6-methylhept-5-en-2-one

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

other company data

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

6-methylhept-5-en-2-one

EC Number:

203-816-7

EC Name:

6-methylhept-5-en-2-one

Cas Number:

110-93-0

Molecular formula:

C8H14O

IUPAC Name:

6-methylhept-5-en-2-one

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): 6-Methylhept-5-en-2-one (Methylheptenon)

- Test substance No.: 00/0874-1
- Physical state: colorless liquid
- Analytical purity: 99.1%
- Purity test date: December 28, 2000
- Lot/batch No.: continuous production
- Storage condition of test material: refrigerator (protected from light, N2 conditions)

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

400 mg/kg bw/day (nominal)

Doses / concentrations 3

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

Examinations

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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).