Linalool

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 5, 1993 - November 16, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Test was conducted comparable to OECD Test Guideline No. 476, under GLP Standards, and QA.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK) gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: culture medium: RPMT 1640 medium; treatment medium: Fischer's medium
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
- Periodically "cleansed" against high spontaneous background: Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 (from Aroclor 1254 treated male Sprague Dawley rats)

Test concentrations with justification for top dose:

Without metabolic activation: 1) 12.5, 25, 50, 100, 200, 250 µg/ml; 2) 25, 50, 100, 145, 200, 224, 274 µg/ml
With metabolic activation: 1) 12.5, 25, 50, 100, 200, 250 µg/ml; 2) 25, 50, 100, 145, 200, 224 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

media (unexposed) (± S9 mix)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (± S9 mix)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: S9: Methyl methanesulfonate; +S9: 3-Methylcholanthrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days
- Fixation time (start of exposure up to fixation or harvest of cells): 10 - 14 days

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT) ( Thymidine analog)

NUMBER OF REPLICATIONS: One culture per dose level; 3 disks per culture

NUMBER OF CELLS EVALUATED: 10*6

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth: Reduction in cell growth relative to the concurrent negative control cultures

Evaluation criteria:

The minimum criterion considered necessary to demonstrate mutagenicity for any given treatment is a mutant frequency that is equal or greater 2 times the concurrent background mutant frequency. The background mutant frequency is defined as the average mutant frequency of the negative control cultures.
To evaluate a test material as a mutagen the following test results must be obtained for either activation or nonactivation conditions:
- A dose-related or toxicity-related increase in mutant frequency should be observed.
- A mutagenic response in one mutation assay should be confirmed in the second mutation assay.
- If the mutant frequency obtained for a single dose at or near the highest testable toxicity is about four times the concurrent background mutant frequency or greater, the trial is considered mutagenic. However, for the test article to be evaluated as positive, the increase must be repeatable in the second trial.
- For some test materials, the correlation between toxicity and applied concentration is poor. Therefore either parameter, applied concentrations or toxicity (percent relative growth), is used to establish whether the increase in mutant frequency is related to an increase in effective treatment.
A test article is evaluated as nonmutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to toxicity causing ten to twenty percent relative growth or in the case of relatively nontoxic materials, a range of applied concentrations extending to the maximum of 5 mg/ml.

Statistics:

No data

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=125 µg/mL based on the range finding experiment; concentations >=200 µg/mL were toxic in the main experiment in the presence and absence of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The test material was tested in the preliminary rangefinding cytotoxicity assay both with and without S9 metabolic activation. The cytotoxicity assays were initiated with treatments from 1.95 µg/mL to 998 µg/mL. With and without metabolic activation, total cellkilling was observed at 499 µg/mL. The 250 µg/mL treatments were highly toxic under both testing conditions (10.8 % and 11.8 % relative survivals). Lower concentrations induced moderate to no toxicity. These results were used to select dose levels for the mutation assays.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes. The average vehicle control mutant frequency was in the acceptable range in all trials. The positive control treatments induced mutant frequencies that were greatly in excess of the background and met assay acceptance criteria.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- S9 Mutation assays: in Trial 1 the six analyzed treatments (12.5 to 250 µg/mL) induced moderate to no toxicity (236.8% to 30.2% relative growth).
In Trial 2 the six analyzed treatments (25.0 to 224 µg/mL) induced relative growths that ranged from 80.2% to 23.4%; > 274 µg/mL was lethal.
+S9 Mutation assays: in Trial 1 the six analyzed treatments (12.5 to 250 µg/mL) induced a wide range of toxicities (134.9% to 10.8% relative growths). In Trial 2 the five analyzed treatments (25.0 to 200 µg/mL) induced a wide range of toxicities (143.5% to 17.6% relative growth); >= 224 µg/mL was lethal.

Not relevant

Conclusions:

Negative with and without metabolic activation

Linalool was not mutagenic with and without metabolic activation in the mouse lymphoma forward mutation assay under the conditions of testing. It was concluded that linalool does not need to be classified as mutagenic according to Annex I of Regulation (EC) No. 1272/2008.

Executive summary:

The objective of this in vitro assay was to evaluate the ability of Linalool to induce forward mutations at the thymidine kinase (TK) locus in the mouse lymphoma L5178Y cell line, using Trifluorothymidine (TFT) as selection agent. The test material was soluble in Dimethylsulfoxide (DMSO) at approximately 100 mg/mL. In the preliminary cytotoxicity assays, cells were exposed to the test material for four hours in the presence and absence of rat liver S9 metabolic activation. The test material produced dose-related toxicity, starting at 200 µg/mL with and without activation. The test material was soluble in mediums up to approximately 200 µg/mL and the pH was maintained at or above 7.0.

 

Under non activation conditions, eight treatments from 12.5 µg/mL to 500 µg/mL were initiated in the first trial and the 350 µg/mL and 500 µg/mL doses were terminated because of excessive toxicity. The remaining six treatments were nontoxic to moderately toxic. In the next nonactivation assay, ten treatments from 25.0 µg/mL to 399 µg/mL were initiated and seven treatments from 25.0 µg/mL to 274 µg/mL were cloned for mutant analysis. One treatment induced less than 10 % relative growth and the remaining six treatments induced moderate to high toxicities. Relative growths ranged from 80.2% to 23.4%. No acceptable treatment in either trial induced a mutant frequency that exceeded the minimum criterion for a positive response. The test material was considered negative without activation in this assay.

 

In the presence of metabolic activation, small increases at 200 and 250 µg/mL were observed in Trial I that just exceeded the minimum criterion for a positive response, but the increases in the mutant frequencies were not observed in Trial 2. In the first activation assay, eight treatments from 12.5 µg/mL to 500 µg/mL were initiated and six of the eight treatments were cloned for mutant analysis. A wide range of toxic actions was induced. The increases occurred at insoluble dose levels. In addition, both positive treatments were very toxic the day after treatment and were not split back. In the second activation assay, ten treatments from 25.0 µg/mL to 399 µg/mL were initiated and treatments above 250 µg/mL were terminated because of excessive toxicity. One treatment at 224 µg/mL had a percent relative growth that was less than 10 %. The remaining six treatments induced a good range of toxic action. None of the acceptable treatments induced a mutant frequency that exceeded the minimum criterion for a positive response. Because of the lack of a repeatable response, Linalool was considered negative with activation in this assay.

 

Linalool was evaluated as negative with and without metabolic activation in the L5178Y mouse lymphoma forward mutation assay under the conditions used in this assay. It was concluded that linalool does not need to be classified as mutagenic according to Annex I of Regulation (EC) No. 1272/2008.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2, 1983 - March 24, 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Test was conducted similar to OECD Test Guideline No. 473, under early GLP Standards, no report summary, acceptable basic data.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

only 100 cells per concentration scored

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not relevant

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

No applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (no further data)

Test concentrations with justification for top dose:

Without S9-mix: a) 16.7, 50.0, and 167.0 nL/mL; b) 100, 150, 200, 250, and 300 nL/mL
With S9-mix: a) 16.7, 50.0, and 167.0 nL/mL; b) 150, 200, 250, 300, and 400 nL/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

untreated (medium)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Mitomycin C (direct-acting mutagen) and Cyclophosphamide (promutagen)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION: No data

SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 100

DETERMINATION OF CYTOTOXICITY
- Method: % reduction in confluence; suppression of mitosis. Whenever possible the top dose at which results were available was chosen.

Evaluation criteria:

No data

Statistics:

No data

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic at 300 nl/ml (-S9 mix) and at 400 nl/ml (+S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: At 1.67 µL/mL and higher concentrations there were visible globules of compound in cultures, but at 0.5 µL/mL and higher concentration an initial cloudy precipitate cleared rapidly so that the compound was apparently in solution. At 0.5, 1.67, and 5.0 µL/mL survival ranged from severely reduced to complete lethality.

COMPARISON WITH HISTORICAL CONTROL DATA: The aberration levels in the negative and solvent controls were normal for this laboratory. The frequencies of breaks were within the normal historical control level for this laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test was repeated in an attempt to obtain results in a narrow dose range at toxic levels of B105. Without S9 mix the doses tested were a closely-spaced series from 100 to 400 nL/mL. At 400 nL/mL and 350 nL/mL, there was severe toxicity with many dead cells and no dividing cells so that the highest dose at which results were available was 300 nL/mL. A this dose there were very few dividing cells although confluence was reduced by only about 25-30%. Mitosis was also suppressed at doses of 200 nL/mL or more.
With S9 mix the dose range tested was from 100-500 nL/mL. There was complete lethality at 500 nl/ml, and at 400 nL/mL confluence was reduced by about 70% compared with the negative and solvent controls and mitosis was greatly suppressed. Confluence was reduced also at 200-300 nl/mL but there was no marked suppression of mitosis. Little toxicity was apparent at 100 and 150 nL/mL.

Not relevant

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

With and without metabolic activation there was no significant increase in aberrations, and no evidence for a dose relation. Linalool is therefore considered negative in the chromosome aberration test under the conditions of these assays. It was concluded that linalool does not need to be classified as mutagenic according to Annex I of Regulation (EC) No. 1272/2008.

Executive summary:

Linalool (B105) was assessed for its ability to induce chromosomal aberrations in cultured CHO cells in vitro. Without metabolic activation doses between 16.7 nL/mL and 300 nL/mL were tested. In presence of a metabolic activation mix doses between 16.7 nL/mL and 400 nL/mL were tested. Linalool was toxic at 300 nL/mL (-S9 mix) and at 400 nL/mL (+S9 mix). The sensitivity of the test system and the activity of the metabolic activation were demonstrated by using the direct acting mutagen Mitomycin C and the promutagen Cyclophosphamide as positive controls. Both substances increased significantly the rate of structural chromosome aberrations.

Exposure of the CHO cells to Linalool with and without metabolic activation did not result in statistically significant increases of the rate of structural chromosome aberrations, and there was no evidence for a dose relation. Linalool is therefore considered negative in the chromosome aberration test under the conditions of these assays. It was concluded that linalool does not need to be classified as mutagenic according to Annex I of Regulation (EC) No. 1272/2008.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2, 1983 - June 8, 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Test was conducted similar to OECD Test Guideline No. 471, under early GLP Standards and QA, no report summary, no second experiment using pre-incubation method was performed. Acceptable basic data.

Principles of method if other than guideline:

Test was conducted similar to OECD Test Guideline No. 471, under early GLP Standards and QA, no report summary, no second experiment using pre-incubation method was performed. Acceptable basic data.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

His-gene: Amino acid histidine

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, and TA 1538

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: all strains: rfa, ¿uvrB; TA 98, TA 100: pKM101

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 induced by Aroclor 1254

Test concentrations with justification for top dose:

Plate incorporation assay: 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, and 10 µL/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Remarks:

absence of untreated test strain cultures

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: TA1535 and TA100 (- S9): Sodium Azide; TA98 and TA 1538 (- S9): 2-Nitrofluorene; TA 1537 (-S9): 9-Aminoacridine; all strains (+ S9): 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Selection time: Approx. 2 days

SELECTION AGENT: Overlay agar containing Histidine

NUMBER OF REPLICATIONS: 3 plates per concentration

NUMBER OF CELLS EVALUATED: Not applicable

DETERMINATION OF CYTOTOXICITY
- Method: In a preliminary toxicity assay determination of reduction in the revertant colony number, appearance of microcolonies, and/or observation of thinning or absence of the background lawn. Each test substance dose (14 doses of 0.02 to 150 µL/plate), as well as the appropriate solvent control, was evaluated in strain TA100 in the standard plate incorporation assay. In TA 100 the test material was completely toxic to revertant colonies at 9.38 µL/plate.

Evaluation criteria:

The criteria used to determine positive effects were based primarily on a historical data base. Most data sets were evaluated using the following criteria:
If the solvent control value is within the normal range, a test material producing a positive response equal to three times the solvent control value is considered mutagenic for strains TA 1535, TA 1537 and TA 1538, and equal to twice the solvent control value for strains TA 98 and TA 100.
The following ranges of revertants for solvent controls are generally considered acceptable:
TA 1535: 8-30; TA 1537: 4-30; TA 1538: 10-35; TA 98: 20-75; TA 100: 80-250.

Statistics:

Mean values and standard deviation (SD).

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic to revertant colonies at 10 µL/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic to revertant colonies at 10 µL/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic to revertant colonies at 10 µL/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic to revertant colonies at 10 µL/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic to revertant colonies at 10 µL/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Not relevant

Not relevant

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

The test material, B105, did not exhibit genetic activity in any of the assays conducted for this evaluation and was considered not mutagenic under these test conditions. It was concluded that 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:

The test compound was examined for mutagenic activity in a series of in vitro microbial plate incorporation assays employing five Salmonella typhimurium strains (TA1535, TA 1537, TA1538, TA98, and TA100). The compound was tested directly and in presence of liver microsomal enzyme preparations from Aroclor-induced rats. A negative control consisting of the solvent (DMSO) used for preparing the stock solution and subsequent dilutions of the test material, and specific positive compounds were assayed concurrently with the test material.

In a preliminary toxicity study conducted at 14 doses of 0.02 µL to150.00 µL per plate using strain TA 100, the test material was completely toxic to revertant colonies at 9.38 µL/plate. The mutagenicity assays were conducted at 7 doses of 0.005 µL to 10.00 µL per plate.

The results of the test conducted on B105 in the absence and presence of a rat liver metabolic activation system were negative. The test material, B105, did not exhibit genetic activity in any of the assays conducted for this evaluation and was considered not mutagenic under these test conditions. It was concluded that linalool does not need to be classified as mutagenic according to the criteria outlined in Annex I of Regulation (EC) No. 1272/2008.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

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

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.

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

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

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.

Dose / conc.:

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

Dose / conc.:

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

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

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

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

For this endpoint 4 tests (3 in vitro and 1 in vivo test), performed according/similar to OECD test guidelines and under GLP conditions, were selected as key studies:

- A Bacterial Reverse Mutation Assays (Ames) performed similar to OECD 471. The test (plate-incorporation) in the absence and presence of a rat liver metabolic activation system was negative. Linalool did not exhibit genetic activity in any of the assays conducted and was considered not mutagenic. (William and Degraff, 1983)

- Linalool was assessed for its ability to induce chromosomal aberrations in cultured CHO cells in vitro, with and without metabolic activation. The test was performed similar to OECD 473. Exposure of the CHO cells to Linalool with and without metabolic activation did not result in statistically significant increases of the rate of structural chromosome aberrations, and there was no evidence for a dose relation. Linalool is therefore considered negative in the in vitro chromosome aberration test. (Galloway, 1983)

- In an in vitro Mammalian cell gene mutation assay, conducted similar to OECD 476, Linalool was evaluated for the ability to induce forward mutations at the thymidine kinase (TK) locus in the mouse lymphoma L5178Y cell line, using Trifluorothymidine (TFT) as selection agent. None of the acceptable treatments induced a mutant frequency that exceeded the minimum criterion for a positive response. Linalool was evaluated as nonmutagenic with and without metabolic activation in the mouse lymphoma forward mutation assay. (Cifone, 1994)

- Linalool was tested in the Micronucleus test in mice, according to OECD 474, to evaluate its genotoxic effect on erythrocytes in bone marrow of mice. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with Linalool. It is concluded that Linalool is not mutagenic in this micronucleus test. (Meerts, 2001)

 

The following three in vitro tests are considered supporting studies:

- Bacterial Reverse Mutation Assay (Ames test). Linalool was tested in the pre-incubation assay according to the method of Ames B. N., McCann J. & Yamasaki E. (1975), using 6 Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, TA92, and TA94, with and without metabolic activation. At the maximum, highest dose of 1.0 mg/plate the result was negative. (Ishidate et al., 1984)

- In vitro Mammalian Chromosome Aberration Test, according to the method of Ishidate M., Jr & Odashima S. (1977). Linalool was tested directly without metabolic activation at three different doses with a maximum dose of 0.25 mg/ml at exposure times of 24 and 48 hours in V79 cells. The incidence of polyploid cells at 48 hours after treatment was 3%. The incidence of cells with structural chromosome aberrations at 48 hours after treatment was 1.0%. This was comparable to the negative control. The results of the test conducted on Linalool in the absence of a metabolic activation system were therefore considered negative at all three concentrations tested in this assay. (Ishidate et al., 1984)

- In vitro Mammalian Cell Gene Mutation Test (similar to OECD 476). The ability of Linalool to induce forward mutations was evaluated at the thymidine kinase (TK) locus in the mouse lymphoma L5178Y cell line, using Trifluorothymidine (TFT) and 5-bromo-2-deoxyuridine (BrdU) as selection agents. Under non activation conditions no repeatable increases in the mutant frequency were induced. In the presence of metabolic activation, small, but repeatable, increases in the mutant frequency were induced. This positive response in presence of S9 is however considered equivocal as it may be related to the cytotoxicity induced. (Cifone, 1982)

Short description of key information:
In vitro
- Gene mutation in bacteria (Bacterial Reverse Mutation Assay/Ames)
not mutagenic (OECD 471).
- Cytogenicity test (Mammalian Chromosome Aberration)
not clastogenic (OECD 473).
- Mammalian cell gene mutation assay (Mouse Lymphoma Forward Mutation)
not mutagenic (OECD 476)
In vivo
- Cytogenicity test (Mammalian Erythrocyte Micronucleus Test)
not mutagenic (OECD 474).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In the 4 key studies and 3 supporting studies Linalool did not show any genotoxic potential. Therefore, it can be concluded that the substance is not mutagenic and therefore does not need to be classified for mutagenicity according to the criteria outlined in Regulation (EC) No 1272/2008 (CLP/EU-GHS), as amended for fifteenth time in Regulation (EU) No 2020/1182.