemented top agar and S9-mix used in this experiment were shown to be sterile.

Remarks on result:

other:

Remarks:

In both Experiment 1 and 2

 Table 7.6.1/2. Mutagenic and cytotoxic effect of the test material.

Strain	S9-mix	Test concentration range (µg/plate)	Lowest mutagenic concentration (µg/plate)	Lowest cytotoxic concentration (µg/plate)
TA100	-	1.5 - 5000	None	None
	+	1.5 - 5000	None	None
TA1535	-	1.5 - 5000	None	1500
	+	1.5 - 5000	None	1500
WP2uvrA	-	1.5 - 5000	None	None
	+	1.5 - 5000	None	None
TA98	-	1.5 - 5000	None	None
	+	1.5 - 5000	None	None
TA1537	-	1.5 - 5000	None	1500
	+	1.5 - 5000	None	1500

Conclusions:

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item diluted in DMSO both in the presence and absence of metabolic activation system(10% liver S9 in standard co-factors) using the Ames plate incorporation and pre‑incubation methods in Experiment 1 and Experiment 2, respectively.

Experiment 1 (Plate Incorporation Method):

1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix

Experiment 2 (Pre-Incubation Method):

- Salmonella strains TA100 and TA98 and Escherichia coli strain WP2uvrA (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate.

- Salmonella strains TA1535 and TA1537 (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.

 

Vehicle (dimethyl sulphoxide) and positive control groups were also included in mutagenicity tests.

 

The test item was cytotoxic in Salmonella strains TA1535 and TA1537 at and above 1500 μg/plate and from 500 μg/plate in Experiments 1 and 2, respectively, in both the presence and absence of metabolic activation (S9-mix). No toxicity was noted to Salmonella strains TA100 and TA98 and Escherichia coli strain WP2uvrA.

 

In both experiments 1 and 2, there were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

In Experiment 2, small, statistically significant increases in revertant colony frequency were observed in the second mutation test at 5000 μg/plate (TA98) and 0.5 μg/plate (TA1537) in the absence of S9-mix only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.9 times the concurrent vehicle controls.

 

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.

 

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD test guideline No 471 and in compliance with GLP with the substance (Harlan, 2016, rel. 1). S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E.coli strain WP2 uvrA were exposed the test material diluted in DMSO both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors). Based on the results of experiment 1 (plate incorporation method), the test material was tested up to the maximum recommended dose level of 5000 µg/plate for Salmonella strains TA98 and TA100 and for Escherichia strain WP2uvrA, and up to 1500 µg/plate for Salmonella strains TA1535 and TA1537 in the Experiment 2 (pre-incubation method).

In both experiments 1 and 2, there were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix). In Experiment 2, small, statistically significant increases in revertant colony frequency were observed at 5000 μg/plate (TA98) and 0.5 μg/plate (TA1537) in the absence of S9-mix only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.9 times the concurrent vehicle controls.

Therefore the test material does not induce gene mutations in bacteria under the test conditions whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies.

The test material is therefore considered as non-mutagenic according to the Ames test.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.