6-methyloct-5-en-2-one

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo 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

IUPAC Name:

6-methylhept-5-en-2-one

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 (e.g. ad libitum): ad libitum, Standardized pelleted feed (Kliba Haltungsdiät, Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): 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

ANALYISIS OF FEED, WATER AND BEDDING
- The feed used in the study was assayed for chemical and microbial contaminants.
- The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and the Department of Water Chemnistry and the Technical Services of BASF Aktiengesellschaft as well as for microorganisms by a contract laboratory.
- The bedding is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).

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 (if gavage or dermal): 10 ml/kg bw
- Purity: type Ph.Eur./DAB

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

Post exposure period:

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

Doses / concentrations

Remarks:

Doses / Concentrations:
200, 400 and 800 mg/kg bw
Basis:
nominal conc.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Cyclophosphamide (CPP): 20 mg CPP (Endaxan®, ASTIA MVEDICA, Reg. Nr. E 432-1 )/kg body weight for clastogenic effects
- Vincristine Sulphate (VCR): 0,15 mg VCR (SIGMA - V 8879)/kg body weight for aneugenic effects
- Route of administration: intraperitoneally
- Doses: both control substances were dissolved in purified water were administered to male animals once each in a volume of 10 mI/kg bw

Examinations

Tissues and cell types examined:

- bone marrow: polychromatic and normo chromatic 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:
The bone marrow was prepared according the method described by Schmid (1976) and Salamone (1980):
- The two femora were prepared by dissection and removing all soft tissues.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/femur).
- The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µl fresh FCS.
- 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette.
Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.

The slides were stained in eosin and methylene blue solution for 5 minutes (May Grünwald solution modified = Wrights solution), rinsed in purified water and then placed in fresh purified water for 2 or 3 minutes.
They were finally stained in 7.5% Giemsa solution for 15 minutes.
After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.


METHOD OF ANALYSIS:
In general, 2,000 polychromatic erythrocytes (PCEs) from each of the male animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) which occur are also scored. The following parameters are recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a ohromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
- Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the target.
Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.
- Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D =cell diameter)
The size of micronuclei may indicate the possible mode of action of the test substance, i.e. a clastogenic or a spindle poison effect.
Slides were coded before microscopic analysis.
Since the absolute values shown have been rounded off but the calculations were made
using the unedited values, deviations in the given relative values can occur.

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:

The statistical evaluation was carried out using the program system MUKERN (BASF AG):
The number of micronuclei in polychromatic erythrocytes was analyzed.
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.
If the results of this test were significant, labels (* for p:5 0.05, ** for p •5 0.01) were printed with the group means in the tables.
This test was performed one-sided.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Vehicle controls validity:

valid

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 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.

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)

STATISTICAL EVALUATION
The administration of the test substance did not lead to any
statistically significant increase in the number of
polychromatic erythrocytes containing either small or large
micronuclei. The rate of micronuclei was nearly the range of
the concurrent negative control in all dose groups and
within the range of the historical control data.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the experimental conditions chosen, the test substance
did not have a 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.