Linalool

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

Study period:

3 July 2001 - 2 September 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Test was conducted according to OECD Test Guideline No. 474, 1997, under GLP Standards, and QA.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Linalool

EC Number:

201-134-4

EC Name:

Linalool

Cas Number:

78-70-6

Molecular formula:

C10H18O

IUPAC Name:

3,7-dimethylocta-1,6-dien-3-ol

Details on test material:

- Name of test material (as cited in study report): Linalool
- Physical state: liquid
- Stability under test conditions: stable
- Storage condition of test material: In refrigerator protected from light under nitrogen

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-8 weeks
- Weight at study initiation: Males: 29.2 ± 1.3 gr. to 31.6 ± 1.5 gr. ; Females: 24.0 ± 1.6 gr. to 25.2 ± 2.3 gr.
- Assigned to test groups randomly: Yes
- Fasting period before study: 3-4 hours
- Housing: Air-conditioned room; group housing of 5 animals per sex per cage in labelled polycarbonate cages containing purified sawdust as bedding material
- Diet: Ad libitum standard pelleted laboratory animal diet
- Water: Ad libitum tap-water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: Corn oil
- Concentration of test material in vehicle: 50, 100, 150 mg/mL for dose levels 500, 1000, 1500 mg/kg bw, respectively

Details on exposure:

No data

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

Once (single dose treatment)

Post exposure period:

24 or 48 hours after application of Linalool animals were sacrificed, 24 hours after dosing of the vehicle and 48 hours after dosing of the positive control.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Five male and five female mice were used per sampling time in each treatment group

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Single oral intubation
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:

Bone-marrow; micronucleated polychromatic erythrocytes and ratio polychromatic erythrocytes (PCE)/normochromatic erythrocytes (NCE)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The dose of 1500 mg/kg bw is the highest applicable one as determined by preliminary experiments (mortality and systemic toxic signs-maximum tolerated dose).

TREATMENT AND SAMPLING TIMES: Two sampling times (24 and 48hours) after oral administration of the high dose (1500 mg/kg) and one sampling time (24 hours) for the lower doses (500 and 1000 mg/kg)

DETAILS OF SLIDE PREPARATION: The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal. The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer. The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.

METHOD OF ANALYSIS: The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.

Evaluation criteria:

A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.
A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups separately.

Statistics:

Averages and standard deviations were calculated and the Wilcoxon Rank Sum Test was used; two-sided test at P < 0.05

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

lethargy and ataxia in the 1st hour after dosing in all animals of the mid and high dose group; same clinical signs in 1 male and 2 females of the low dose

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 and 1500 mg/kg bw
- Clinical signs of toxicity in test animals: Ataxia and lethargy on day 1 after treatment within 5 min, 1.5 h and 2.5 h. 1 Sacrifice at 2000 mg/kg bw for humane reasons.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of Linalool treated animals compared to the vehicle treated animals.
- Ratio of PCE/NCE (for Micronucleus assay): The animals of the groups which were treated with Linalool showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis.
- Statistical evaluation: None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups separately.

Applicant's summary and conclusion

Conclusions:

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of Linalool treated animals compared to the vehicle treated animals. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. It is therefore concluded that this test is valid and that Linalool is not mutagenic in the micronucleus test under the experimental conditions described in this report. Linalool does not need to be classified as mutagenic according to the criteria outlined in Annex I of Regulation (EC) No. 1272/2008.

Executive summary:

Linalool was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow. Four groups each comprising 5 males and 5 females, received a single oral intubation. Two groups were dosed with 1500 mg/kg body weight, one group was dosed with 1000 mg/kg body weight and one group was dosed with 500 mg/kg body weight. A vehicle treated group served as negative control, a group treated with a single oral intubation of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control. Bone marrow of the groups treated with Linalool was sampled 24 or 48 hours after dosing. Bone marrow from the negative control group was harvested at 24 hours after dosing only and bone marrow from the positive control group was harvested at 48 hours after dosing only. After dosing all animals treated with 1500 and 1000 mg/kg Linalool showed the following toxic signs: lethargy and ataxia. Of the group treated with 500 mg/kg Linalool, one male and two female animals showed lethargy and ataxia. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with Linalool. The groups that were treated with Linalool showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls. It is concluded that Linalool is not mutagenic in the micronucleus test under the experimental conditions described in this report. Linalool does not need to be classified as mutagenic according to the criteria outlined in Annex I of Regulation (EC) No. 1272/2008.