6-methylhept-5-en-2-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD TG 471): negative

Gene mutation in mammalian cells (OECD TG 490): negative

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF Aktiengesellschaft Experimentelle Toxikogie und Ökologie Ludwigshafen

Type of assay:

bacterial reverse mutation assay

Target gene:

His, Trp gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

20 - 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: TA1535, TA 100, TA 1537, TA 98: 2-aminoanthracene (2.5 µg/plate); E. coli WP2 uvrA: 2-aminoanthracene (60 µg/plate)

Remarks:

with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Positive control substance:

other: TA 1535, TA 100: N-methyI-N'-nitro-N-nitrosoguanidine (5 µg/plate); TA 98: 4-nitro-o-phenylendiamine (10 µg/plate); TA 1537: 9-aminoacridine (100 µg/plate); E. coli: 4-nitroquinoline-N-oxide (5 µg/plate)

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:

1ST EXPERIMENT: in agar (plate incorporation)
(SPT)DURATION
- Exposure duration: 48 - 72 h, 37°C

2ND and 3RD EXPERIMENT: preincubation
(PIT)DURATION
- Preincubation period: 20 min, 37°C
- Exposure duration: 48 - 72 h, 37°C

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth), reduction in the titer

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.A test substance is generally considered nonmutagenic in this test if:- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other:

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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 examined

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- A weak bacteriotoxic effect (slight decrease in the number of revertants and/or slight reduction in the titer) was occasionally abserved in the standard plate test depending on the strain and test conditions from about 2,500 µg/plate onward.
- In the preincubation assay bacteriotoxicity (reduced background growth, decrease in the number of revertants, reduction in the titer) was observed depending an the strain and test conditions from about 1,000 µg - 2,000 µg/plate onward.
- Precipitation: No test substance precipitation was found.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

An increase in the number of revertants was not observed both in the  standard plate test and in the preincubation test either without S-9 mix  or after the addition of a metabolizing system.
Thus, under the experimental conditions chosen here, it is concluded that 6-Methylhept-5-en-2-one is not a mutagenic agent in a bacterialreverse mutation test.

Conclusions:

An increase in the number of revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions chosen here, it is concluded that 6-Methylhept-5-en-2-one (Methylheptenon) is not a mutagenic agent in a bacterial reverse mutation test and does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

The substance 6-Methylhept-5-en-2-one (Methylheptenon) was tested for its mutagenic potential based on the ability to induce point mutations in selected loc of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The strains tested were TA 1535, TA 100, TA 1537, TA 98 and E. coIi WP2 uvrA. These were tested in a standard plate test (SPT) and preincubation test (PIT) boths with and without metabolic activation (Aroclor-induced rat livers S-9 mix). Bacterial strains were exposed to 20 µg - 5,000 µg / plate (SPT), and 20 µg - 5,000 µg/plate (PIT), no precipitation of the test substance was found. A bacteriotoxic effect was observed under all test conditions, but no increase in the number of his+ or trp+ revertants was observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system. Thus, according to the results of the present study, the test substance methylheptenon is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen and does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09-2015 to 25-11-2015

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:

July 28, 2015

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

May 30, 2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian Cell Gene Mutation Test

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: obtained from Dr. J. Cole of the MRC Cell Mutation Unit in Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time. Large stocks of the cleansed L5178Y cell line are stored in liquid nitrogen in the cell bank of Envigo CCR allowing the repeated use of the same cell culture batch in experiments.
- Suitability of cells: The L5178Y cell line has been used successfully in in vitro experiments for many years.
- Cell cycle length, doubling time or proliferation index: L5178Y cells are characterized by a high proliferation rate (doubling time 10 - 12 h in stock cultures) and cloning efficiency of untreated cells of usually more than 50% both necessary for the appropriate performance of the study.
- Number of passages if applicable: the cells were subcultured two times prior to treatment.
- Methods for maintenance in cell culture if applicable: thawed stock cultures were propagated in plastic flasks in RPMI 1640 complete culture medium. The cell cultures were incubated at 37 ± 1.5°C in a humidified atmosphere with 4.5% carbon dioxide and 95.5% ambient air.
- Modal number of chromosomes: the cells have a stable karyotype with a near diploid (40 ± 2) chromosome number.
- Normal (negative control) cell cycle time: doubling time 10 - 12 h in stock cultures)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Prior to mutagenicity testing the amount of spontaneous mutants is reduced by growing the cells for one day in RPMI 1640-HAT medium. The incubation of the cells in HAT-medium is followed by a recovery period of 2 days in RPMI 1640 medium. After this incubation the L5178Y cells are returned to normal RPMI 1640 medium (complete culture medium). 4.5% carbon dioxide and 95.5% ambient air is used.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: [yes/no]

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

liver microsome preparations (S9 mix)

Test concentrations with justification for top dose:

Doses applied in Experiment 1 (4h with and without S9 mix): 40.2, 80.4, 160.8, 321.5, 643.0, 964.5,1286.0 µg/mL
Doses applied in Experiment 2 (24h with and without, and 4h with S9 mix): 80.4, 160.8, 321.5, 482.3, 643.0, 964.5,1286.0 µg/mL

In a pre-experiment the concentrations between 10.1 µg/mL and 1286.0 µg/mL were used.
The maximum test concentration was 1286 μg/mL, equal to approximately 10 mM, based on the molecular weight (126.2 g/mol) and the purity (98.1%) of the test substance.

The concentrations used in the first experiment were selected based on cytotoxicity observed in the pre-experiment. The concentration range of the second experiment was based on toxicity observed in the pre-experiment (without metabolic activation) and in the first experiment (with metabolic activation). In both main experiments the individual concentrations were generally spaced by a factor of 2.0. A narrower spacing was used at high concentrations to cover the cytotoxic range more closely. To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (final concentration 0.5% v/v)
- Justification for choice of solvent/vehicle: DMSO was chosen as solvent based on its solubility properties, and at this concentration it is tolerated by the cells.

Untreated negative controls:

no

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: in suspension
- Cell density at seeding (if applicable): 4×10^3 cells per microtiter plate well

DURATION
- Preincubation period: RPMI 1640-HAT medium for 1 day, followed by 2 day recovery in RPMI 1640 supplemented with Hypoxantine and Thimidine
- Exposure duration: 4h or 24 h in RPMI medium with 3% horse serum (or 15% for the 24h exposure).
- Expression time (cells in growth medium): 48h
- Selection time (if incubation with a selection agent): 10 - 15 days

SELECTION AGENT 5 μg/mL TFT in RPMI 1640 (complete culture medium)
NUMBER OF REPLICATIONS: one culture per dose level; 2 microtiter plates per culture
NUMBER OF CELLS EVALUATED: 10^7

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth of less than 50% in both parallel cultures
- Any supplementary information relevant to cytotoxicity: The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures (I.e. the relative total growth – RTG- should be greater than 10% of the concurrent selective control group).

Rationale for test conditions:

According to OECD guidance

Evaluation criteria:

Follows the criteria in OECD guidance
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10^6 cells above the corresponding solvent control.
(dose-dependent, in parallel cultures, relative total growth is more than 10% of vehicle control unless IWGT exception criteria are fulfilled)
When a test item is considered mutagenic, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

Statistics:

Linear regression (least squares) using R Script LM.Rnw statistics software. A trend was judged as significant whenever the p-value (probability value) was below 0.05. However, both, biological relevance and statistical significance were considered together.

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Phase separation occurred at 643.0 μg/mL and above in the presence and absence of metabolic activation after 4 hours treatment. After 24 hours of treatment no precipitation or phase separation occurred.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment was performed in the presence and absence of metabolic activation with a treatment time of 4 hours and in the absence of metabolic activation for 24 hours. Test substance concentrations between 10.1 μg/mL and 1286 μg/mL were used. The maximum concentration of the pre-experiment was equal to a molar concentration of approximately 10 mM, based on the molecular weight (126.2 g/mol) of the test substance and the purity (98.1%). A relevant toxic effect indicated by a relative suspension growth (RSG) below 50% was solely observed at the maximum concentration of 1286.0 μg/mL with all of the experimental parts of the pre-experiment.

HISTORICAL CONTROL DATA: Yes. The average vehicle control mutant frequency was in the acceptable range of 49 up to 157 mutant colonies per 10^6 cells. The mean of both parallel positive control treatments showed a distinct increase in induced mutant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Relevant cytotoxic effects were observed at starting at 643.0 μg/mL without metabolic activation and at 964.5 μg/ml with metabolic activation (4 hours of exposure). Cytotoxicity was noted at 482.3 μg/ml in the absence of metabolic activation (24 hours of exposure). The recommended cytotoxic range of approximately 10-20% relative total growth was covered with and without metabolic activation.
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Conclusions:

Under the conditions of the test, methylheptenone did not induce mutations in the absence and presence of metabolic activation. It was concluded that methylheptenone does not need to be classified as mutagenic according to the criteria outlined in Annex I of the CLP Regulation (EC) No. 1272/2008.

Executive summary:

The objective of this in vitro assay was to evaluate the potential of Methylheptenone (6-methyl-5-hepten-2-one) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in two independent experiments, in acoordance with OECD TG 490, using two parallel cultures each. The first experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 4 hours with and 24 hours without metabolic activation.

The maximum concentration of the test item in the pre-experiment and both main experiments was 1286 μg/mL, equal to approximately 10 mM, based on the molecular weight (126.2 g/mol) and the purity (98.1%) of the test substance. No substantial and reproducible dose dependent increases in mutant colony numbers were observed in the main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid. Under the conditions of the test, Methylheptenone did not induce mutations in the absence and presence of metabolic activation. Thus it was concluded that methylheptenone does not need to be classified as mutagenic according to the criteria outlined in Annex I of Regulation (EC) No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Cytogenicity in mammalian cells (OECD TG 474): negative

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: 5 - 8 weeks according to the information from the breeder
- Weight at study initiation: ca. 28 g
- Identification: cage cards- Housing: individually, Makrolon cages, type MI,
- Diet: ad libitum, Standardized pelleted feed (Kliba Haltungsdiät, Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum drinking water from bottles- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, olive oil Ph.Eur./DAB was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available
- Concentration of test material in vehicle: 2, 4, 8 g/100 ml- Amount of vehicle: 10 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:All test substance formulations were prepared immediately before administration.The amount of substance or volume to be administered was related to the specific weight of the individual animals on the day of the 1st administration.

Duration of treatment / exposure:

2 injections at a 24-hour interval; positive control group was treated only once

Frequency of treatment:

2 injections at a 24-hour interval; positive control group was treated only once

Post exposure period:

samples of bone marrow were taken 24 hours after the last treatment

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

Dose / conc.:

800 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- Cyclophosphamide (CPP): 20 mg/kg body weight
- Vincristine Sulphate (VCR): 0.15 mg /kg body weight
- Route of administration: intraperitoneally
- Doses: both control substances were dissolved in purified water were administered once each in a volume of 10 mI/kg bw

Tissues and cell types examined:

- bone marrow: polychromatic and normochromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In a pretest for the determination of the acute intraperitoneal toxicity death were observed at a dose of 1,000 mg/kg body weight. 800 mg/kg body weight were survived by all animals but led to evident signs of toxicity such as staggering, abdominal position, squatting posture and the general state of the animals was poor, however, showing no distinct symptomatic differences between the male and female animals. Thus, only male animals were used for the cytogenetic investigations. Therefore, a dose of 800 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 400 mg/kg and 200 mg/kg body weight were administered as further doses.

DETAILS OF SLIDE PREPARATION:
- two femora used
- bone marrow was flushed (about 2 ml/femur).
- Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained (May Grünwald solution modified = Wrights solution and Giemsa solution).

METHOD OF ANALYSIS: 2,000 polychromatic erythrocytes (PCEs) were investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) which occur are also scored. Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D =cell diameter) were assessed.

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:- A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes was observed.- The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range.A test substance is generally be considered negative in this test system if:- There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies.- The frequencies of cells containing micronuclei were within the historical control range.

Statistics:

A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used. This test was performed one-sided.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 800, 1000 mg/kg bw
- Clinical signs of toxicity in test animals: death were observed at a dose of 1000 mg/kg body weight. 800 mg/kg body weight were survived by all animals but led to evident signs of toxicity such as staggering, abdominal position, squatting posture and the general state of the animals was poor, however, showing no distinct symptomatic differences between the male and female animals.

RESULTS OF MAIN STUDY:

MORTALITY
No mortality occurred in all groups.

CLINICAL SIGNS
The administration of the test substance at 2 x 800 mg/kg bw
led to evident signs of toxicity in all treated animals
(poor general state, abdominal position, squatting posture,
staggering) which were reversible after 2 days. At the 2
lower doses only minor signs of clincal toxicity were
observed after 1 hour of administration of the test
substance (squatting posture).

EFFECT ON PCE/NCE RATIO
No inhibition of erythropoiesis, determined from the PCE/NCE ratio was detected. The vehicle and the the positive control substances, CPP and VCR, caused no evident signs of toxicity.

Mean number of PCEs and NCEs (Interval: 24 hrs)

PCEs NCEs
vehicle 10,000 2,746
200 mg/kg bw 10,000 2,348
400 mg/kg bw 10,000 2,750
800 mg/kg bw 10,000 2,627
CPP (20 mg/kg bw) 10,000 4,129
VCR (0.15 mg/kg bw) 10,000 4,212

GENOTOXIC EFFECTS
Mean number of PCEs containing MN per 1,000 PCE at 24 hrs:

vehicle: 0.3
200 mg/kg bw: 1.0
400 mg/kg bw: 0.9
800 mg/kg bw: 0.7
CPP (20 mg/kg bw): 16.0 (p < = 0.01)
VCR (0.15 mg/kg bw): 52.9 (p < = 0.01)

Conclusions:

Under the conditions of the test, methylheptenone does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo. Methylheptenone does not need to be classified as mutagenic according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

Methylheptenone was tested for clastogenicity and for the ability to induce aneugenic activity in NMRI mice using the micronucleus test method. The test substance, dissolved in olive oil, was administered twice intraperitoneally, with a 24-hour interval between administrations, to male animals at dose levels of 200 mg/kg, 400 mg/kg and 800 mg/kg body weight in a volume of 10 ml/kg body weight. Olive oil (selected vehicle) was administered to male mice as a negative control, and as positive controls: cyclophosphamide was given for clastogenic effects 20 mg/kg body weight, and vincristine for induction of aneugenic activity 0.15 mg/kg body weight. 24 h after exposure, the animals were sacrificed and bone marrow was prepared of two femora. 2000 polychromatic erythrocytes per animal were evaluated for micronuclei, normocytes were also registered. The negative control did not produce toxicity or any effects other than within the historical control range. Both positive controls, did not cause clinical signs of toxicity but led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. The 2 administration of Methylheptenone did not lead to any increase in the number of polychromatic erythrocytes containing either small or large nuclei compared to the negative control and historical control data. No inhibition of erythropoiesis was detected either. Thus, under the experimental conditions chosen here, Methylheptenone does not have any clastogenic effect, and there were no indications of aneugenic activity in bone marrow cells in vivo. Based on this result, the substance does not need to be classified as mutagenic according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Gene mutation in bacteria (in vitro)

The substance 6-Methylhept-5-en-2-one (Methylheptenone) was tested for its mutagenic potential based on the ability to induce point mutations in selected loc of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The strains tested were TA 1535, TA 100, TA 1537, TA 98 and E. coIi WP2 uvrA. These were tested in a standard plate test (SPT) and preincubation test (PIT) boths with and without metabolic activation (Aroclor-induced rat livers S-9 mix). Bacterial strains were exposed to 20 µg - 5,000 µg / plate (SPT), and 20 µg - 5,000 µg/plate (PIT), no precipitation of the test substance was found. A bacteriotoxic effect was observed under all test conditions, but no increase in the number of his+ or trp+ revertants was observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system. Thus, according to the results of the present study, the test substance methylheptenone is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen.

 

Gene mutation in mammalian cells (in vitro)

The objective of this in vitro assay was to evaluate the potential of Methylheptenone (6-methyl-5-hepten-2-one) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in two independent experiments, in acoordance with OECD TG 490, using two parallel cultures each. The first experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 4 hours with and 24 hours without metabolic activation.

The maximum concentration of the test item in the pre-experiment and both main experiments was 1286μg/mL, equal to approximately 10 mM, based on the molecular weight (126.2 g/mol) and the purity (98.1%) of the test substance. No substantial and reproducible dose dependent increases in mutant colony numbers were observed in the main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid. Under the conditions of the test, Methylheptenone did not induce mutations in the absence and presence of metabolic activation.

 

Cytogenicity in mammalian cells (in vivo)

Methylheptenone was tested for clastogenicity and for the ability to induce aneugenic activity in NMRI mice using the micronucleus test method. The test substance, dissolved in olive oil, was administered twice intraperitoneally, with a 24-hour interval between administrations, to male animals at dose levels of 200 mg/kg, 400 mg/kg and 800 mg/kg body weight in a volume of 10 ml/kg body weight. Olive oil (selected vehicle) was administered to male mice as a negative control, and as positive controls: cyclophosphamide was given for clastogenic effects 20 mg/kg body weight, and vincristine for induction of aneugenic activity 0.15 mg/kg body weight. 24 h after exposure, the animals were sacrificed and bone marrow was prepared of two femora. 2000 polychromatic erythrocytes per animal were evaluated for micronuclei, normocytes were also registered. The negative control did not produce toxicity or any effects other than within the historical control range. Both positive controls, did not cause clinical signs of toxicity but led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. The 2 administration of Methylheptenone did not lead to any increase in the number of polychromatic erythrocytes containing either small or large nuclei compared to the negative control and historical control data. No inhibition of erythropoiesis was detected either. Thus, under the experimental conditions chosen here, Methylheptenone does not have any clastogenic effect, and there were no indications of aneugenic activity in bone marrow cells in vivo.

Justification for classification or non-classification

Based on the results of the available stsudies, Methylheptenone does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).