Octan-4-olide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test): the substance octan-4-olide did not induce mutagenicity in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence or absence of Aroclor-1254 induced rat liver S9 (OECD 471/GLP).

Gene mutation (mammalian cell gene mutation assay): there was no evidence of induced mutant colonies over background in mouse lymphoma L5178Y cells exposed to octan-4-olide in the presence or absence of phenobarbitone and β-naphthoflavone-induced rat liver S9 metabolic activation (OECD 490/GLP).

Chromosome aberration (mammalian cell cytogenetics assay; micronucleus test): Octan-4-olide did not induce any induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells in the presence or absence of phenobarbitone and β-naphthoflavone-induced rat liver S9 (OECD 487/GLP).

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:

August 13th 200 - September 13th 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Haarmann & Reimer GmbH; 50529270
- Purity: 99.2%

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-1254 indiuced rat liver S9

Test concentrations with justification for top dose:

Main test 1: 0, 15, 50, 150, 500, 1500, 5000 µg/plate
Main test 2: 0, 15, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene: All strains +S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for both experiments

DURATION
- Exposure duration: 48 – 72 hrs

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: background lawn reduction

Statistics:

Estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level, using a Chi squared (X2) test.

Species / strain:

other: S. typhimurium TA 1537, TA 98 and TA 102

Remarks:

Main test 1 & 2

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

other: S. typhimurium TA 1535, TA 100

Remarks:

Main test 1 & 2

Metabolic activation:

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:

not examined

Positive controls validity:

valid

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100

Remarks:

Main tests 1 & 2

Metabolic activation:

with

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:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Remarks:

Main tests 1 & 2

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test compound an the plates was not observed.

RANGE-FINDING/SCREENING STUDIES: According to an initial toxicity test, OCTALACTONE GAMMA was tested in concentrations of 15 to 5000 lag per plate in the presence and absence of S9 (data not shown).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: A historical overview of the revertant frequencies of the strains used in the Freiburger Labor für Mutagenitätsprüfung of the years 1998 to 2000 was provded for positive controls
- Negative historical control data: A historical overview of the revertant frequencies of the strains used in the Freiburger Labor für Mutagenitätsprüfung of the years 1998 to 2000 was provided for spontaneous and solvent controls.

Conclusions:

In conclusion, these results indicate that octan-4-olide, under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

Executive summary:

In a reverse gene mutation assay in bacteria (2000073), strains of S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1022 were exposed to octan-4-olide (99.2%) in DMSO at concentrations of 0, 15, 50, 150, 500, 1500, 5000 µg/plate (plate incorporation, both experiments) in the presence and absence of mammalian metabolic activation (Aroclor-1254 induced rat liver S9).

Octan-4-olide was tested up to limit concentrations (5000 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 January 2018 - 11 May 2018

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)

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Chongqing Zoteq Aroma Chemical Co. Ltd.; 170833910
- Expiration date of the lot/batch: 13 December 2019
- Purity: 99.14%

Target gene:

TK+/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Eurofins Munich stock cultures
- Doubling time: 10-12 h
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: The cells obtain a near diploid karyotype (40 ± 2 chromosomes).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 complete medium; 5% CO2/95% humidified air.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and ß-naphthoflavone-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary test (+ and -S9): 0.2, 0.5, 2.5, 5.0, 7.5 and 10 mM
Main test (4 hrs, + and -S9): 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 10 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: None

Untreated negative controls:

yes

Remarks:

RPMI cell culture medium (+ 5% Horse Serum)

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hrs (with and without S9)
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): 12 days

SELECTION AGENT (mutation assays): Trifluorothymidine (5 µg/mL)

NUMBER OF REPLICATIONS: Single cell cultures

DETERMINATION OF CYTOTOXICITY
Preliminary test: Relative Suspension Growth
Main test: Relative Total Growth

Evaluation criteria:

The test item is considered mutagenic if the following criteria are met:

- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 cells and
- dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.

According to the OECD guideline, the biological relevance is considered first for the interpretation of results.

A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.

Statistics:

Statistical significance at the 5% level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Species / strain:

mouse lymphoma L5178Y cells

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:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: The pH-value detected with the test item and the osmolality were within the physiological range.
- Precipitation: No precipitation of the test item was noted in the preliminary test or the main experiment.

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined in a pre-experiment up to a maximum concentration of 10 mM. Six concentrations [0.2, 0.5, 2.5, 5.0, 7.5 and 10 mM] were tested without and with metabolic activation. The experimental conditions in these pre-experiments were the same as described below in the paragraph experimental performance. After a 2-day growth period the relative suspension growth (RSG) of the treated cell cultures was calculated (Tables 2, 3).

HISTORICAL CONTROL DATA:
Historical data for mutant frequencies are shown in Table 12 (Appendix). All mutant frequencies for negative and positive controls were found within the historical range of the test facility Eurofins Munich (January 2011 to December 2016).

Conclusions:

In conclusion, in the described mutagenicity test under the experimental conditions reported, octan-4-olide is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Executive summary:

In a mammalian cell gene mutation assay [MLA: TK+/-; 180397], mouse lymphoma L5178Y cells cultured in vitro were exposed to octan-4-olide (99.14%) for 4 hours at concentrations of 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 10 mM in the presence and absence of phenobarbital and ß-naphthoflavone-induced rat liver S9 metabolic activation.

Octan-4-olide was tested up to limit concentrations (i.e. 10 mM).  The positive controls induced the appropriate response.  There was no evidence of induced mutant colonies over background.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 490 for in vitro mutagenicity (Mammalian Cell Gene Mutation Assay: MLA - TK+/-) data.  

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Chongqing Zoteq Aroma Chemical Co. Ltd; 170833910
- Expiration date of the lot/batch: 13 December 2019
- Purity test date: 99.14%; Decebmer 14th 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in cell culture medium, processed by ultrasonication for 1 min at 37°C and diluted within 1 hour prior to treatment.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC, CCL-93
- Suitability of cells: These cells were chosen because of their stable karyotype and their low spontaneous induction rate of micronucleus formation under standardized culture conditions. These facts were necessary for the appropriate performance of the study.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM (minimum essential medium); 5% carbon dioxide atmosphere (95% air).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Routine checking of mycoplasma infections were carried out before freezing.

Cytokinesis block (if used):

cytochalasin B.

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital / β-naphthoflavone-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity test: 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5.0, 7.5 and 10 mM

Experiment I: without and with metabolic activation: 1.0, 2.5, 5, 6, 7, 8, 9 and 10 mM

Experiment II: without metabolic activation: 0.10, 0.25, 0.50, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25 and 2.5 mM

The following concentrations were selected for the microscopic analyses of micronuclei frequencies:
Experiment I with short-term exposure (4 h): without metabolic activation: 6, 9 and 10 mM; with metabolic activation: 5, 7 and 10 mM

Experiment II with long-term exposure (24 h): without metabolic activation: 0.5, 1.0 and 1.5 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none
- Justification for choice of solvent/vehicle: The test item was dissolved in cell culture medium, processed by ultrasonication for 1 min at 37°C and diluted within 1 hour prior to treatment. The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Remarks:

cell culture medium

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

other: 0.16 and 2.0 μg/mL Colchicine -S9

Details on test system and experimental conditions:

DURATION
- Exposure duration: 4 hrs +/-S9 and 24 hrs -S9
- Expression time (cells in growth medium): 20 hrs (experiment 1); 23 hrs (experiment 2)

SPINDLE INHIBITOR (cytogenetic assays): 1.5 μg/mL cytochalasin B

STAIN (for cytogenetic assays): acridine orange

NUMBER OF REPLICATIONS: Duplicate cultures were performed at each concentration level except for the pre-experiment

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the cultivation, the complete culture medium was removed. Subsequently, cells were trypsinated and resuspended in about 9 ml complete culture medium. The cultures were transferred into tubes and incubated with hypotonic solution (0.4% KCl) for some minutes at room temperature. Prior to this an aliquot of each culture was removed to determine the cell count by a cell counter (ALSystems). After the treatment with the hypotonic solution the cells were fixed with methanol + glacial acetic acid (3+1). The cells were resuspended gently and the suspension was dropped onto clean glass slides. Consecutively, the cells were dried on a heating plate. Finally, the cells were stained with acridine orange solution.

NUMBER OF CELLS EVALUATED: All slides, including those of positive and negative controls were independently coded before microscopic analysis. For each experimental point, at least 2000 binucleated cells per concentration (1000 binucleated cells per slide) were analysed for micronuclei.


CRITERIA FOR MICRONUCLEUS IDENTIFICATION: According to the criteria of Fenech i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered.

DETERMINATION OF CYTOTOXICITY
- Method: The cytokinesis block proliferation index (CBPI) was used to calculate the cytostasis (cytostasis [%] = 100 - CBPI relative [%]). Cytostasis was used to describe cytotoxicity.

Evaluation criteria:

A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative/solvent control data (e.g. Poisson-based 95% control limits).

When all of these criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.

A test item is considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above are met.

Statistics:

Statistical significance at the 5% level (p < 0.05) was evaluated by the non-parametric χ² test. The p value was used as a limit in judging for significance levels in comparison with the concurrent
negative control.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment 1 (4 hrs)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

10 mM

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment 1 (4 hrs)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

Experiment 2 (24 hrs)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1.5 mM

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: For the maximum concentration without metabolic activation (10mM) thepH value was determined and the substance did not adversely affect the pH.
- Effects of osmolality: For the maximum concentration without metabolic activation (10mM) the osmolality (in comparison to negative control) was determined and was not significantly different.
- Precipitation: The test item was dissolved in cell culture medium (MEM medium). No precipitate of the test item was noted in any concentration evaluated in experiment I and II

RANGE-FINDING/SCREENING STUDIES:
According to the used guideline the highest recommended concentration is 10 mM. The test item was dissolved in cell culture medium. No precipitate of the test item was noted. The highest dose
group evaluated in the pre-experiment was 10 mM. The concentrations evaluated in the main experiment were based on the results obtained in the pre-experiment (Table 3).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:
The χ² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I with metabolic activation and in experiment II without metabolic activation, but in
experiment I without metabolic activation a statistical significant increase was noted (Table 14). This concentration-related increase was considered as not biologically relevant since it based on the increased micronucleus frequency observed at the highest test concentration in this experimental condition that was also considered as not biologically relevant (see discussion).

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: Duplicate cultures were set up and 1000 binucleated cells per culture were scored for micronuclei.
- Indication whether binucleate or mononucleate where appropriate: Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes (2012-2017) (Appendix 1)
- Negative (solvent/vehicle) historical control data: Yes (2012-2017) (Appendix 1)

Conclusions:

In an in vitro mammalian cell micronucleus test in Chinese hamster V79 cells, octan-4-olide did not induce structural and/or numerical chromosomal damage with or without metabolic activation.

Executive summary:

In an in vitro mammalian cell micronucleus test (180398), Chinese hamster V79 cells were exposed to octan-4-olide, (99.14%), in cell culture medium at concentrations of 0, 1.0, 2.5, 5, 6, 7, 8, 9 and 10 mM (first experiment; 4 hrs) with and without metabolic activation (phenobarbital/β-naphthoflavone-induced rat liver S9) and 0, 0.10, 0.25, 0.50, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25 and 2.5 mM (second experiment; 24 hrs) without metabolic activation.

Octan-4-olide was tested up to cytotoxic concentrations. Positive controls induced the appropriate response.  

In experiment I without metabolic activation the micronucleated cell frequency of the negative control (0.50%) was within the historical control limits of the negative control (0.37% – 1.37%. The mean values of micronucleated cells found after treatment with the test were 0.95% (6 mM), 0.60% (9 mM) and 1.85% (10 mM). The numbers of micronucleated cells found at 6 and 9 mM were within the historical control limits of the negative control did not show a biologically relevant increase compared to the concurrent negative. At the highest test item concentration of 10 mM a sudden and statistically significant increase of the micronucleus frequency above the upper historical control limit was observed. However, since there was a large variability between both cultures with culture 1 showing a micronucleus frequency within the historical control limits (1.05%) and culture 2 showing a strongly increased micronucleus frequency (2.65%) and since strong cytotoxicity was observed at this concentration (56% cytostasis), the effect was considered as inconclusive and possibly artefactual.

In experiment I with metabolic activation the micronucleated cell frequency of the negative control (0.95%) was within the historical control limits of the negative control (0.42% – 1.64%). The mean values of micronucleated cells found after treatment with the test item were 1.00% (5 mM), 1.15% (7 mM) and 1.20% (10 mM). The numbers of micronucleated cells were within the historical control limits of the negative control and did not show a biologically relevant increase compared to the concurrent negative control.

In experiment II without metabolic activation the micronucleated cell frequency of the negative control (1.30%) was within the historical control limits of the negative control (0.37% – 1.37%). The mean values of micronucleated cells found after treatment with the test item were 1.05% (0.5 mM), 0.50% (1.0 mM) and 0.85% (1.5 mM). The numbers of micronucleated cells were within the historical control limits of the negative control and did not show a biologically relevant increase compared to the concurrent negative control.

Since the increase in the micronucleus frequency observed in experiment I without metabolic activation in a single culture at a high cytotoxicity level could not be reproduced in experiment II, the effect was considered as artefactual and not biologically relevant. Therefore octan-4-olide did not induce structural and/or numerical chromosomal damage with or without metabolic activation in Chinese hamster V79 cells.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 487 (in vitro mammalian cell micronucleus test) for in vitro cytogenetic mutagenicity data.  

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test);

There is one in vitro gene mutation study (reverse gene mutation assay/Ames test in bacteria) available.

In a reverse gene mutation assay in bacteria (OECD 471/GLP), strains of S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were exposed to octan-4-olide (99.2%) in DMSO at concentrations of 0, 15, 50, 150, 500, 1500, 5000 µg/plate (plate incorporation, both experiments) in the presence and absence of mammalian metabolic activation (Aroclor-1254 induced rat liver S9). Octan-4-olide was tested up to limit concentrations (5000 µg/plate).

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

Chromosome aberration (mammalian cell cytogenetics assay; micronucleus test):

There is one in vitro mammalian cell micronucleus test available.

In an in vitro mammalian cell micronucleus test (OECD 487/GLP), Chinese hamster V79 cells were exposed to octan-4-olide, (99.14%), in cell culture medium at concentrations of 0, 1.0, 2.5, 5, 6, 7, 8, 9 and 10 mM (first experiment; 4 hrs) with and without metabolic activation (phenobarbital/β-naphthoflavone-induced rat liver S9) and 0, 0.10, 0.25, 0.50, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25 and 2.5 mM (second experiment; 24 hrs) without metabolic activation. Octan-4-olide was tested up to cytotoxic concentrations. Positive controls induced the appropriate response. In experiment I without metabolic activation the micronucleated cell frequency of the negative control (0.50%) was within the historical control limits of the negative control (0.37% – 1.37%. The mean values of micronucleated cells found after treatment with the test were 0.95% (6 mM), 0.60% (9 mM) and 1.85% (10 mM). The numbers of micronucleated cells found at 6 and 9 mM were within the historical control limits of the negative control did not show a biologically relevant increase compared to the concurrent negative. At the highest test item concentration of 10 mM a sudden and statistically significant increase of the micronucleus frequency above the upper historical control limit was observed. However, since there was a large variability between both cultures with culture 1 showing a micronucleus frequency within the historical control limits (1.05%) and culture 2 showing a strongly increased micronucleus frequency (2.65%) and since strong cytotoxicity was observed at this concentration (56% cytostasis), the effect was considered as inconclusive and possibly artefactual. In experiment I with metabolic activation the micronucleated cell frequency of the negative control (0.95%) was within the historical control limits of the negative control (0.42% – 1.64%). The mean values of micronucleated cells found after treatment with the test item were 1.00% (5 mM), 1.15% (7 mM) and 1.20% (10 mM). The numbers of micronucleated cells were within the historical control limits of the negative control and did not show a biologically relevant increase compared to the concurrent negative control. In experiment II without metabolic activation the micronucleated cell frequency of the negative control (1.30%) was within the historical control limits of the negative control (0.37% – 1.37%). The mean values of micronucleated cells found after treatment with the test item were 1.05% (0.5 mM), 0.50% (1.0 mM) and 0.85% (1.5 mM). The numbers of micronucleated cells were within the historical control limits of the negative control and did not show a biologically relevant increase compared to the concurrent negative control.

Since the increase in the micronucleus frequency observed in experiment I without metabolic activation in a single culture at a high cytotoxicity level could not be reproduced in experiment II, the effect was considered as artefactual and not biologically relevant. Therefore octan-4-olide did not induce structural and/or numerical chromosomal damage with or without metabolic activation in  Chinese hamster V79 cells.

Gene mutation (mammalian cell gene mutation assay):

There is one gene mutation (mammalian cell gene mutation assay) available.

In a mammalian cell gene mutation assay [MLA: TK+/-] (OECD 490/GLP), mouse lymphoma L5178Y cells cultured in vitro were exposed to octan-4-olide (99.14%) for 4 hours at concentrations of 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 10 mM in the presence and absence of phenobarbital and ß-naphthoflavone-induced rat liver S9 metabolic activation. Octan-4-olide was tested up to limit concentrations (i.e. 10 mM).  The positive controls induced the appropriate response.  There was no evidence of induced mutant colonies over background.

The results from these studies are acceptable to use in the human health risk assessment.

Justification for classification or non-classification

Based on the available information in the dossier, the substance octan-4-olide (CAS No. 104-50-7) does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.