Lavender, Lavandula angustifolia, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: non mutagenic (OECD 471, GLP, K, rel. 1)

Micronucleus test in human lymphocytes: non clastogenic (OECD 487, GLP, K, rel. 1).

HPRT test in L5178Y cells: non mutagenic (OECD 476, GLP, K, rel. 1 and read-across with Lavandin oil, equivalent to OECD 476, GLP, K, rel. 2)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Ames mutagenicity test was performed according to Maron and Ames (1983).

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Lavandula angustifolia was of commercial origin, bought in the market of Thessaloniki
- The air-dried plant material (leaves only) was cut in small pieces, and the essential oil was isolated after hydrodistillation for 2 h.

Target gene:

No data

Species / strain / cell type:

S. typhimurium, other: TA97, TA98, TA100, and TA102

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not specified

Test concentrations with justification for top dose:

250, 500, 1000, and 2000 ppm

Vehicle / solvent:

No data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

No data

Evaluation criteria:

No data

Statistics:

No data

Key result

Species / strain:

S. typhimurium, other: TA97, TA98, TA100, and TA102

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No mutagenic activity observed at any concentration tested.

None

Conclusions:

Under the test condition, test substance is not mutagenic in S. typhimurium strains (TA97, TA98, TA100, and TA102).

Executive summary:

In a reverse gene mutation assay in bacteria, strains of Salmonella typhimurium (TA97, TA98, TA100, and TA102) was exposed to test substance (250, 500, 1000, and 2000 ppm).

No significant increases in the number of revertant colonies were detected in any S. typhimurium strains.

 

Under the test conditions, test substance is not considered as mutagenic in this bacterial system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01-22 August 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test guideline No. 471 without deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Research Institute for Fragrance Materials, Inc. / 130050
- Physical state: Clear colorless liquid
- Date of manufacture: 26 March 2014
- Date of receipt: 09 July 2014
- Expiration date of the lot/batch: 27 September 2015
- Purity test date: 30 June 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature, protected from light
- Stability: Test substance was considered stable through 27 September 2015

Target gene:

Histidine and tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10 % S9: S9-mix from the livers of male Sprague-Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Initial toxicity-mutation assay: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (TA 98, TA 100, TA 1535 and WP2uvrA) with and without metabolic activation
Retest of the initial toxicity-mutation assay: 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (TA 1537) with and without metabolic activation
Confirmatory mutagenicity assay: 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (TA 98, TA 100, TA 1535, TA 1537 and WP2uvrA) with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under yellow light.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: methyl methanesulfonate

Remarks:

Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

TEST SYSTEM: Salmonella tester strains (TA98, TA100, TA1535 and TA1537) were derived from Dr.Bruce Ames’ cultures; E. coli tester strains (WP2 uvrA) were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-72 h at 37 ± 2 °C

NUMBER OF REPLICATIONS:
Duplicate plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification.

- OTHER:
- Solubility Test: A solubility test was conducted using sterile water and DMSO to determine the vehicle, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration up to 50 mg/mL for aqueous solvents and up to 500 mg/mL for organic solvents.
- Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity.
- Sterility of the test substance and the vehicle, all test substance dose levels and the vehicle used in the initial toxicity-mutation and confirmatory mutagenicity assays were plated on selective agar with an aliquot volume equal to that used in the assay. These plates were incubated under the same conditions as the assay.

Evaluation criteria:

The following criteria must be met for the initial toxicity-mutation and the confirmatory mutagenicity assays to be considered valid.
All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.
All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2 uvrA, 10 - 60.
To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x10^9 cells/mL.
The mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control.
A minimum of four non-toxic dose levels is required to evaluate assay data.
A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn (background code 3, 4 or 5).

Statistics:

None

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
-Solubility: DMSO was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test.
- Precipitation: No precipitate was observed.

MUTATION ASSAY
Initial Toxicity-Mutation Assay: In Experiment B1 (Initial Toxicity-Mutation Assay), the maximum dose tested was 5000 μg/plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate. No positive mutagenic responses were observed with tester strains TA98, TA100, TA1535 or WP2 uvrA in either the presence or absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 1500 or at 5000 μg/plate with all Salmonella tester strains. Due to confluent bacterial growth, tester strain TA1537 was not evaluated for mutagenicity but was retested in Experiment B2 based on the precipitate and toxicity profile observed (i.e., toxicity at 5000 μg/plate and no precipitate). Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the retest and confirmatory mutagenicity assays was 5000 μg/plate.
In Experiment B2 (Retest of the Initial Toxicity-Mutation Assay), no positive mutagenic responses were observed with tester strain TA1537 in either the presence or absence of S9 activation. The dose levels tested were 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate. No precipitate was observed. Toxicity was observed at 5000 μg/plate.
Confirmatory Mutagenicity Assay: In Experiment B3 (Confirmatory Mutagenicity Assay), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate. No precipitate was observed. Toxicity was observed beginning at 1500 or at 5000 μg/plate with all Salmonella tester strains.

OTHERS:
Sterility results: No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

None

Conclusions:

Under the test condition, test substance is not mutagenic with and without metabolic activation in S.typhimurium strains (TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E.coli strain WP2 uvrA were exposed to test substance both in the presence and absence of metabolic activation system (10% liver S9-mix) using the plate incorporation method. The first phase, the initial toxicity- mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance.

 

Initial toxicity-mutation assay: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (TA 98, TA 100, TA 1535 and WP2uvrA) with and without metabolic activation

Retest of the initial toxicity-mutation assay: 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (TA 1537) with and without metabolic activation

Confirmatory mutagenicity assay: 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (TA 98, TA 100, TA 1535, TA 1537 and WP2uvrA) with and without metabolic activation

Vehicle (DMSO) and positive control groups were also included in mutagenicity assay.

 

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg/plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate. No positive mutagenic responses were observed with tester strains TA98, TA100, TA1535 or WP2uvrA in either the presence or absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 1500 or at 5000 μg/plate with all Salmonella tester strains. Due to confluent bacterial growth, tester strain TA1537 was not evaluated for mutagenicity but was retested based on the precipitate and toxicity profile observed (i.e., toxicity at 5000 μg/plate and no precipitate).

Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the retest and confirmatory mutagenicity assays was 5000 μg/plate.

 

In the retest of the initial toxicity-mutation assay, no positive mutagenic responses were observed with tester strain TA1537 in either the presence or absence of S9 activation. The dose levels tested were 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate. No precipitate was observed. Toxicity was observed at 5000 μg/plate.

 

In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate. No precipitate was observed. Toxicity was observed beginning at 1500 or at 5000 μg/plate with all Salmonella tester strains.

 

Under the test condition, test substance is not mutagenic with and without metabolic activation in S.typhimurium strains (TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP)..

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

purity of test item; strains lacking, no. of bacterial cells per culture, individual/mean plate count and positive controls, positive control data not reported. Only plate incorporation method without pre-incubation

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Janousec, Muggia, Trieste, Italy / JL055000
- Lavender oil predominantly contained Linalyl acetate (43.1%), Linalool (32.7%), Caryophyllene (4.9%) and Terpinen-4-ol (3.1%), 2-Octanone/myrcene (2.4%), Trans-ocimene (1.5%), A-Terpineol (1.0%), Borneol (0.8%), B-Farnesene (0.8%), 1,8-Cineole (0.6%), Camphor (0.5%), Caryophyllene oxide (0.5%), A-Humulene (0.4%), Limonene (0.3%)

Target gene:

histidine- or tryptophan

Species / strain / cell type:

S. typhimurium, other: TA 98 and TA 100

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10 % S9: S9-mix from the liver of rats induced with phenobarbital/5,6-benzoflavone

Test concentrations with justification for top dose:

No data

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

methylmethanesulfonate

Remarks:

Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

Details on test system and conditions
TEST SYSTEM: S. typhimurium strain TA98, TA100 and E. coli WP2 uvrA strain were provided by the Research Toxicological Centre (Pomezia, Rome, Italy).

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72 h at 37 °C

NUMBER OF REPLICATIONS:
Triplicate plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: In the preliminary experiment, toxicity was assessed either as a reduction in the number of revertant colonies and as a change in the auxotrophic background growth (background lawn). To ensure that cytotoxicity did not interfere with inhibitory responses, in all subsequent assays, the upper limit of the test-concentration range was either the highest non-toxic concentration or the lowest toxic concentration determined in the preliminary toxicity test.

OTHERS:
The experiments were repeated at least twice and each concentration was determined in triplicate.

Evaluation criteria:

A positive response in the main test was defined as an increase (at least two-fold above the control), in histidine- or tryptophan-independent revertant colonies in every strain, with or without metabolic activation.

Statistics:

All values are expressed as mean ± SE. An analysis of variance (ANOVA), followed by Holm-Sidak method when appropriate, was used to verify the significance of a positive response. p Values less than or equal to 0.05 were considered to indicate statistical significance.

Key result

Species / strain:

bacteria, other: S.typhimurium TA98, TA100 and E.coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TOXICITY:
Lavender oil was toxic at 2.78 mg/plate in TA 98 strain with or without metabolic activation. Lavender oil at the highest concentration tested (2.5 mg/plate) induced no toxicity on E. coli WP2 uvrA strain.

MUTAGENICITY:
No mutagenicity was observed either with or without metabolic activation in any of the strains.

None

Conclusions:

Under the test condition, test substance is not mutagenic with and without metabolic activation in S.typhimurium strains (TA98 and TA100) and E.coli WP2 uvrA.

Executive summary:

In a reverse gene mutation assay , S. typhimurium strains TA98 and TA100 and E.coli strain WP2 uvrA were exposed to test substance both in the presence and absence of metabolic activation system (10% liver S9-mix) using the plate incorporation method. Vehicle (DMSO) and positive control groups were also included in mutagenicity assay.

 

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

Test substance was toxic at 2.78 mg/plate in TA 98 strain with or without metabolic activation. No toxicity was observed at up to 2.5 mg/plate in E. coli WP2 uvrA. No mutagenicity was observed either with or without metabolic activation in any of the strains.

 

Under the test conditions, test substance is not considered as mutagenic in these bacterial systems.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 September to 12 November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test guideline No. 487. Read-across substance

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Research Institute for Fragrance Materials, Inc. / 130050
- Physical state: Clear colorless liquid
- Date of manufacture: 26 March 2014
- Date of receipt: 09 July 2014
- Expiration date of the lot/batch: 27 September 2015
- Purity test date: 30 June 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature, protected from light
- Stability: Test substance was considered stable through 27 September 2015

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Peripheral blood lymphocytes were obtained from a healthy non-smoking 26-year-old adult female on 30 September 2014 for the preliminary toxicity assay and from another 27-year-old adult female on 13 October 2014 for the micronucleus assay.
- The donors had no recent history of radiotherapy, viral infection or the administration of drugs, and who had abstained from alcohol for at least 12 hours prior to blood donation.

MEDIA USED
- Peripheral blood lymphocytes were cultured in complete medium (RPMI-1640 containing 15% fetal bovine serum, 2 mM L-glutamine, 100 units penicillin, 100 μg/mL streptomycin) by adding 0.5 mL heparinized blood to a centrifuge tube containing 5 mL of complete medium with 2% phytohemagglutinin. The cultures were incubated under standard conditions (37 ± 1 °C in a humidified atmosphere of 5 ± 1% CO2 in air) for 44-48 hours.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B (cytoB) was used at 6 μg/mL concentration to block cytokinesis.

Metabolic activation:

with and without

Metabolic activation system:

2 % S9: Aroclor 1254-induced rat liver S9 was used as the metabolic activation system

Test concentrations with justification for top dose:

Preliminary Toxicity Test
4 hours exposure with 20 hours recovery time: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/mL, with and without metabolic activation
24 hours exposure without recovery: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/mL, with and without metabolic activation

Main test:
4 hours exposure with 20 hours recovery time: 10, 25, 50, 60, 70, 85, 100, 125 and 150 μg/mL, without metabolic activation
4 hours exposure with 20 hours recovery time: 50, 100, 150, 225, 250, 275, 300, 325, 350, 375, 400 and 450 μg/mL, with metabolic activation
24 hours exposure without recovery: 10, 25, 50, 60, 70, 85, 100 and 125 μg/mL, without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under yellow light.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Vinblastine (5, 7.5, and 10 ng/mL) was used as positive control for aneugenicity.

Details on test system and experimental conditions:

SOLUBILITY TEST:
A solubility test was conducted to determine the vehicle. The test was conducted using sterile water and DMSO to determine the vehicle, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration up to 50 mg/mL for aqueous solvents and up to 500 mg/mL for organic solvents.

METHOD OF APPLICATION: Peripheral blood lymphocytes were cultured in complete medium (RPMI-1640 containing 15% fetal bovine serum, 2 mM L-glutamine, 100 units penicillin, 100 μg/mL streptomycin) by adding 0.5 mL heparinized blood to a centrifuge tube containing 5 mL of complete medium with 2% phytohemagglutinin. The cultures were incubated under standard conditions (37 ± 1 °C in a humidified atmosphere of 5 ± 1% CO2 in air) for 44-48 hours.

DURATION
- Exposure duration: In the preliminary toxicity and the micronucleus assays, Human peripheral blood lymphocytes cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system.
- Fixation time (start of exposure up to fixation or harvest of cells): All cells were harvested 24 hours after treatment initiation.

CYTOKINESIS:
After the 4 hour treatment in the non-activated and the S9-activated studies, the cells were incubated with complete medium containing cytoB at 6.0 μg/mL. For the 24 hour treatment in the non-activated study, cytoB (6.0 μg/mL) was added at the beginning of the treatment.

NUMBER OF REPLICATIONS:
Preliminary toxicity test: Single culture/dose
Main test: Duplicate cultures/dose

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Cells were collected after being exposed to cyto B for 24 hours (± 30 minutes), 1.5 to 2 normal cell cycles, to ensure identification and selective analysis of micronucleus frequency in cells that have completed one mitosis evidenced by binucleated cells (Fenech and Morley, 1986). The cyto B exposure time for the 4 hour treatment in the non-activated and the S9-activated studies was 20 hours (± 30 minutes). Cells were collected by centrifugation, swollen with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 25:1 v/v), capped and may be stored overnight or longer at 2-8 °C. To prepare slides, the cells were collected by centrifugation and the cells were resuspended in fresh fixative. The suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with acridine orange for evaluation.

STAIN (for cytogenetic assays): Acridine orange staining

NUMBER OF CELLS EVALUATED:
Cell Cycle Kinetics Scoring (Preliminary Toxicity Test and Definitive Assay): For the preliminary toxicity test, at least 500 cells were evaluated to determine the CBPI at each dose level and the control. For the micronucleus assay, at least 1,000 cells (500 cells per culture) were evaluated to determine the CBPI at each dose level and the control.
Micronucleus Scoring (Definitive Assay):
A minimum of 2000 binucleated cells from each concentration (1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Micronuclei in a binucleated cell (MN-BN) were recorded if they meet the following criteria:
- the micronucleus should have the same staining characteristics as the main nucleus.
- the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges).
- the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus.

DETERMINATION OF CYTOTOXICITY
CBPI = (1X Mononucleated cells + 2 x Binucleated cells + 3 x Multinucleated cells)/Total number of cells scored
% Cytostasis (cytotoxicity) = 100 -100 {(CBPIt-1) /(CBPIc-1)}
t = test substance treatment culture
c = vehicle control culture

Evaluation criteria:

The test substance would be considered positive if it induced a statistically significant and dose-dependent increase the frequency of MN-BN cells (p ≤ 0.05). If only one criterion was met (statistically significant OR dose-dependent increase), the result was considered equivocal. If neither criterion was met, the results were considered to be negative.

Statistics:

Statistical analysis of the percentage of micronucleated cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent micronucleated cells of each treatment group with that of the vehicle control. Due to negative results, Cochran-Armitage test was not required to measure dose-responsiveness.

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: DMSO was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.
- Effects of pH: The pH of the highest concentration of test substance in treatment medium was 7.5.
- Effects of osmolality: The osmolality in treatment medium of the highest dose level tested, 5000 μg/mL, was 345 mmol/kg. The osmolality in treatment medium of the lowest precipitating dose level, 500 μg/mL, was 427 mmol/kg. The osmolality in treatment medium of the highest soluble dose level, 150 μg/mL, was 428 mmol/kg. The osmolality of the vehicle (DMSO) in the treatment medium was 434 mmol/kg. The osmolality of the test substance dose levels in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 20%.
- Precipitation: Yes

PRELIMINARY TOXICITY TEST
The test substance was soluble in DMSO at all concentrations tested. Visible precipitate was observed in treatment medium at dose levels ≥ 500 μg/mL, while dose levels ≤ 150 μg/mL were soluble in treatment medium at the beginning of the treatment period. At the conclusion of the treatment period, visible precipitate was observed in treatment medium at dose levels ≥ 1500 μg/mL, while dose levels ≤ 500 μg/mL were soluble in treatment medium in all treatment conditions. Also at the conclusion of the treatment period, hemolysis was observed at dose levels ≥ 500 μg/mL in the non-activated 4-hour exposure group, and at dose levels ≥ 1500 μg/mL in the S9-activated 4-hour and the non-activated 24-hour exposure groups.
Substantial cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels ≥ 150 μg/mL in the non-activated 4 and 24-hour exposure groups, and at dose levels ≥ 500 μg/mL in the S9-activated 4-hour exposure group.

MAIN TEST
In the micronucleus assay, the test substance was soluble in DMSO at all concentrations tested. Visible precipitate was observed in treatment medium at dose levels ≥ 225 μg/mL, while dose levels ≤ 150 μg/mL were soluble in treatment medium at the beginning of the treatment period. At the conclusion of the treatment period, in the S9-activated 4-hour exposure group, visible precipitate was observed in treatment medium at dose levels ≥ 225 μg/mL, while dose levels ≤ 150 μg/mL were soluble in treatment medium. In the non-activated 4 and 24-hour exposure groups, all dose levels were soluble in the treatment medium at the conclusion of the treatment period. The pH of the highest concentration of test substance in treatment medium was 7.5.
4 hours exposure with 20 hours recovery time, without metabolic activation: The dose levels selected for analysis of micronucleus were 50, 100, and 150 μg/mL. At the highest test concentration, 150 μg/mL, cytotoxicity was 53% relative to the vehicle control. The percentage of cells with micronuclei in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of micronucleated cells in the MMC (positive control) group (3.2%) was statistically significant (p ≤ 0.01, Fisher's Exact test).
4 hours exposure with 20 hours recovery time, with metabolic activation: The dose levels selected for analysis of micronucleus were 50, 150, and 225 μg/mL. At the highest test concentration, 225 μg/mL, cytotoxicity was 5% relative to the vehicle control. The percentage of cells with micronuclei in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of micronucleated cells in the CP (positive control) group (1.3%) was statistically significant (p ≤ 0.01, Fisher's Exact test).
24 hours exposure without recovery, without metabolic activation: The dose levels selected for analysis of micronucleus were 25, 50, and 70 μg/mL. At the highest test concentration, 70 μg/mL, cytotoxicity was 58% relative to the vehicle control. The percentage of cells with micronuclei in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of micronucleated cells in the VB (positive control) group (2.2%) was statistically significant (p ≤ 0.01, Fisher's Exact test).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation
- Positive historical control data: Mitomycin C (range: 1.2-9.0%, mean: 4.120 ± 1.797%); Cyclophosphamide (range: 0.8-2.9%, mean: 1.496 ± 0.420%)
- Negative (solvent/vehicle) historical control data: without metabolic activation (range: 0.1-1.6%, mean: 0.364 ± 0.254%); with metabolic activation (range: 0.0-1.5%, mean: 0.345 ± 0.249%)

None

Conclusions:

Under the test conditions, Lavender Oil was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.

Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured Human peripheral blood lymphocytes were exposed to test substance in the presence and absence of a metabolic activation system.

 

In the preliminary toxicity and the micronucleus assays, HPBL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation.

 

Preliminary Toxicity Test

4 hours exposure with 20 hours recovery time: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/mL, with and without metabolic activation

24 hours exposure without recovery: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/mL, with and without metabolic activation

 

Main test:

4 hours exposure with 20 hours recovery time: 10, 25, 50, 60, 70, 85, 100, 125 and 150 μg/mL, without metabolic activation

4 hours exposure with 20 hours recovery time: 50, 100, 150, 225, 250, 275, 300, 325, 350, 375, 400 and 450 μg/mL, with metabolic activation

24 hours exposure without recovery: 10, 25, 50, 60, 70, 85, 100 and 125 μg/mL, without metabolic activation

 

Dimethyl sulfoxide (DMSO) was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

 

In the preliminary toxicity assay, substantial cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels ≥ 150 μg/mL in the non-activated 4 and 24-hour exposure groups, and at dose levels ≥ 500 μg/mL in the S9-activated 4-hour exposure group. Based on these findings, the doses chosen for the micronucleus assay ranged from 10 to 150 μg/mL for the non-activated 4-hour exposure group, from 50 to 450 μg/mL for the S9-activated 4-hour exposure group, and from 10 to 125 μg/mL for the non-activated 24-hour exposure group.

 

In the micronucleus assay, substantial cytotoxicity was observed at 150 μg/mL in the non-activated 4-hour exposure group, at dose levels ≥ 275 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 70 μg/mL in the non-activated 24-hour exposure group. The highest dose analyzed under each treatment condition either exceeded the limit of solubility in treatment medium at the conclusion of the treatment period or produced 50 to 60% reduction in CBPI, which met the dose limit as recommended by testing guidelines for this assay. A minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei.

 

The percentage of cells with micronucleated binucleated cells in the test substance-treated groups was not statistically significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher’s Exact test). The results for the positive and negative controls indicate that all criteria for a valid assay were met.

Under the test conditions, Lavender Oil was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See read-across justification in section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration tested (1250 μg/mL), compared to the concurrent vehicle controls.
- Precipitation: Yes
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 39.06 to 1250 μg/mL (a precipitating concentration based on data previously generated in a solubility assessment). Upon addition of the test item to the cultures and following the 3 hour treatment incubation period, precipitate was observed at 1250 μg/mL in the absence and presence of S-9. The highest concentrations to give >10% relative survival (RS) were 78.13 μg/mL in the absence of S-9 and 312.5 μg/mL in the presence of S-9, which gave 60% and 96% RS, respectively

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g
. 95%)
The historical control ranges for the last 20 experiments performed in this laboratory are as follows:
- Negative (solvent/vehicle) historical control data:
Vehicle Controls
In the absence of S-9
Mean: 4.52 mutants per 10^6 viable cells, Range* = 0.80 to 8.24 mutants per 10^6 viable cells.
In the presence of S-9
Mean: 5.26 mutants per 10^6 viable cells, Range* = 1.30 to 9.22 mutants per 10^6 viable cells.
*Range = Mean ± 2 x SD.
- Positive historical control data:
NQO 0.15 μg/mL in the absence of S-9:
Mean: 43.04 mutants per 10^6 viable cells, Range* = 1.08 to 84.99 mutants per 10^6 viable cells.
NQO 0.2 μg/mL in the absence of S-9:
Mean: 56.23 mutants per 10^6 viable cells, Range* = 7.85 to 104.62 mutants per 10^6 viable cells.
B[a]P 2 μg/mL in the presence of S-9:
Mean: 28.32 mutants per 10^6 viable cells, Range* = 0 to 58.85 mutants per 10^6 viable cells.
B[a]P 3 μg/mL in the presence of S-9:
Mean: 42.66 mutants per 10^6 viable cells, Range* = 8.06 to 77.25 mutants per 10^6 viable cells.
*Range = Mean ± 2 x SD.

MUTATION EXPERIMENT
- In the Mutation Experiment twelve concentrations, ranging from 40 to 200 μg/mL, in the absence of S-9 and ranging from 50 to 500 μg/mL in the presence of S-9, were tested. Upon addition of the test item to the cultures, precipitate was observed at 220 μg/mL and above in the presence of S-9. However, no post-treatment precipitation was observed in either the absence or presence of S-9. Seven days after treatment, the highest seven concentrations in the absence of S-9 (130 to 200 μg/mL) and the highest four concentrations in the presence of S-9 (300 to 500 μg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition intermediate concentrations of 220 and 260 μg/mL in the presence of S-9 were not selected due to either excessive heterogeneity/marked toxicity. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 120 μg/mL in the absence of S-9 and 280 μg/mL in the presence of S-9, which gave 10% and 13% RS, respectively.
- When tested up to toxic concentrations, no statistically significant increases in MF were observed following treatment with test item at any concentration analysed in the absence and presence of S-9 and there were no statistically significant linear trends, indicating a clear negative result.

Table 7.6.1/1: Range-finder experiment - 3 h treatment in the absence and presence of S-9

 

Concentration (μg/mL)	%RS (Percent Relative Survival)
	3 hour treatment –S-9	3 hour treatment +S-9
0	100	100
39.06	94	99
78.13	60	75
156.3	3	87
312.5	0	96
625	0	0
1250 P, PP	0	0

P Precipitation noted at time of treatment

PP Precipitation noted at end of treatment incubation period

 

Table 7.6.1/2: Mutation experiment - 3 h treatment in the absence and presence of S-9

 

3 hour treatment –S-9			3 hour treatment +S-9
Concentration (μg/mL)	%RS (Percent Relative Survival)	MF §	Concentration (μg/mL)	%RS (Percent Relative Survival)	MF §
0	100	8.78	0	100	6.01
40	103	8.46 NS	50	106	6.37 NS
60	85	10.81 NS	100	104	4.90 NS
80	80	7.02 NS	150	92	5.67 NS
100	78	3.26 NS	200	70	8.09 NS
120	10	4.38 NS	240 P	25	6.81 NS
			280 P	13	9.03 NS
NQO 0.15	37	56.79	B[a]P 2	30	61.14
NQO 0.2	36	50.38	B[a]P 3	5	71.35

Linear trend: Not Significant (negative trend) – 3 hour absence of S-9

Linear trend: Not Significant – 3 hour presence of S-9

Conclusions:

Under the test conditions, test item did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of toxicity, in the absence and presence of a rat liver metabolic activation system. Therefore the registered substance is also not considered as mutagenic in L5178Y mouse lymphoma cells.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, L5178Y tk+/-(3.7.2C) mouse lymphoma cells were exposed to test item for 3 h at the following concentrations:

Range-Finder Experiment: 39.06, 78.13, 156.3, 312.5, 625 and 1250 μg/mL, with and without S9 mix

Mutation Experiment:

Without S9: 40, 60, 80, 100, 120, 130, 140, 150, 160, 170, 180 and 200 μg/mL

With S9: 50, 100, 150, 200, 220, 240, 260, 280, 300, 350, 400 and 500 μg/mL

Vehicle and positive control groups were also included in each mutagenicity test. Metabolic activation system used in this test was 2 % S9 mix (final concentration). S9 fraction was prepared from liver homogenates of rats treated with Aroclor 1254.

 

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 39.06 to 1250 μg/mL (a precipitating concentration based on data previously generated in a solubility assessment). Post-treatment precipitation was observed at 1250 μg/mL in the absence and presence of S-9. The highest concentrations to give>10% relative survival (RS) were 78.13 μg/mL in the absence of S-9 and 312.5 μg/mL in the presence of S-9, which gave 60% and 96% relative survival (RS), respectively.

 

In the Mutation Experiment twelve concentrations, ranging from 40 to 200 μg/mL, in the absence of S-9 and ranging from 50 to 500 μg/mL in the presence of S-9, were tested. No post-treatment precipitation was observed. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 120 μg/mL in the absence of S-9 and 280 μg/mL in the presence of S-9, which gave 10% and 13% RS, respectively.

 

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mean mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

 

When tested up to toxic concentrations, no statistically significant increases in MF were observed following treatment with test item at any concentration analysed in the absence and presence of S-9 and there were no statistically significant linear trends, indicating a clear negative result.

 

Under the test conditions, test item did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of toxicity, in the absence and presence of a rat liver metabolic activation system. Therefore the registered substance is also not considered as mutagenic in L5178Y mouse lymphoma cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD test guideline No 471 with Lavender oil. No significant increases in the frequency of revertant colonies were recorded for

any of the bacterial strains, with any dose, either in the presence or absence of metabolic activation.

Lavender oil does not induce gene mutations in bacteria under the test conditions whereas the positive control chemical (with and without metabolic activation) induced significant increase of colonies.

A Bacterial Reverse mutation Assay (Ames test) was performed, on Salmonella typhimurium (TA97, TA98, TA100 and TA 102) with Lavender oil. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose (250, 500, 1000 and 2000 ppm), either in the presence or absence of metabolic activation

In a reverse gene mutation assay , S. typhimurium strains TA98 and TA100 and E.coli strain WP2 uvrA were exposed to test substance both in the presence and absence of metabolic activation system (10% liver S9-mix) using the plate incorporation method. Vehicle (DMSO) and positive control groups were also included in mutagenicity assay. No mutagenicity was observed either with or without metabolic activation in any of the strains. Under the test conditions, Lavender oil is not considered as mutagenic in these bacterial systems.

In anin vitromicronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured Human peripheral blood lymphocytes were exposed to Lavender oil in the presence and absence of a metabolic activation system.

 In the preliminary toxicity and the micronucleus assays, HPBL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation.

 Dimethyl sulfoxide (DMSO) was used as the vehicle based on the solubility of the test substance and compatibility with the target cells.

In the preliminary toxicity assay, substantial cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels ≥ 150 μg/mL in the non-activated 4 and 24-hour exposure groups, and at dose levels ≥ 500 μg/mL in the S9-activated 4-hour exposure group. Based on these findings, the doses chosen for the micronucleus assay ranged from 10 to 150 μg/mL for the non-activated 4-hour exposure group, from 50 to 450 μg/mL for the S9-activated 4-hour exposure group, and from 10 to 125 μg/mL for the non-activated 24-hour exposure group.

In the micronucleus assay, substantial cytotoxicity was observed at 150 μg/mL in the non-activated 4-hour exposure group, at dose levels ≥ 275 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 70 μg/mL in the non-activated 24-hour exposure group. The highest dose analyzed under each treatment condition either exceeded the limit of solubility in treatment medium at the conclusion of the treatment period or produced 50 to 60% reduction in CBPI, which met the dose limit as recommended by testing guidelines for this assay. A minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei.

The percentage of cells with micronucleated binucleated cells in the test substance-treated groups was not statistically significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher’s Exact test). The results for the positive and negative controls indicate that all criteria for a valid assay were met.

Under the test conditions, Lavender Oil was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in thein vitromammalian micronucleus test using human peripheral blood lymphocytes.Therefore, the registered substance Lavandin oil is not considered as clastogenic in mammalian cells.

In anin vitromammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, L5178Y tk+/-(3.7.2C) mouse lymphoma cells were exposed to test item for 3 h at the following concentrations:

Range-Finder Experiment: 39.06, 78.13, 156.3, 312.5, 625 and 1250 μg/mL, with and without S9 mix

Mutation Experiment:

Without S9: 40, 60, 80, 100, 120, 130, 140, 150, 160, 170, 180 and 200 μg/mL

With S9: 50, 100, 150, 200, 220, 240, 260, 280, 300, 350, 400 and 500 μg/mL

Vehicle and positive control groups were also included in each mutagenicity test. Metabolic activation system used in this test was 2 % S9 mix (final concentration). S9 fraction was prepared from liver homogenates of rats treated with Aroclor 1254.

In the range-finding experiment, post-treatment precipitation was observed at 1250μg/mL in the absence and presence of S-9. The highest concentrations to give>10% relative survival (RS) were 78.13μg/mL in the absence of S-9 and 312.5μg/mL in the presence of S-9, which gave 60% and 96% relative survival (RS), respectively.

In the Mutation Experiment twelve concentrations, ranging from 40 to 200μg/mL, in the absence of S-9 and ranging from 50 to 500μg/mL in the presence of S-9, were tested. No post-treatment precipitation was observed. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 120μg/mL in the absence of S-9 and 280μg/mL in the presence of S-9, which gave 10% and 13% RS, respectively.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mean mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

When tested up to toxic concentrations, no statistically significant increases in MF were observed following treatment with test item at any concentration analysed in the absence and presence of S-9 and there were no statistically significant linear trends, indicating a clear negative result.

Under the test conditions, Lavandin oil did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of toxicity, in the absence and presence of a rat liver metabolic activation system.

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No.1272/2008.

Self-classification:

Based on the available information, no classification is proposed.