Linalool

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

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

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): B276
- Physical state: liquid
- Lot/batch No.: confidential

Method

Target gene:

Thymidine kinase (TK) gene

Species / strain

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

Controls

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

Results and discussion

Test results

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.

Any other information on results incl. tables

Not relevant

Applicant's summary and conclusion

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.