L-menthan-3-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

in vitro (Ames test): negative (±S9 mix) in vitro (HPRT test): negative (±S9 mix) in vivo (Micronucleus assay): negative

Link to relevant study records

Reference

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

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

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

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

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

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

- in vitro:

The Ames test (Sokolowski 2012) was performed to investigate the potential of Menthone-L/Isomenthone-D to induce gene mutations in the plate incorporation test (Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and the Escherichia coli strain WP2uvrA: 3-5000 µg/plate) and the preincubation test (TA1535, TA1537, TA100: 1-2500 µg/plate, TA98, WPuvrA: 3-5000 µg/plate). The assay was performed in two independent experiments, both with and without liver microsomal activation. No substantial increase in revertant colony members of any of the five tester strains was observed following treatment with Menthone-L/Isomenthone-D at any dose level, neither in the presence nor absence of metabolic activation (S9mix).

The HPRT test (Wollny, 2013) was performed in order to investigate the mutagenic potential in mammalian Chinese hamster V79 cells. The assay was performed in two independent experiments with and without liver microsomal activation. The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

-in vivo:

In the study (Honarvar 2009) the potential of L-menthone to induce micronuclei in polychromatic erytrocytes (PCE) in the bone marrow of the mouse was investigated. After single oral administration of 10 mL/kg bw of test item, the bone marrow cells were collected at 24 and 48 h for micronuclei analysis. After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PECs of the vehicle control thus indicating that L-menthone did not exert any cytotoxic effects in the bone marrow.

Justification for selection of genetic toxicity endpoint
in vivo GLP study according to OECD TG 474

Justification for classification or non-classification

Based on the above stated assessment of the genotoxic potential of L-menthan-3-one the substance is considered to be non-genotoxic and classification according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU is not proposed.