L-menthan-3-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: Valid and conclusive guideline study under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

The relative humidity in the animal rooms ranged between 30-114 % instead of 30 and 70 %

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

L-Menthone

IUPAC Name:

L-Menthone

Constituent 2

Reference substance name:

L-menthan-3-one

EC Number:

237-926-1

EC Name:

L-menthan-3-one

Cas Number:

14073-97-3

Molecular formula:

C10H18O

IUPAC Name:

(2S,5R)-5-methyl-2-(propan-2-yl)cyclohexan-1-one

Details on test material:

- Name of test material (as cited in study report): L-menthone
- Analytical purity: 99.1 % (mixture of isomers)
- Impurities (identity and concentrations): no data

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories GmbH, 33178 Borchen, Germany
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: males mean value 35.7 g, females mean value 31.4 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: single
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 114
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: 50, 100, 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg
- Lot/batch no. (if required): no data
- Purity: no data

Details on exposure:

not applicable (gavage)

Duration of treatment / exposure:

once

Frequency of treatment:

once

Post exposure period:

24 and 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
actual ingested
nominal dose

No. of animals per sex per dose:

12

Control animals:

yes

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: oral (gavage)
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:

bonne marrow, polychromatic erythrocytes (PCE)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: the highest dose was the maximum tolerated dose of 2000 mg/kg. A total tree adequately spaced dose levels spaced by a factor of 2 were chosen.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): treatment once and sampling time 24 and 48 hours post-dose.

DETAILS OF SLIDE PREPARATION: the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 390 x g for 10 minutesand the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merk, 64293 Darmstadt, Germany)/Giemsa (Merk, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from bone marrow sample.

METHOD OF ANALYSIS: NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes.

Evaluation criteria:

The study was considered valid as the following criteria are met:
-the negative and positive controls are in the range of historical control data.
-at least 5 animals per group and sex can be evaluated.
-PCE to erythrocyte ratio should not be less than 20 % of the negative control.

Statistics:

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erytrocytes in a single dose group. A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutatgenic in this system. Nonparametric Mann-Whitney test.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
L-menthone is considered to be non-mutagenic in this micronucleous assay.

Executive summary:

The study was performed to investigate the potential of L-menthone to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was formulated in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurence of micronuclei. At least 2000 PCEs per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erytrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test item were investigated:

24 h preparation interval: 500, 1000 and 2000 mg/kg bw

48 h preparation interval: 2000 mg/kg bw

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable. In the main study 1 male died after treatment with this dose. After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that L-menthone did not exert any cytotoxic effects in the bone marrow.