Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames (OECD TG 471): negative

- Mouse lymphoma assay (similar to OECD TG 476): negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1994
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA1535, TA97, TA98, TA100, and TA102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/B-naphthoflavone induced rat liver S-9 mix.
Test concentrations with justification for top dose:
Justification top dose: Slight toxic effects (weak reduction in the background growth) was observed at the top concentration of 5000 µg/plate in the preliminary test.

Concentrations main study:
Experiment 1: All strains (with and without metabolic activation) 0, 50, 166, 500, 1666, and 5000 µg/plate
Experiment 2: All strains (with and without metabolic activation) 0, 50, 166, 500, 1666, and 5000 µg/plate
Experiment 3: TA1535 / TA98 / TA100 / TA102 (without metabolic activation) 0, 0.5, 1.6, 5, 16.6, and 50 µg/plate and TA98 / TA100 (with metabolic acitvation) 0, 5, 16.6, 50, 166, and 500 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The compound was soluble in dimethylsulfoxide (DMSO).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene (all strains; 4 µg/plate) / ICR191 (TA97; 1µg/plate)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1 in agar (plate incorporation) and experiment 2 and 3 preincubation.
- Cell density at seeding: 1.0 - 2.7 x 10^8 plated per plate.

DURATION
Plate incorporation (experiment 1):
- Exposure duration: 48 hours

Preincubation experiments (2 and 3):
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 plates per concentration and two plates for positive controls.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (reduction in background lawns)


Evaluation criteria:
The study director was responsible for the ultimate decision in the evaluation of the results.
Key result
Species / strain:
S. typhimurium, other: TA1535, TA98, TA97, TA100 and TA102
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:
RANGE-FINDING/SCREENING STUDIES:
A preliminary test was performed using strain TA100 and 0, 1, 3, 10 , 33, 100, 333, 1000, 3333, and 5000 µg/plate test substance. Slight toxic effects (weak reduction in the background growth) was observed at the top concentration of 5000 µg/plate in the preliminary test.

HISTORICAL CONTROL DATA
The mutant frequencies of the controls were in the range of the historical controls and the data published in the literature (Maron and Ames, 1983; Levin et at., 1982a, 1982b). The positive controls induced significant increases in the mutant frequencies verifying the sensitivity of the strains used. For TA100 the spontaneous rates were at the low range. Nevertheless the experiments were considered acceptable, since the positive controls verified the responsiveness of the cultures used. Neither in the standard plate incorporation nor in the preincubation assay any increase in the mutant frequency was observed for the five tester strains after treatment with the test substance

ADDITIONAL INFORMATION ON CYTOTOXICITY:
For the main experiments the concentration range 50 to 5000 µg/plate, the generally recommended highest test concentration for non toxic compounds, was selected. Upon addition to the aqueous medium, increasingly milky suspensions were observed at 166 (standard assay) and 50 µg/plate preincubation assay). In addition an oily precipitate was noticed starting at 1666 and 500 µg/plate for the standard and preincubation assay, respectively. Using the standard plate incorporation assay no toxicity was observed. Using the preincubation modification, known to be more sensitive for several class of compounds, toxic effects were pronounced starting for some strains (-S9) at 50 µg/plate. Therefore a repeat experiment using the preincubation method was performed in the concentration range 0.5 to 50 µg/plate with strains TA1535, TA98, TA100, and TA102 without S9 and in the dose range 5 to 500 µg/plate with strains TA98, and TA100 with S9.
Remarks on result:
other: results for plate incorporation and preincubation method
Conclusions:
Under the conditions of the test, Tetrahydrolinalyl acetate was found to be not mutagenic. Therefore, the substance does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

An Ames test was performed according to OECD 471 and in compliance with GLP. A standard plate incorporation and a preincubation modification assay in absence and in presence of metabolic activation system (S9) were performed. Five Salmonella typhimurium tester strains (TA1535, TA97, TA98, TA100, and TA102) were exposed to concentrations ranging from 50 – 5000 µg/plate. Experiments were performed in triplicate. The activity of the S9-mix and the responsiveness of the tester strains were verified by including appropriate controls into each experiment. It was concluded, that neither the test substance per se, nor any of its metabolites formed under the experimental conditions, induced genetic damage in the Ames test. Therefore Tetrahydrolinalyl acetate was concluded to be not mutagenic. Based on this result, the substance does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test was conducted comparable to OECD Test Guideline No. 476, under GLP Standards, and QA. Due to the read-across purpose it was given a Klimisch 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
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 ug/ml; 2) 25, 50, 100, 145, 200, 224, 274 ug/mlWith metabolic activation: 1) 12.5, 25, 50, 100, 200, 250 ug/ml; 2) 25, 50, 100, 145, 200, 224 ug/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 mediumDURATION- 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 daysSELECTION AGENT (mutation assays): Trifluorothymidine (TFT) ( Thymidine analog)NUMBER OF REPLICATIONS: one culture per dose level; 3 disks per cultureNUMBER OF CELLS EVALUATED: 10*6DETERMINATION 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 ug/ml based on the range finding experiment; concentations >=200 ug/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 ug/ml to 998 ug/ml. With and without metabolic activation, total cellkilling was observed at 499 ug/ml. The 250 ug/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 ug/ml) induced moderate to no toxicity (236.8% to 30.2% relative growth).In Trial 2 the six analyzed treatments (25.0 to 224 ug/ml) induced relative growths that ranged from 80.2% to 23.4%; > 274ug/ml was lethal.+S9 Mutation assays: in Trial 1 the six analyzed treatments (12.5 to 250 ug/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 ug/ml) induced a wide range of toxicities (143.5% to 17.6% relative growth); >= 224 ug/ml was lethal.

Not relevant

Conclusions:
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 ug/ml with and without activation. The test material was soluble in mediums up to approximately 200 ug/ml and the pH was maintained at or above 7.0.

Under non activation conditions, eight treatments from 12.5 ug/ml to 500 ug/ml were initiated in the first trial and the 350 ug/ml and 500 ug/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 ug/ml to 399 ug/ml were initiated and seven treatments from 25.0 ug/ml to 274 ug/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 ug/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 ug/ml to 500 ug/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 ug/ml to 399 ug/ml were initiated and treatments above 250 ug/ml were terminated because of excessive toxicity. One treatment at 224 ug/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.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached justification
Reason / purpose:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>=125 ug/ml based on the range finding experiment; concentations >=200 ug/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 ug/ml to 998 ug/ml. With and without metabolic activation, total cellkilling was observed at 499 ug/ml. The 250 ug/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 ug/ml) induced moderate to no toxicity (236.8% to 30.2% relative growth).In Trial 2 the six analyzed treatments (25.0 to 224 ug/ml) induced relative growths that ranged from 80.2% to 23.4%; > 274ug/ml was lethal.+S9 Mutation assays: in Trial 1 the six analyzed treatments (12.5 to 250 ug/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 ug/ml) induced a wide range of toxicities (143.5% to 17.6% relative growth); >= 224 ug/ml was lethal.

Not relevant

Conclusions:
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. This result was used for read-across to tetrahydrolinalyl acetate.
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 ug/ml with and without activation. The test material was soluble in mediums up to approximately 200 ug/ml and the pH was maintained at or above 7.0.

Under non activation conditions, eight treatments from 12.5 ug/ml to 500 ug/ml were initiated in the first trial and the 350 ug/ml and 500 ug/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 ug/ml to 399 ug/ml were initiated and seven treatments from 25.0 ug/ml to 274 ug/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 ug/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 ug/ml to 500 ug/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 ug/ml to 399 ug/ml were initiated and treatments above 250 ug/ml were terminated because of excessive toxicity. One treatment at 224 ug/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. This result was used for read-across to tetrahydrolinalyl acetate.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- Micronucleus assay (OECD TG 474): negative

Link to relevant study records

Referenceopen allclose all

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test was conducted according to OECD Test Guideline No. 474, 1997, under GLP Standards, and QA. Due to the read-across purpose it was given a Klimisch 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID.
Reason / purpose:
reference to same study
Qualifier:
according to
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 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 daysENVIRONMENTAL 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(s)/solvent(s) 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.
Remarks:
Doses / Concentrations:500, 1000, 1500 mg/kg bwBasis:actual ingestedoral intubation
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 ( in addition to information in specific fields): 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
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:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached justification
Reason / purpose:
read-across source
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. This result was used for read-across to tetrahydrolinalyl acetate.
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. This result was used for read-across to tetrahydrolinalyl acetate.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

An Ames test in accordance with OECD 471 and in compliance with GLP was performed for Tetrahydrolinalyl acetate. A standard plate incorporation and a preincubation modification assay in absence and in presence of metabolic activation system (S9) were performed. Five Salmonella typhimurium tester strains (TA1535, TA97, TA98, TA100, and TA102) were exposed to concentrations ranging from 50 – 5000 µg/plate. Experiments were performed in triplicate. The activity of the S9-mix and the responsiveness of the tester strains were verified by including appropriate controls into each experiment. It was concluded, that neither the test substance per se, nor any of its metabolites formed under the experimental conditions, induced genetic damage in the Ames test. This result was supported by a negative Ames test for the read-across substance linalool. Also for linalool, an in vitro genetic toxicity test was performed similar to OECD 476 and in compliance with GLP. The objective of this in vitro assay was to evaluate the ability of Linalool to induce forward mutations at the thymidine kinase locus in the mouse lymphoma L5178Y cell line, using Trifluorothymidine as selection agent. Preliminary cytotoxicity assays were performed in the presence and absence of rat liver S9 metabolic activation. In the main study, no acceptable treatment in either of the two trials without metabolic activation induced a mutant frequency that exceeded the minimum criterion for a positive response. In the activation assays, small increases were observed in Trial I that just exceeded the minimum criterion for a positive response, but these increases in the mutant frequencies were not observed in Trial 2. 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. Another study for linalool was performed in accordance with OECD TG 474 and in compliance with GLP. This in vivo micronucleus test in mice evaluates the 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 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. Cyclophosphamide 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. Based on these results is concluded that Linalool is not mutagenic in the micronucleus test. This result is supported by an available in vitro chromosome aberration test for linalool.

Justification for classification or non-classification

Based on the results of the available in vitro and in vivo studies, Tetrahydrolinalyl acetate does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).