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Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was not performed according to GLP or according to a specific guideline. However, the study gives detailed information on the enzymatic transformation of linalool in rats and guinea pigs.
Reason / purpose for cross-reference:
reference to same study
Objective of study:
metabolism
Qualifier:
no guideline available
Principles of method if other than guideline:
The study was designed to investigate the substrate specifities of the isofunctional Phase II enzyme UDP-glucuronosyltransferase (UDPGT) and to determine possible differences in the regulation. Liver microsomes were prepared from male Wistar and Gunn rats as well as guinea pigs. Animals were either used untreated or induced with phenobarbital or 3-methyl-cholanthrene. The conjugation of several monoterpenoid alcohols, including linalool, was studied in both control and phenobarbital-induced animals. Other studied chemical families were the hydroxycoumarins and alkyl or arylphenols.
GLP compliance:
no
Radiolabelling:
no
Species:
other: microsome fraction of rat and guinea pig
Strain:
other: Wistar rat, Gunn rat, "tricolores" guinea pig
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS- Source: Rats were obtained from IFFA-CREDO (Saint-Germain sur l'abresle, France), guinea pigs were obtained from the Centre d'Elevage d'Animaux de Laboratoire (Ardenay, France)- Weight at study initiation: rats 200g, guinea pigs 350-400 g- Diet (e.g. ad libitum): free access to food and waterENVIRONMENTAL CONDITIONSNot applicable, experiment was performed in vitro with microsomes from rats and guinea pigs
Route of administration:
other: not relevant, in vitro experiment
Vehicle:
not specified
Details on exposure:
Not relevant
Duration and frequency of treatment / exposure:
Measurement of UDPGT activity: 10 minutes
Remarks:
Doses / Concentrations:Experiment with Wistar rats: 0.25 mM linaloolExperiment with Gunn rats: not reportedExperiment with guinea pigs: 0.175 mM
No. of animals per sex per dose / concentration:
For the preparation of microsomes, four male rats were assigned to each treatment (Wistar and Gunn rats: induction with phenobarbital, corresponding control receiving saline solution; Wistar rats: induction with 3-methylcholanthrene, corresponding control receiving corn oil), and four guinea pigs were treated with phenobarbital.
Control animals:
yes
Positive control reference chemical:
No data
Details on study design:
UDPGT activities were measured by a modification of the method described by Mulder and van Doorn (1975, Biochemical Journal, 151, 131)
Details on dosing and sampling:
Not relevant
Statistics:
Specific activity was calculated by linear regression. Each value is given as a mean of four determinations.
Preliminary studies:
Not relevant
Details on absorption:
Not relevant
Details on distribution in tissues:
Not relevant
Details on excretion:
Not relevant
Metabolites identified:
not specified
Details on metabolites:
Not relevant

UDPGT specific activity for linalool in liver microsomes from Wistar rats induced by either phenobarbital or 3 -methylcholanthrene, and in livermicrosomes from guinea pigs induced by phenobarbital are summarized in the table below.

 

UDPGT specific activities (nmol/min/mg)

Control (NaCl)

Phenobarbital-induced

Control (oil)

3-methylcholanthrene-induced

Wistar rats

7.4± 0.3

9.7± 0.1

7.5± 0.1

6.8±0.2

Gunn rats

No results reported for linalool

Guinea pigs

4.0± 0.3

6.3± 0.1

Not studied

Not studied

Conclusions:
Linalool is conjugated with glucuronic acid to the respective glucuronid derivative. The activity of the phase II enzyme UDP-glucuronosyltransferase towards linalool was enhanced by induction with phenobarbital in microsomes of both Wistar rats and guinea pigs. Induction with 3-methylcholanthrene in Wistar rats did not result in an enhancement of activity.
Executive summary:

In this study, the substrate specifities of the isofunctional Phase II enzyme UDP-glucuronosyltransferase (UDPGT) and possible differences in the regulation were investigated. Liver microsomes were prepared from male Wistar and Gunn rats as well as guinea pigs, and induced with phenobarbital, 3-methylcholanthrene or with vehicle only (controls). The conjugation of several monoterpenoid alcohols, including linalool, was studied in both control and phenobarbital-induced guinea pigs. Other studied chemical families were the hydroxycoumarins and alkyl or arylphenols.

In Wistar rats, induction with phenobarbital enhanced UDPGT activity, whereas induction with 3 -methylcholanthrene did not. In guinea pigs, UDPGT activity was also enhanced by phenobarbital induction. Linalool is conjugated with glucuronic acid to respective glucuronid derivative

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See attached justification
Reason / purpose for cross-reference:
read-across source
Preliminary studies:
Not relevant
Details on absorption:
Not relevant
Details on distribution in tissues:
Not relevant
Details on excretion:
Not relevant
Metabolites identified:
not specified
Details on metabolites:
Not relevant

UDPGT specific activity for linalool in liver microsomes from Wistar rats induced by either phenobarbital or 3 -methylcholanthrene, and in livermicrosomes from guinea pigs induced by phenobarbital are summarized in the table below.

 

UDPGT specific activities (nmol/min/mg)

Control (NaCl)

Phenobarbital-induced

Control (oil)

3-methylcholanthrene-induced

Wistar rats

7.4± 0.3

9.7± 0.1

7.5± 0.1

6.8±0.2

Gunn rats

No results reported for linalool

Guinea pigs

4.0± 0.3

6.3± 0.1

Not studied

Not studied

Conclusions:
Linalool is conjugated with glucuronic acid to the respective glucuronid derivative. The activity of the phase II enzyme UDP-glucuronosyltransferase towards linalool was enhanced by induction with phenobarbital in microsomes of both Wistar rats and guinea pigs. Induction with 3-methylcholanthrene in Wistar rats did not result in an enhancement of activity. This study was used for read-across to ethyllinalyl acetate.
Executive summary:

In this study, the substrate specifities of the isofunctional Phase II enzyme UDP-glucuronosyltransferase (UDPGT) and possible differences in the regulation were investigated. Liver microsomes were prepared from male Wistar and Gunn rats as well as guinea pigs, and induced with phenobarbital, 3-methylcholanthrene or with vehicle only (controls). The conjugation of several monoterpenoid alcohols, including linalool, was studied in both control and phenobarbital-induced guinea pigs. Other studied chemical families were the hydroxycoumarins and alkyl or arylphenols.

In Wistar rats, induction with phenobarbital enhanced UDPGT activity, whereas induction with 3 -methylcholanthrene did not. In guinea pigs, UDPGT activity was also enhanced by phenobarbital induction. Linalool is conjugated with glucuronic acid to respective glucuronid derivative. This study was used for read-across to ethyllinalyl acetate.

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Not performed according to GLP. An equivalent or similar guideline to OECD 428 is used.
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
yes
Remarks:
temperature 37°C instead of 32°C
GLP compliance:
no
Radiolabelling:
no
Species:
human
Strain:
other: not relevant
Sex:
female
Details on test animals or test system and environmental conditions:
Not relevant
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Duration of exposure:
1, 2, and 4 hours
Doses:
- Nominal dose: 500 mg
No. of animals per group:
Not relevant
Control animals:
no
Remarks:
not relevant
Details on study design:
DOSE PREPARATIONTest article applied undilutedTEST SITESkin extracts of 0.65 cm2REMOVAL OF TEST SUBSTANCE- Washing procedures and type of cleansing agent: Skin was rinsed shortly with methanol- Time after start of exposure: 1, 2 or 4 hoursSKIN PREPARATION AFTER EXPOSUREAfter 1, 2, or 4 h, the stratum corneum layers were separated, and every fraction of the stratum corneum as well as the remaining skin were extracted with methanol and stored for analysis. The acceptor medium was also collected for analysis.ANALYSIS - Method type(s) for identification: GC-MS
Details on in vitro test system (if applicable):
SKIN PREPARATION- Source of skin: 40-year old woman- Ethical approval if human skin: Yes, ethics committee of the Medical University of Gdansk- Type of skin: Thorax - Storage conditions: At -20 degrees Celsius- Justification of species, anatomical site and preparative technique: PRINCIPLES OF ASSAY- Diffusion cell: flow-through Teflon diffusion cells- Receptor fluid: isotonic phosphate buffer (pH 7.3) preserved with 0.005% sodium azide- Solubility of test substance in acceptor fluid: 1.34 mg/ml - Flow-through system: yes, constant rate of 10 ml/h- Test temperature: 37 +/- 0.5 degrees Celsius- Humidity: No data- Occlusion: Donor compartment occluded with parafilm
Signs and symptoms of toxicity:
not examined
Remarks:
(not relevant)
Dermal irritation:
not examined
Remarks:
(not relevant)
Absorption in different matrices:
At 1 hour after application (ug/cm2):- Stratum corneum (tape strips): 78 +/- 4.1- Skin preparation (epidermis + dermis): 827.0 +/- 66.5- Acceptor fluid: 0At 2 hours after application (ug/cm2):- Stratum corneum (tape strips): 109.1 +/- 5.6- Skin preparation (epidermis + dermis): 1083.5 +/- 106.1- Acceptor fluid: 0At 4 hours after application (ug/cm2):- Stratum corneum (tape strips): 478.9 +/- 18.8- Skin preparation (epidermis + dermis): 1343.0 +/- 127.1- Acceptor fluid: 0
Total recovery:
- Total recovery: 0.23% (1821.9/(769.2*1000)- Recovery of applied dose acceptable: No, not within 100 +/- 10% as stated in the OECD guideline- Limit of detection (LOD): No data
Conversion factor human vs. animal skin:
Not relevant

Linalool easily penetrated into the skin, however it was not detected in the acceptor fluid.

The percutaneous absorption of linalool into the skin layers (ug/cm2), is summarized in the following table. The applied dose was 500 mg/0.65 cm2 (= 769.2 mg/cm2).

Skin layer

Time

1h

2h

4h

Stratum corneum SC1

34.3±8.8

65.9±36.5

242.6±36.4

Stratum corneum SC2

22.4±3.5

22.4±4.7

144.3±13.2

Stratum corneum SC3

21.6±3.7

20.8±6.1

92.0±12.9

Total Stratum corneum

78.3±4.1

109.1±5.6

478.9±18.8

Remaining skin (epidermis and dermis)

827.0±66.5

1083.5±106.1

1343.0±127.1

Skin total

905.3±64.7

1192.6±104.1

1821.9±135.8

Conclusions:
Under the conditions of this in vitro penetration study in human skin, 0.17% of the applied dose could be recovered in epidermis and dermis.
Executive summary:

The in vitro skin absorption and elimination of linalool was studied by applying linalool (500 mg/0.65 cm2) to human skin and determine the amount of linalool in the stratum corneum and epidermis and dermis after 1, 2, and 4h. Linalool was analysed using gas chromatography.

Linalool easily penetrated into the skin, however it was not detected in the acceptor fluid. Additionally, 1343 +/- 127.1 ug linalool/cm2 was found in epidermis and dermis. The percentage of applied dose in epidermis and dermis is therefore calculated to be 0.17%. Considering results of another in vitro penetration study conducted by Green et al (2007), it is likely that most of the applied material is evaporated.

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
See attached justification
Reason / purpose for cross-reference:
read-across source
Signs and symptoms of toxicity:
not examined
Remarks:
(not relevant)
Dermal irritation:
not examined
Remarks:
(not relevant)
Absorption in different matrices:
At 1 hour after application (ug/cm2):- Stratum corneum (tape strips): 78 +/- 4.1- Skin preparation (epidermis + dermis): 827.0 +/- 66.5- Acceptor fluid: 0At 2 hours after application (ug/cm2):- Stratum corneum (tape strips): 109.1 +/- 5.6- Skin preparation (epidermis + dermis): 1083.5 +/- 106.1- Acceptor fluid: 0At 4 hours after application (ug/cm2):- Stratum corneum (tape strips): 478.9 +/- 18.8- Skin preparation (epidermis + dermis): 1343.0 +/- 127.1- Acceptor fluid: 0
Total recovery:
- Total recovery: 0.23% (1821.9/(769.2*1000)- Recovery of applied dose acceptable: No, not within 100 +/- 10% as stated in the OECD guideline- Limit of detection (LOD): No data
Conversion factor human vs. animal skin:
Not relevant

Linalool easily penetrated into the skin, however it was not detected in the acceptor fluid.

The percutaneous absorption of linalool into the skin layers (ug/cm2), is summarized in the following table. The applied dose was 500 mg/0.65 cm2 (= 769.2 mg/cm2).

Skin layer

Time

1h

2h

4h

Stratum corneum SC1

34.3±8.8

65.9±36.5

242.6±36.4

Stratum corneum SC2

22.4±3.5

22.4±4.7

144.3±13.2

Stratum corneum SC3

21.6±3.7

20.8±6.1

92.0±12.9

Total Stratum corneum

78.3±4.1

109.1±5.6

478.9±18.8

Remaining skin (epidermis and dermis)

827.0±66.5

1083.5±106.1

1343.0±127.1

Skin total

905.3±64.7

1192.6±104.1

1821.9±135.8

Conclusions:
Under the conditions of this in vitro penetration study in human skin, 0.17% of the applied dose could be recovered in epidermis and dermis. This study was used for read-across to ethyllinalyl acetate.
Executive summary:

The in vitro skin absorption and elimination of linalool was studied by applying linalool (500 mg/0.65 cm2) to human skin and determine the amount of linalool in the stratum corneum and epidermis and dermis after 1, 2, and 4h. Linalool was analysed using gas chromatography. Linalool easily penetrated into the skin, however it was not detected in the acceptor fluid. Additionally, 1343 +/- 127.1 ug linalool/cm2 was found in epidermis and dermis. The percentage of applied dose in epidermis and dermis is therefore calculated to be 0.17%. Considering results of another in vitro penetration study conducted by Green et al (2007), it is likely that most of the applied material is evaporated. This study was used for read-across to ethyllinalyl acetate.

 

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was not performed according to GLP. Study was similar to OECD417, metabolisation and enzyme induction were studied.
Reason / purpose for cross-reference:
reference to same study
Objective of study:
other: metabolism and enzyme induction
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
yes
Remarks:
only metabolism was studied
Principles of method if other than guideline:
Metabolites in urine were determined, as well as enzyme activities in lung and liver microsomes
GLP compliance:
no
Radiolabelling:
no
Species:
rat
Strain:
other: IISC strain
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS- Weight at study initiation: 160-200 g- Housing: individually- Individual metabolism cages: yes- Diet (e.g. ad libitum): free access to food- Water (e.g. ad libitum): free access to water
Route of administration:
oral: gavage
Vehicle:
other: 1% methyl cellulose soln.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:Suspension in 1% methyl cellulose soln.DIET PREPARATIONVEHICLE- Amount of vehicle (if gavage): 4 ml/kgHOMOGENEITY AND STABILITY OF TEST MATERIAL:Suspension
Duration and frequency of treatment / exposure:
Induction study: once daily for six daysIdentification of metabolites: once daily for 20 days
Remarks:
Doses / Concentrations:Induction study: 600 mg/kg bwIdentification of metabolites: 800 mg/kg bw
No. of animals per sex per dose / concentration:
Not specified
Control animals:
yes, concurrent vehicle
Positive control reference chemical:
Not relevant
Details on study design:
Not relevant
Details on dosing and sampling:
METABOLITE CHARACTERISATION STUDIES- Tissues and body fluids sampled: urine- Time and frequency of sampling: urine collected daily for 20 days following oral administration- Method type(s) for identification: TLC on silica gel G-coated plates,GC, HPLC, and NMRTREATMENT FOR CLEAVAGE OF CONJUGATES: Urine was acidified to pH 3-4 and extracted with ether. The aqueous portion containing conjugated metabolites was refluxed under acid conditions
Statistics:
Not relevant
Preliminary studies:
Not relevant
Details on absorption:
Not relevant
Details on distribution in tissues:
Not relevant
Details on excretion:
Not relevant
Metabolites identified:
yes
Details on metabolites:
The identified metabolites of linalool were 8-hydroxy linalool and 8-carboxy linalool (main metabolites). Several other (minor) metabolites could not be identified.

Administration of linalool to rats resulted in a statistically significant increase (~50%) in the concentration of cytochrome P-450 in the liver microsomes after three days of dosing. After six days, it had decreased to control values. No significant changes in the activities of cytochrome b5, NADPH-cytochrome c reductase and NADH-cytochrome c reductase in the liver microsomes were observed after six days, however, an increase (~20%) in cytochrome b5 was noticed after three days of treatment.

No effects were observed on the activity of measured enzymes in the lung microsomes.

Conclusions:
Linalool is metabolised extensively. The metabolism of linalool after oral exposure to rats results in the metabolites 8-hydroxy linalool and 8-carboxy linalool, indicating that 8-hydroxylation (allylic hydroxylation) is the major pathway. Although liver-microsomal P450 enzymes were elevated after three days of treatment, it decreased to normal values after six days, therefore no conclusion can be drawn on the role of cytochrome P450 in the metabolism of linalool. Linalool did not affect any of the lung-microsomal parameters measured.
Executive summary:

In an induction experiment activities of lung- and liver-microsomal enzymes after six days of exposure to linalool (600 mg/kg bw/day) were measured.

The induction experiment showed that although liver-microsomal P450 enzymes were elevated after three days of treatment, it decreased to normal values after six days. Therefore no conclusion can be drawn on the role of cytochrome P450 in the metabolism of linalool. Furthermore, linalool did not affect any of the lung-microsomal parameters measured.

Metabolism was studied in urine obtained from male rats treated with 800 mg/kg bw/day for 20 days. In this experiment the metabolites 8-hydroxy linalool and 8-carboxy linalool were identified as major metabolites, indicating that 8-hydroxylation is the major pathway. In addition, several other metabolites were found, which could, however, not be identified.

Endpoint:
basic toxicokinetics
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See attached justification
Reason / purpose for cross-reference:
read-across source
Preliminary studies:
Not relevant
Details on absorption:
Not relevant
Details on distribution in tissues:
Not relevant
Details on excretion:
Not relevant
Metabolites identified:
yes
Details on metabolites:
The identified metabolites of linalool were 8-hydroxy linalool and 8-carboxy linalool (main metabolites). Several other (minor) metabolites could not be identified.

Administration of linalool to rats resulted in a statistically significant increase (~50%) in the concentration of cytochrome P-450 in the liver microsomes after three days of dosing. After six days, it had decreased to control values. No significant changes in the activities of cytochrome b5, NADPH-cytochrome c reductase and NADH-cytochrome c reductase in the liver microsomes were observed after six days, however, an increase (~20%) in cytochrome b5 was noticed after three days of treatment.

No effects were observed on the activity of measured enzymes in the lung microsomes.

Conclusions:
Linalool is metabolised extensively. The metabolism of linalool after oral exposure to rats results in the metabolites 8-hydroxy linalool and 8-carboxy linalool, indicating that 8-hydroxylation (allylic hydroxylation) is the major pathway. Although liver-microsomal P450 enzymes were elevated after three days of treatment, it decreased to normal values after six days, therefore no conclusion can be drawn on the role of cytochrome P450 in the metabolism of linalool. Linalool did not affect any of the lung-microsomal parameters measured. This study was used for read-across to ethyllinalyl acetate.
Executive summary:

In an induction experiment activities of lung- and liver-microsomal enzymes after six days of exposure to linalool (600 mg/kg bw/day) were measured.

The induction experiment showed that although liver-microsomal P450 enzymes were elevated after three days of treatment, it decreased to normal values after six days. Therefore no conclusion can be drawn on the role of cytochrome P450 in the metabolism of linalool. Furthermore, linalool did not affect any of the lung-microsomal parameters measured.

Metabolism was studied in urine obtained from male rats treated with 800 mg/kg bw/day for 20 days. In this experiment the metabolites 8-hydroxy linalool and 8-carboxy linalool were identified as major metabolites, indicating that 8-hydroxylation is the major pathway. In addition, several other metabolites were found, which could, however, not be identified. This study was used for read-across to ethyllinalyl acetate.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Only short publication of study available
Reason / purpose for cross-reference:
reference to same study
Objective of study:
absorption
Qualifier:
no guideline followed
Principles of method if other than guideline:
Mice were exposed to linalool by inhalation to assess the possible sedative influence. Gas chromatography/mass spectrometry (GC/MS) with chemical ionization (CI) and slected ion monitoring (SIM), as well as gas chromatography/flame ionization detection (GC/FID) were used to detect, identify and quantify the concentrations of linalool in blood.
GLP compliance:
no
Radiolabelling:
no
Species:
mouse
Strain:
not specified
Sex:
not specified
Details on test animals or test system and environmental conditions:
No data
Route of administration:
inhalation
Vehicle:
not specified
Details on exposure:
No data
Duration and frequency of treatment / exposure:
No data
Remarks:
Doses / Concentrations:5 mg/l
No. of animals per sex per dose / concentration:
No data
Control animals:
yes
Positive control reference chemical:
No data
Details on study design:
No data
Details on dosing and sampling:
METABOLITE CHARACTERISATION STUDIES- Tissues and body fluids sampled: blood- Time and frequency of sampling: immediately after finsh of experiment- From how many animals: GC/MS with blood from one animal, CI, SIM with pooled extracted serum samples. GC/FID no data - Method type(s) for identification GC-FID, GC-MS with CI (chemical ionization) and SIM (selected ion monitoring)
Statistics:
Not relevant
Preliminary studies:
Not relevant
Details on absorption:
Not relevant
Details on distribution in tissues:
Not relevant
Details on excretion:
Not relevant
Metabolites identified:
no
Details on metabolites:
Not relevant

The inhalation of linalool (5 mg/l) leads to significant reduction of the motility of exposed mice (down to 30 - 40%) with respect to the control group. The amount of linalool detected in blood samples was 7 -9 ng/ml, determined by GC/MS with CI and SIM, and GC/FID.

Conclusions:
Mice that were exposed to 5 mg linalool/l by inhalation showed blood concentrations of 7 -9 ng/ml immediately after termination of exposure. The exposure lead to a significant reduction of motility of 30 - 40% compared to control values.
Executive summary:

Mice were exposed to linalool by inhalation to assess the possible sedative influence. Gas chromatography/mass spectrometry (GC/MS) with chemical ionization (CI) and slected ion monitoring (SIM), as well as gas chromatography/flame ionization detection (GC/FID) were used to detect, identify and quantify the concentrations of linalool in blood.

The inhalation of linalool (5 mg/l) leads to significant reduction of the motility of exposed mice (down to 30 - 40%) with respect to the control group. The amount of linalool detected in blood samples was 7 -9 ng/ml, determined by GC/MS with CI and SIM, and GC/FID.

Endpoint:
basic toxicokinetics in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See attached justification
Reason / purpose for cross-reference:
read-across source
Preliminary studies:
Not relevant
Details on absorption:
Not relevant
Details on distribution in tissues:
Not relevant
Details on excretion:
Not relevant
Metabolites identified:
no
Details on metabolites:
Not relevant

The inhalation of linalool (5 mg/l) leads to significant reduction of the motility of exposed mice (down to 30 - 40%) with respect to the control group. The amount of linalool detected in blood samples was 7 -9 ng/ml, determined by GC/MS with CI and SIM, and GC/FID.

Conclusions:
Mice that were exposed to 5 mg linalool/l by inhalation showed blood concentrations of 7 -9 ng/ml immediately after termination of exposure. The exposure lead to a significant reduction of motility of 30 - 40% compared to control values. This study was used for read-across to ethyllinalyl acetate.
Executive summary:

Mice were exposed to linalool by inhalation to assess the possible sedative influence. Gas chromatography/mass spectrometry (GC/MS) with chemical ionization (CI) and slected ion monitoring (SIM), as well as gas chromatography/flame ionization detection (GC/FID) were used to detect, identify and quantify the concentrations of linalool in blood.

The inhalation of linalool (5 mg/l) leads to significant reduction of the motility of exposed mice (down to 30 - 40%) with respect to the control group. The amount of linalool detected in blood samples was 7 -9 ng/ml, determined by GC/MS with CI and SIM, and GC/FID. This study was used for read-across to ethyllinalyl acetate.

Endpoint:
dermal absorption in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This human study was reported with limited information on test substance composition and applied concentration. Test was performed in human with one male subject and no details on health history were reported. Furthermore, no other effects of dermal exposure were monitored. However, the methods and results are reported concise but clear.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline available
Principles of method if other than guideline:
One male subject was treated with massage oil (containing 0.5% linalool and 0.6% linalyl acetate) on the skin. Blood samples were taken at 0, 5, 10, 15, 20, 30, 45, 60, 75, and 90 minutes thereafter to obtain the quantity of linalool and linalyl acetate (main components of lavender oil) present in the blood, using GC-FID and GC-MS. For statistics, test was repeated 3 times in the same subject.
GLP compliance:
no
Radiolabelling:
no
Species:
human
Strain:
other: Not relevant
Sex:
male
Details on test animals or test system and environmental conditions:
TEST SUBJECT- Age at study initiation: 34- Weight at study initiation: 60 kg
Type of coverage:
open
Vehicle:
other: peanut oil
Duration of exposure:
10 minutes
Doses:
Linalool- Nominal doses: 0.5% or 7.437 mg linalool (calculated as: 1.5 g massage oil * 2% lavender oil * 24.79% linalool)- Actual doses: 0.12 mg/kg linalool (pharmacokinetic model)Linalyl acetate- Nominal doses: 0.6% or 8.877 mg (calculated as: 1.5 g massage oil * 2% lavender oil * 29.59% linalyl acetate)- Actual doses: 0.144 mg/kg linalyl acetate (pharmacokinetic model)
No. of animals per group:
1 human subject
Control animals:
no
Details on study design:
APPLICATION OF DOSE: Massage oil was spread on skin and gently massaged into the skin. The remaining oil was completely removed.VEHICLE- Amount(s) applied (weight with unit): 1.5 g massage oilTEST SITE- Area of exposure: Defined skin area of the stomach- Coverage: 376 cm2 of the bodyREMOVAL OF TEST SUBSTANCE- Washing procedures and type of cleansing agent: Completely removal of test substance- Time after start of exposure: 10 minSAMPLE COLLECTION- Collection of blood: Blood samples were drawn from the left cubital vein at 0, 5, 10, 15, 20, 30, 45, 60, 75 and 90 minutes after finishing the massage.SAMPLE PREPARATION- Storage procedure: Heparine was added, plasma centrifuged, and the samples stored at -20 Degrees Celsius until chromatographic and spectroscopic investigations.- Preparation details: Plasma extracted by solid-phase-extraction.ANALYSIS - Method type(s) for identification: GC-FID, GC-MS (for confirmation of GC-FID results)- Validation of analytical procedure and quantification by internal standard: tiglinic acid benzyl ester (ST)
Details on in vitro test system (if applicable):
Not relevant
Signs and symptoms of toxicity:
not specified
Dermal irritation:
not specified
Absorption in different matrices:
- Blood: Linalool was absorbed in the skin and detected in blood within 5 minutes after finishing the massage. Almost all linalool was eliminated from the blood after 90 minutes.Invasion rate constant: 0.11/minElimination rate constant: 0.46/minHalf life of invasion: 6.16 minBiological half life: 13.76 minPeak plasma concentration: 19 minMean plasma concentration: 100 ng/mlAUC (0-90 min): 4927.25 (mg/ml)min- Blood: Linalyl acetate was absorbed in the skin and detected in blood within 5 minutes after finishing the massage. Almost all linalool was eliminated from the blood after 90 minutes.Invasion rate constant: 0.11/minElimination rate constant: 0.48/minHalf life of invasion: 6.29 minBiological half life: 14.30 minPeak plasma concentration: 19 minMean plasma concentration: 121 ng/mlAUC (0-90 min): 4174.50 (mg/ml)min
Total recovery:
No data
Conversion factor human vs. animal skin:
Not relevant

Plasma concentration of linalool was significantly higher than that of linalyl acetate (represented by a larger AUC)

Conclusions:
Under the conditions of this test, it can be concluded that linalool is absorbed in the blood after dermal application in humans, but is also readily excreted.
Executive summary:

The dermal absorption of lavender oil (in massage oil) containing linalool was studied in one male human subject. Massage oil was massaged onto the skin for ten minutes. In total 7.23 mg linalool was applied, which corresponds with a dose of 0.12 mg/kg bw. Linalyl acetate dose was 8.64 in total, representing 0.144 mg/kg bw. Subsequently, the concentration of linalool and linalyl acetate in the blood was determined at several time points by GC-FID and GC-MS. The experiment was repeated three times in the same subject for statistical evaluation. Toxicokinetic parameters were estimated with an open two-compartment standard pharmacokinetic model.

The results indicate that linalool and linalyl acetate can be detected in the blood after absorption through skin within 5 minutes after application. The mean plasma concentration (100 ng/ml linalool, 121 ng/ml linalyl acetate) is reached after 19 minutes. Within 90 minutes linalool has been eliminated almost completely. Calculated AUC of linalool is 4927.25 (ng/ml)min, for linalyl acetate this was 4174.50 (ng/ml)min, indicating that the concentration of linalool is significantly higher than that of linalyl acetate. Under the conditions of this test, it can be concluded that linalool and linalyl acetate are absorbed in the blood after dermal application in humans, but are also readily excreted.

Endpoint:
dermal absorption in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
See attached justification
Reason / purpose for cross-reference:
read-across source
Signs and symptoms of toxicity:
not specified
Dermal irritation:
not specified
Absorption in different matrices:
- Blood: Linalool was absorbed in the skin and detected in blood within 5 minutes after finishing the massage. Almost all linalool was eliminated from the blood after 90 minutes.Invasion rate constant: 0.11/minElimination rate constant: 0.46/minHalf life of invasion: 6.16 minBiological half life: 13.76 minPeak plasma concentration: 19 minMean plasma concentration: 100 ng/mlAUC (0-90 min): 4927.25 (mg/ml)min- Blood: Linalyl acetate was absorbed in the skin and detected in blood within 5 minutes after finishing the massage. Almost all linalool was eliminated from the blood after 90 minutes.Invasion rate constant: 0.11/minElimination rate constant: 0.48/minHalf life of invasion: 6.29 minBiological half life: 14.30 minPeak plasma concentration: 19 minMean plasma concentration: 121 ng/mlAUC (0-90 min): 4174.50 (mg/ml)min
Total recovery:
No data
Conversion factor human vs. animal skin:
Not relevant

Plasma concentration of linalool was significantly higher than that of linalyl acetate (represented by a larger AUC)

Conclusions:
Under the conditions of this test, it can be concluded that linalool is absorbed in the blood after dermal application in humans, but is also readily excreted. This study was used for read-across to ethyllinalyl acetate.
Executive summary:

The dermal absorption of lavender oil (in massage oil) containing linalool was studied in one male human subject. Massage oil was massaged onto the skin for ten minutes. In total 7.23 mg linalool was applied, which corresponds with a dose of 0.12 mg/kg bw. Linalyl acetate dose was 8.64 in total, representing 0.144 mg/kg bw. Subsequently, the concentration of linalool and linalyl acetate in the blood was determined at several time points by GC-FID and GC-MS. The experiment was repeated three times in the same subject for statistical evaluation. Toxicokinetic parameters were estimated with an open two-compartment standard pharmacokinetic model.

The results indicate that linalool and linalyl acetate can be detected in the blood after absorption through skin within 5 minutes after application. The mean plasma concentration (100 ng/ml linalool, 121 ng/ml linalyl acetate) is reached after 19 minutes. Within 90 minutes linalool has been eliminated almost completely. Calculated AUC of linalool is 4927.25 (ng/ml)min, for linalyl acetate this was 4174.50 (ng/ml)min, indicating that the concentration of linalool is significantly higher than that of linalyl acetate. Under the conditions of this test, it can be concluded that linalool and linalyl acetate are absorbed in the blood after dermal application in humans, but are also readily excreted. This study was used for read-across to ethyllinalyl acetate.

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study is well-documented in this publication, as methods and results are documented extensively. Design is proper and results are validated.
Reason / purpose for cross-reference:
reference to same study
Objective of study:
metabolism
Qualifier:
no guideline available
Principles of method if other than guideline:
Cytochrome P450-mediated enzymatic assays were used to study the metabolism of linalool (substrate) by two recombinant enzymes (CYP2D6- and CYP2C19) normally present in human skin. Dependency on time, linalool or enzyme concentration was also determined.
GLP compliance:
no
Radiolabelling:
no
Details on test animals or test system and environmental conditions:
Not relevant
Details on exposure:
Not relevant
Duration and frequency of treatment / exposure:
Not relevant
Remarks:
Doses / Concentrations:Not relevant
No. of animals per sex per dose / concentration:
Not relevant
Positive control reference chemical:
Not relevant
Details on study design:
Not relevant
Details on dosing and sampling:
Not relevant
Statistics:
Not relevant
Preliminary studies:
Not relevant
Details on absorption:
Not relevant
Details on distribution in tissues:
Not relevant
Details on excretion:
Not relevant
Metabolites identified:
yes
Details on metabolites:
Enzymatic conversion of linalool catalyzed by both CYP2D6 and CYP2C19 resulted in the formation and identification of three different enzymatic products: (R/S)-furanoid-linalool oxide, (R/S)-pyranoid-linalool oxide and (cis/trans)-8-hydroxylinalool. The identity of the first two metabolites was confirmed by reference substances, the latter one was confirmed by calcuation of Kovacs Indices (KI) and interpretation of the EI fragmentation pattern. Dihydrolinalool was also identified, but is an impurity in linalool.

The time dependency of the enzymatic product formation by both CYP was linear for all identified enzymatic products.

Increase of enzyme concentrations by a factor 2 resulted in an increase of the enzymatic activity by a factor of 2.

Linalool concentration dependency of the enzymatic products formation was linear for all identified enzymatic products.

Conclusions:
Linalool is metabolised in vitro by recombinant CYP2D6 and CYP2C19. Under the conditions of this study, it was found that linalool is metabolized in vitro by human skin enzymes CYP2C19 and CYP2D6 to (R/S)-furanoid-linalool oxide, (R/S)-pyranoid-linalool oxide and (cis/trans)-8-hydroxylinalool. A linear relationship was observed between the formation of metabolites and incubation time, enzyme and linalool concentration.
Executive summary:

Cytochrome P450-mediated enzymatic assays were used to study the metabolism of linalool (substrate) by two recombinant enzymes (CYP2D6- and CYP2C19) normally present in human skin. It was also determined if time, linalool or enzyme concentration dependency was present.

It was found that linalool is metabolized in vitro by human skin enzymes CYP2C19 and CYP2D6 to (R/S)-furanoid-linalool oxide, (R/S)-pyranoid-linalool oxide and (cis/trans)-8-hydroxylinalool. A linear relationship was observed between the formation of metabolites and incubation time, enzyme and linalool concentration. The authors indicate that in the formation of the two cyclic ethers an intermediary electrofilic epoxide may be formed.

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See attached justification
Reason / purpose for cross-reference:
read-across source
Preliminary studies:
Not relevant
Details on absorption:
Not relevant
Details on distribution in tissues:
Not relevant
Details on excretion:
Not relevant
Metabolites identified:
yes
Details on metabolites:
Enzymatic conversion of linalool catalyzed by both CYP2D6 and CYP2C19 resulted in the formation and identification of three different enzymatic products: (R/S)-furanoid-linalool oxide, (R/S)-pyranoid-linalool oxide and (cis/trans)-8-hydroxylinalool. The identity of the first two metabolites was confirmed by reference substances, the latter one was confirmed by calcuation of Kovacs Indices (KI) and interpretation of the EI fragmentation pattern. Dihydrolinalool was also identified, but is an impurity in linalool.

The time dependency of the enzymatic product formation by both CYP was linear for all identified enzymatic products.

Increase of enzyme concentrations by a factor 2 resulted in an increase of the enzymatic activity by a factor of 2.

Linalool concentration dependency of the enzymatic products formation was linear for all identified enzymatic products.

Conclusions:
Linalool is metabolised in vitro by recombinant CYP2D6 and CYP2C19. Under the conditions of this study, it was found that linalool is metabolized in vitro by human skin enzymes CYP2C19 and CYP2D6 to (R/S)-furanoid-linalool oxide, (R/S)-pyranoid-linalool oxide and (cis/trans)-8-hydroxylinalool. A linear relationship was observed between the formation of metabolites and incubation time, enzyme and linalool concentration. This study was used for read-across to ethyllinalyl acetate.
Executive summary:

Cytochrome P450-mediated enzymatic assays were used to study the metabolism of linalool (substrate) by two recombinant enzymes (CYP2D6- and CYP2C19) normally present in human skin. It was also determined if time, linalool or enzyme concentration dependency was present.

It was found that linalool is metabolized in vitro by human skin enzymes CYP2C19 and CYP2D6 to (R/S)-furanoid-linalool oxide, (R/S)-pyranoid-linalool oxide and (cis/trans)-8-hydroxylinalool. A linear relationship was observed between the formation of metabolites and incubation time, enzyme and linalool concentration. The authors indicate that in the formation of the two cyclic ethers an intermediary electrofilic epoxide may be formed. This study was used for read-across to ethyllinalyl acetate.

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Only very short publication available, but experimental result consistent with good scientific knowledge.
Reason / purpose for cross-reference:
reference to same study
Objective of study:
absorption
distribution
excretion
metabolism
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
yes
Remarks:
number and sex of animals is not specified
Principles of method if other than guideline:
1,2[14C]linalool was administered in male Wistar rats intragastrically and intraperitoneally in this study. Experiment 1: After intragastric administration of 500 mg/kg bw, urine, faeces and expired air were collected and measured at several intervals over a 72 hours period. Radioactivity was determined. At the end of the experiment, residual radioactivity in brain, lung, liver, heart, spleen, gastro-intestinal tract, kidney, skin and skeletal muscle was determined.Experiment 2: In this experiment intraperitoneal administration was used to determine if biliary excretion occurred. Bile was collected at several intervals after exposure of 2 rats and radioactivity was determined.Experiment 3: In this experiment one rat was intraperitoneally exposed. Bile from this treated animal was introduced into the duodenum of a second animal. Bile of this animal was then collected. Presence of radioactivity was indicative of enterohepatic circulation.
GLP compliance:
no
Radiolabelling:
yes
Remarks:
14C
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS- Age at study initiation: 12 weeks- Individual metabolism cages: yes
Route of administration:
other: intragastric (exp. 1) and intraperitoneal (exp. 2 & 3)
Vehicle:
propylene glycol
Details on exposure:
VEHICLE- Concentration in vehicle: Experiment 1: 25% (w/v)Experiment 2 & 3: 10% (w/v)
Duration and frequency of treatment / exposure:
Single administration
Remarks:
Doses / Concentrations:Experiment 1: 500 mg/kg bwExperiment 2 & 3: 20 mg/animal
No. of animals per sex per dose / concentration:
Experiment 1: Not indicatedExperiment 2 & 3: 2 male animals
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)Experiment 1:- Tissues and body fluids sampled: urine, faeces, expired air- Time and frequency of sampling: collected at intervals over a 72h period- Tissues and body fluids sampled at end of experiment (distribution): brain, lung, liver, heart, spleen, gastro-intestinal tract, kidney, skin and skeletal muscleExperiment 2:- Tissues and body fluids sampled: bile- Time and frequency of sampling: at intervals over a 6h and 11h period (1 animal for each period)Experiment 3:- Tissues and body fluids sampled: bile (from one of the two animals)METABOLITE CHARACTERISATION STUDIESExperiment 2:- Tissues and body fluids sampled: bile- Time and frequency of sampling: at intervals over a 6h and 11h period (1 animal for each period)Experiment 3:- Tissues and body fluids sampled: faeces, bile- Time and frequency of sampling: at intervals over a 12h period- From how many animals: 1
Statistics:
Not relevant
Preliminary studies:
Not relevant
Details on absorption:
Experiment 1: at least 85% of linalool is absorbed (detected in urine and expired air) after 72 hours
Details on distribution in tissues:
Experiment 1 (in total: 3% of dose) after 72 hours:- Liver: 0.5%- Gut: 0.6%- Skin: 0.8%- Skeletal muscle: 1.2%- Other organs: insignificant amounts of radioactivity
Details on excretion:
Experiment 1: approx. 100% of the administered dose is excreted (urine, faeces, expired gas) after 72 hours.- Expired air: 23%- Urine: approx. 60%- Faeces: approx. 15% (delayed, possible biliary excretion)Experiment 2:- Bile: more than 25% of the ip dose detected after 11 hoursExperiment 3:- Bile: 2.5% of ip dose detected in second rat after 12 hours
Metabolites identified:
yes
Details on metabolites:
Experiment 2: No free linalool, but only polar conjugates were detectable in bile (partially hydrolysed by beta-glucuronidase and to a greater extent by a mixture of beta-glucuronidase and sulphatase).Experiment 3: Biliary conjugates detected in bile, non-polar ether-extractable metabolites (5% of dose) detected in faeces.

Experiment 3: Assuming 25% of the ip dose appeared in bile of first animal and also that 25% of conjugates appeared in bile of second animal, it was calculated that 40% of the biliary conjugates are hydrolysed and reabsorbed on the first pass. Extensive enterohepatic circulation is therefore possible. Part of the absorbed linalool is thus excreted by faeces.

Conclusions:
Complete absorption, no bioaccumulation, rapid excretion, glucuronide- and sulfate conjugates, enterohepatic recirculation. Under the conditions of this study, linalool (500 mg/kg bw) was rapidly and completely absorbed in rats after oral (gavage) administration. After 72h, 3% of the dose remained in tissues. Intraperitoneal administration (20 mg/animal) showed that at least 25% of the dose is excreted in bile (faeces). Enterohepatic circulation was detected and biliary conjugates and non-polar ether-extractable metabolites (in faeces) are formed. These are glucuronide- and sulfate conjugates. Within 72 h, 97% are excreted with the majority (ca. 80%) after 36h and 95% after 48h. 60% of administered radioactivity occured in urine, 15% in faeces and 25% in expired air.
Executive summary:

14C labelled linalool was administered orally (gavage) and intraperitoneally to male Wistar rats in this study. For the first exposure route a dose of 500 mg/kg bw was used, for the second route this was 20 mg/animal.

Experiment 1: After intragastric administration, urine, faeces and expired air were collected and measured at several intervals over a 72 hours period. Radioactivity was determined. At the end of the experiment, residual radioactivity in brain, lung, liver, heart, spleen, gastro-intestinal tract, kidney, skin and skeletal muscle was determined.

Experiment 2: In this experiment intraperitoneal administration was used to determine if biliary excretion occurred. Bile was collected at several intervals up to 11 hours after exposure of 2 rats and radioactivity was determined.

Experiment 3: In this experiment one rat was intraperitoneally exposed. Bile from a this treated animal was introduced into the duodenum of a second animal. Bile of this animal was then collected. Presence of radioactivity was indicative of enterohepatic circulation.

Under the conditions of this study, linalool (500 mg/kg bw) was rapidly and completely absorbed in rats after oral (gavage) administration. After 72 h, 3% of the dose could be detected in tissues. Intraperitoneal administration (20 mg/animal) showed that ca. 25% of the dose is excreted in bile (faeces). Enterohepatic circulation is possible and that biliary conjugates and non-polar ether-extractable metabolites (faeces) are formed. These are likely glucuronide and sulfate conjugates. Excretion is rapid; within 36h 80% of radioactivity and within 48h 95% of radioactivity had been excreted. After 72h, majority was excreted via urine (60% of administered radioactivity), faeces (15%), expired air (23%).

Endpoint:
basic toxicokinetics
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See attached justification
Reason / purpose for cross-reference:
read-across source
Radiolabelling:
yes
Remarks:
14C
Preliminary studies:
Not relevant
Details on absorption:
Experiment 1: at least 85% of linalool is absorbed (detected in urine and expired air) after 72 hours
Details on distribution in tissues:
Experiment 1 (in total: 3% of dose) after 72 hours:- Liver: 0.5%- Gut: 0.6%- Skin: 0.8%- Skeletal muscle: 1.2%- Other organs: insignificant amounts of radioactivity
Details on excretion:
Experiment 1: approx. 100% of the administered dose is excreted (urine, faeces, expired gas) after 72 hours.- Expired air: 23%- Urine: approx. 60%- Faeces: approx. 15% (delayed, possible biliary excretion)Experiment 2:- Bile: more than 25% of the ip dose detected after 11 hoursExperiment 3:- Bile: 2.5% of ip dose detected in second rat after 12 hours
Metabolites identified:
yes
Details on metabolites:
Experiment 2: No free linalool, but only polar conjugates were detectable in bile (partially hydrolysed by beta-glucuronidase and to a greater extent by a mixture of beta-glucuronidase and sulphatase).Experiment 3: Biliary conjugates detected in bile, non-polar ether-extractable metabolites (5% of dose) detected in faeces.

Experiment 3: Assuming 25% of the ip dose appeared in bile of first animal and also that 25% of conjugates appeared in bile of second animal, it was calculated that 40% of the biliary conjugates are hydrolysed and reabsorbed on the first pass. Extensive enterohepatic circulation is therefore possible. Part of the absorbed linalool is thus excreted by faeces.

Conclusions:
Complete absorption, no bioaccumulation, rapid excretion, glucuronide- and sulfate conjugates, enterohepatic recirculation. Under the conditions of this study, linalool (500 mg/kg bw) was rapidly and completely absorbed in rats after oral (gavage) administration. After 72h, 3% of the dose remained in tissues. Intraperitoneal administration (20 mg/animal) showed that at least 25% of the dose is excreted in bile (faeces). Enterohepatic circulation was detected and biliary conjugates and non-polar ether-extractable metabolites (in faeces) are formed. These are glucuronide- and sulfate conjugates. Within 72 h, 97% are excreted with the majority (ca. 80%) after 36h and 95% after 48h. 60% of administered radioactivity occured in urine, 15% in faeces and 25% in expired air. This study was used for read-across to ethyllinalyl acetate.
Executive summary:

14C labelled linalool was administered orally (gavage) and intraperitoneally to male Wistar rats in this study. For the first exposure route a dose of 500 mg/kg bw was used, for the second route this was 20 mg/animal.

Experiment 1: After intragastric administration, urine, faeces and expired air were collected and measured at several intervals over a 72 hours period. Radioactivity was determined. At the end of the experiment, residual radioactivity in brain, lung, liver, heart, spleen, gastro-intestinal tract, kidney, skin and skeletal muscle was determined.

Experiment 2: In this experiment intraperitoneal administration was used to determine if biliary excretion occurred. Bile was collected at several intervals up to 11 hours after exposure of 2 rats and radioactivity was determined.

Experiment 3: In this experiment one rat was intraperitoneally exposed. Bile from a this treated animal was introduced into the duodenum of a second animal. Bile of this animal was then collected. Presence of radioactivity was indicative of enterohepatic circulation.

Under the conditions of this study, linalool (500 mg/kg bw) was rapidly and completely absorbed in rats after oral (gavage) administration. After 72 h, 3% of the dose could be detected in tissues. Intraperitoneal administration (20 mg/animal) showed that ca. 25% of the dose is excreted in bile (faeces). Enterohepatic circulation is possible and that biliary conjugates and non-polar ether-extractable metabolites (faeces) are formed. These are likely glucuronide and sulfate conjugates. Excretion is rapid; within 36h 80% of radioactivity and within 48h 95% of radioactivity had been excreted. After 72h, majority was excreted via urine (60% of administered radioactivity), faeces (15%), expired air (23%). This study was used for read-across to ethyllinalyl acetate.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was not conducted according to a guideline or under GLP conditions. A proper design was used, however, the study is reported concise in a publication.
Reason / purpose for cross-reference:
reference to same study
Objective of study:
other: interaction with hepatic drug-metabolism enzymes
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male rats were exposed to linalool or vehicle (control group) by intragastric intubation (gavage) for 64 days. Body and liver weight were observed. Liver homogenates and microsomal fractions were prepared from the livers of sacrificed rats at several timepoints during the dosing period. Biphenyl-4-hydroxylase, 4-methylumbelliferone glucuronyltransferase and alchohol dehydrogenase activity and cytochrome P-450, cytochrome b5 and microsomal protein were determined.
GLP compliance:
no
Radiolabelling:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS- Age at study initiation: 4 weeks
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
VEHICLE- Concentration in vehicle: 25% (w/v)- Amount of vehicle (if gavage): 2 ml/kg (calculated by: 0.5 (g/kg bw/day) / 0.25 (concentration))
Duration and frequency of treatment / exposure:
64 days, once daily exposure
Remarks:
Doses / Concentrations:500 mg/kg bw day
No. of animals per sex per dose / concentration:
24 (4 animals per timepoint)
Control animals:
yes, concurrent vehicle
Positive control reference chemical:
Not relevant
Details on study design:
No data
Details on dosing and sampling:
ENZYME INDUCTION STUDY- Tissues sampled: Liver (homogenates and microsomal fractions)- Time and frequency of sampling: at 0, 3, 7, 14, 30 and 64 days after dosing- From how many animals: 4 animals/interval (control and test group)
Statistics:
No data
Preliminary studies:
Not relevant
Details on absorption:
Not relevant
Details on distribution in tissues:
Not relevant
Details on excretion:
Not relevant
Metabolites identified:
not measured
Details on metabolites:
Presence and activity of enzymes was evaluated (see field "Any other information on results incl. tables")

Parameter Effect
Body weight gain Unaffected over 64 days  
Absolute liver weight Unaffected until 30 days Significantly increased after 64 days
Relative liver weight Unaffected until 30 days Significantly increased after 64 days
Microsomal protein concentration Unaffected up to 14 days Elevated from day 30 (20%) up to 64 days
Cytochrome P-450 Biphasic response Depressed on day 7, increased (50%) on day 30-64
b5 concentration Biphasic response Depressed on day 7, increased on day 30 (50%) up to day 64 (70%)
Biphenyl 4-hydroxylase Unaffected over 64 days  
4-Methylumbelliferone glucuronyltransferase Dramatical increase Day 3: 17%, day 64: 150%)
Alcohol dehydrogenase Biphasic response Depressed on day 3 (33%), increased on day 7 (36%), unaffected from day 14
Conclusions:
Repeated administration of linalool results in increased liver weights and increased liver enzyme activities (mainly glucuronidation). Under the conditions of this study, it was found that 64-day oral exposure of rats to 500 mg/kg bw/day linalool induces liver effects. Liver weight was increased, as well as the activity of liver enzymes responsible for metabolism of linalool. It was concluded that the observed effects represent a physiological adaptation to linalool exposure.
Executive summary:

Male Wistar rats were exposed to 500 mg/kg bw/day linalool or a similar volume of propylene glycol (control group) by intragastric intubation (gavage) for 64 days. Body and liver weight were observed. Liver homogenates and microsomal fractions were prepared from the livers of sacrificed rats at several timepoints during the dosing period (4 animals/timepoint). Biphenyl-4-hydroxylase, 4-methylumbelliferone glucuronyltransferase and alchohol dehydrogenase activity and cytochrome P-450, cytochrome b5 and microsomal protein were determined.

Under the conditions of this study, it was found that 64-day oral exposure of rats to 500 mg/kg bw/day linalool induces liver effects. Liver weight was increased, as well as the activity of liver enzymes responsible for metabolism of linalool (cytochrome P-450, cytochrome b5, biphenyl-4 -hydroxylase and 4 -methylumbelliferone glucuronyltransferase) after 64 days. Alcohol dehydrogenase activity was normal during the latter part (14-64 days) of the study period. It was concluded that the observed effects represent a physiological adaptation to linalool exposure.

Endpoint:
basic toxicokinetics in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
See attached justification
Reason / purpose for cross-reference:
read-across source
Radiolabelling:
no
Preliminary studies:
Not relevant
Details on absorption:
Not relevant
Details on distribution in tissues:
Not relevant
Details on excretion:
Not relevant
Metabolites identified:
not measured
Details on metabolites:
Presence and activity of enzymes was evaluated (see field "Any other information on results incl. tables")

Parameter Effect
Body weight gain Unaffected over 64 days  
Absolute liver weight Unaffected until 30 days Significantly increased after 64 days
Relative liver weight Unaffected until 30 days Significantly increased after 64 days
Microsomal protein concentration Unaffected up to 14 days Elevated from day 30 (20%) up to 64 days
Cytochrome P-450 Biphasic response Depressed on day 7, increased (50%) on day 30-64
b5 concentration Biphasic response Depressed on day 7, increased on day 30 (50%) up to day 64 (70%)
Biphenyl 4-hydroxylase Unaffected over 64 days  
4-Methylumbelliferone glucuronyltransferase Dramatical increase Day 3: 17%, day 64: 150%)
Alcohol dehydrogenase Biphasic response Depressed on day 3 (33%), increased on day 7 (36%), unaffected from day 14
Conclusions:
Repeated administration of linalool results in increased liver weights and increased liver enzyme activities (mainly glucuronidation). Under the conditions of this study, it was found that 64-day oral exposure of rats to 500 mg/kg bw/day linalool induces liver effects. Liver weight was increased, as well as the activity of liver enzymes responsible for metabolism of linalool. It was concluded that the observed effects represent a physiological adaptation to linalool exposure. This study was used for read-across to ethyllinalyl acetate.
Executive summary:

Male Wistar rats were exposed to 500 mg/kg bw/day linalool or a similar volume of propylene glycol (control group) by intragastric intubation (gavage) for 64 days. Body and liver weight were observed. Liver homogenates and microsomal fractions were prepared from the livers of sacrificed rats at several timepoints during the dosing period (4 animals/timepoint). Biphenyl-4-hydroxylase, 4-methylumbelliferone glucuronyltransferase and alchohol dehydrogenase activity and cytochrome P-450, cytochrome b5 and microsomal protein were determined.

Under the conditions of this study, it was found that 64-day oral exposure of rats to 500 mg/kg bw/day linalool induces liver effects. Liver weight was increased, as well as the activity of liver enzymes responsible for metabolism of linalool (cytochrome P-450, cytochrome b5, biphenyl-4 -hydroxylase and 4 -methylumbelliferone glucuronyltransferase) after 64 days. Alcohol dehydrogenase activity was normal during the latter part (14-64 days) of the study period. It was concluded that the observed effects represent a physiological adaptation to linalool exposure. This study was used for read-across to ethyllinalyl acetate.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 dec 2017 - 03 dec 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Objective of study:
toxicokinetics
Qualifier:
according to guideline
Guideline:
other:
Version / remarks:
ICH S3a. Toxicokinetics: The Assessment of Systemic Exposure in Toxicity Studies,
October 1994.
Deviations:
not applicable
GLP compliance:
yes
Specific details on test material used for the study:
No correction factor required
Radiolabelling:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species
for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/
developmental historical data in this species from the same strain and source. This animal model has
been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (males) and 13 weeks (females)
- Weight at study initiation: 261 - 295 g (males) and 209 and 241 g (females)
- Fasting period before study: No
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in Macrolon cages.
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon cages.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad
libitum . The feed was analyzed by the supplier for nutritional components and environmental contam
inants.
- Water: tap water, ad libitum. During motor activity measurements, animals had no access to water f
or a maximum of 2 hours. Periodic analysis of the water was performed.
- Acclimation period: 5 days
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily as a solution and dosed within 4 hours after adding the test item to the vehicle protected from light.
The test item was directly mixed with the vehicle to prevent any loss; therefore vehicle was weighed prior to the test item.
Test item dosing formulations were kept at room temperature protected from light until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.
Dose volume: 5 mL/kg body weight.
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
No. of animals per sex per dose / concentration:
10
Control animals:
yes, concurrent vehicle
Positive control reference chemical:
no
Details on study design:
On Day 10 of treatment, blood samples (~0.4 mL using K2-EDTA tubes) were collected from TK animals in all dose groups at pre-dose and at 0.25, 0.5, 1, 2, 4, 8 and 24 hours postdose by sampling from the jugular vein.
Details on dosing and sampling:
- On Day 10 of treatment, blood samples (~0.4 mL using K2-EDTA tubes) were collected from thejugular vein from 6 animals/sex/group of Groups 1-4. Animals were restrained during blood collecti on.
- Samples were collected at 0 (before dosing) and at 15 min, 30 min, 1, 2, 4, 8 and 24 hours (after dosing).
- Parameters that were calculated included tlast, tmax, Cmax, AUClast, dose effect, sex effect and exposure to metabolite versus parent. The t1/2 value could only be calculated in females at the high dose. For low- and mid-dose females and all groups of males t1/2 values could not be calculated since no log linear regression was possible.
Statistics:
Descriptive statistics (means and standard deviation) for concentration data, using appropriate grouping and sorting variables, were generated using Phoenix. Concentration and TK parameter values were tabulated and concentration vs time graphs were generated. Dose effect, sex differences and parent versus metabolite evaluations of Cmax and/or AUC values were evaluated where appropriate. To conclude on dose-proportional exposure, group means should be roughly within a 2-fold of the dose increment. To conclude on comparable exposure, respective groups mean should be roughly within a factor of 2.
Type:
absorption
Results:
Ethyllinalyl Acetate was absorbed from the gastrointestinal track and was metabolized to Ethyllinalool.
Details on absorption:
The toxicokinetic evaluation showed the following: Ethyllinalyl Acetate was absorbed from the gastrointestinal track and was metabolized to Ethyllinalool. The peak blood concentration, Cmax, of test item was reached at 2-4 h. Tlast ranged from 8 to 24 h post-dose and increased with increasing dose levels. The apparent terminal half-life of the test item for females at 1000 mg/kg and was 3.2 h. Cmax of the metabolite was 4 h post-dose.
Observation:
not determined
Toxicokinetic parameters:
half-life 1st: 3.2h
Remarks:
could only be measured in females 1000mg/kg
Toxicokinetic parameters:
AUC: see table
Toxicokinetic parameters:
Cmax: see table
Remarks:
At 200 mg/kg no clear sex differences were noted for Cmax and AUClast. At 1000 mg/kg higher exposures, in terms of Cmax and AUClast, were noted in females compared to males.
Toxicokinetic parameters:
Tmax: see table
Metabolites identified:
yes
Remarks:
Ethyllinalool
Details on metabolites:
Blood was analysed for the concentrations of the test item and a defined metabolite i.e. Ethyllinalool.
Ethyllinalyl Acetate was absorbed from the gastrointestinal track and was metabolized to Ethyllinalool. Cmax of the metabolite was 4 h post-dose. Tlast of the metabolite ranged from 4 to 24 h post-dose and increased with increasing dose levels.
Conclusions:
Ethyllinalyl Acetate was absorbed from the gastrointestinal track and was metabolized to Ethyllinalool.
Executive summary:

The toxicokinetic evaluation showed the following: Ethyllinalyl Acetate was absorbed from the gastrointestinal track and was metabolized to Ethyllinalool. The peak blood concentration, Cmax, of test item was reached at 2-4 h. Tlast ranged from 8 to 24 h post-dose and increased with increasing dose levels. The apparent terminal half-life of the test item for females at 1000 mg/kg and was 3.2 h. Cmax of the metabolite was 4 h post-dose. Tlast of the metabolite ranged from 4 to 24 h post-dose and increased with increasing dose levels. In males, both Cmax and systemic exposure of the test item expressed as AUClast increased less than dose-proportionally. In females, Cmax increased dose-proportionally and AUClast increased more than dose-proportionally. Sex differences in Cmax and AUClast were observed at 1000 mg/kg for the test item and its metabolite. Cmax and AUClast were about 5-fold higher in females than in males. Systemic exposure to the metabolite, in terms of Cmax and AUClast, was lower than that to the unchanged test item. In males at 200 and 1000 mg/kg systemic exposure to the metabolite was about 10 times lower. In females at 1000 mg/kg exposure to the metabolite was about three times lower.

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
See attached justification
Reason / purpose for cross-reference:
read-across source
Signs and symptoms of toxicity:
not examined
Remarks:
in vitro study
Dermal irritation:
not examined
Remarks:
in vitro study
Absorption in different matrices:
See field "Remarks on results including tables and figures"
Total recovery:
- Total recovery (mean +/- SD): Unoccluded: 8.01 +/- 2.1%Occluded: 36.3 +/- 10.1%Control: no radiolabel contamination was detected in any of the compartmentsOverall evaporative loss: ~97%Permeation benzoic acid: 89.6 +/- 3.0%, indicating a valid test system- Recovery of applied dose acceptable: No, recovery should be 100 +/- 10%- Results adjusted for incomplete recovery of the applied dose: No, but evaporative loss is determined
Key result
Time point:
24 h
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 12.7 %
Remarks on result:
other: Occluded conditions (excl. epidermis)
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 0.7 %
Remarks on result:
other: 0.5 h
Remarks:
Open conditions (excl. epidermis)
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 1.3 %
Remarks on result:
other: 1 h
Remarks:
Open conditions (excl. epidermis)
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 2.7 %
Remarks on result:
other: 6 h
Remarks:
Open conditions (excl. epidermis)
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 2.9 %
Remarks on result:
other: 12 h
Remarks:
Open conditions (excl. epidermis)
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 3 %
Remarks on result:
other: 24 h
Remarks:
Open conditions (excl. epidermis)
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 1.3 %
Remarks on result:
other: 0.5 h
Remarks:
Occluded conditions (excl. epidermis)
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 3.4 %
Remarks on result:
other: 1 h
Remarks:
Occluded conditions (excl. epidermis)
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 10.1 %
Remarks on result:
other: 6 h
Remarks:
Occluded conditions (excl. epidermis)
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 11.7 %
Remarks on result:
other: 12 h
Remarks:
Occluded conditions (excl. epidermis)
Conversion factor human vs. animal skin:
Not applicable

Distribution of linalool and percentage absorption in different compartments (see Appendix 2):

Mean (+/- SD) distribution of linalool in all compartments (ug/cm2) (after 24 hours) 24h wipe Donor chamber Strip 1* Strips 2-3 Strips 4-6 Strips 7-10 Epidermis Filter paper Receptor phase
Unoccluded conditions Mean 5.41 2.16 0.399 0.553 0.295 0.172 0.65 0.609 5.95
  SD 1.06 0.9 0.256 0.443 0.164 0.097 0.448 0.191 3.17
Occluded conditions Mean 6.32 36.7 0.317 0.312 0.227 0.161 1.02 2.39 25.6
  SD 1.83 18.1 0.232 0.204 0.13 0.098 0.51 0.71 7.5

* Skin membranes were stripped 10 times and the strips were grouped

Absorption (%) of linalool in all compartments (after 24 hours) 24h wipe Donor chamber Strip 1* Strips 2-3 Strips 4-6 Strips 7-10 Epidermis Filter paper Receptor phase
Unoccluded conditions Mean 2.67 1.07 0.198 0.274 0.146 0.085 0.321 0.301 2.95
  SD 0.52 0.44 0.128 0.221 0.082 0.049 0.223 0.095 1.58
Occluded conditions Mean 3.14 18.2 0.158 0.155 0.113 0.08 0.506 1.19 12.7
  SD 0.91 9.0 0.116 0.101 0.065 0.049 0.253 0.35 3.7

* Skin membranes were stripped 10 times and the strips were grouped

Conclusions:
Under the conditions of this test, linalool was found to penetrate through human skin. Occlusion of the test system resulted in a higher absorption rate after 24 hours (3.0% after open application versus 12.7% after occlusion). However, a large portion of the applied dose evaporated within 24 hours.
Executive summary:

This study was conducted to determine the skin penetrating potential of linalool. An in vitro diffusion cell test system was used (methods equivalent or similar to OECD guideline 428). Prepared human skin membranes were exposed open and occluded to 40.2 mg/ml (201 ug/cm2, 1.2 cm2 cell) radiolabelled linalool (in 70/30% ethanol/water) for 24 hours and the amount of test substance left on the skin (by wipe), in the donor chamber, stratum corneum (tape strips), epidermis, filter paper and receptor chamber were determined by liquid scintillation counting. One control skin membrane was used (unoccluded) where only the vehicle was added. Evaporation loss was also determined in an additional experiment. A reference substance (benzoic acid) was used to test the validity of the test sytem.

Linalool was found to penetrate through skin and after 24 hours 3.0% of the applied dose was recoveredin receptor fluid in the open test system, versus 12.7% in the receptor fluid in the occluded test system. It was found that about 97% of the applied linalool dose evaporated within 24 hours, resulting in a very low total recovery in the open test system. Benzoic acid was found to penetrate rapidly through skin, total recovery was 89.6%. The test system is therefore shown to be valid.

Under the conditions of this test, linalool was found to penetrate through human skin. Occlusion of the test system resulted in a higher absorption rate after 24 hours (3.0% versus 12.7%). However, a large portion (97%)of the applied dose evaporated within 24 hours.

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was not performed according to GLP. An equivalent or similar guideline to OECD 428 is used. Study is reported extensively.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
Principles of method if other than guideline:
Not relevant
GLP compliance:
no
Radiolabelling:
yes
Remarks:
[1,2-14C]Linalool
Species:
human
Strain:
other: Not relevant
Sex:
female
Details on test animals or test system and environmental conditions:
Not relevant
Type of coverage:
other: Open and occlusive (greased coverslip to diffusion cell donor chamber)
Vehicle:
other: 70:30 ethanol/water (v/v)
Duration of exposure:
24 hours
Doses:
- Actual doses: 40.2 mg/ml (4.02%)- Dose volume: 5 ul/cm2 = 201 ug/cm2
No. of animals per group:
12 replicates/condition (open/occlusive) of 7 different donors
Control animals:
yes
Remarks:
one control cell (open, only vehicle), and benzoic acid as reference control
Details on study design:
DOSE PREPARATION- Method for preparation of dose suspensions: 199.7 mg linalool in 5 ml ethanol/water (70/30 v/v). 0.53 mg labelled linalool added to 2 ml of this solution.VEHICLE- Amount(s) applied (volume or weight with unit): 5 ul/cm2 * 1.2 cm2 = 6 ul- Concentration (if solution): 4.02%- Purity ethanol: 99.7-100%REMOVAL OF TEST SUBSTANCEAfter 24 hours with dry cotton budSAMPLE PREPARATION- Storage procedure: In tightly sealed vials- Preparation details: Extraction from media with different solvents (acetonitrile, optisolve for tape strips, methanol)ANALYSIS - Method type for identification: Liquid scintillation counting
Details on in vitro test system (if applicable):
SKIN PREPARATION- Source of skin: 7 different donors- Type of skin: Breast and abdominal- Preparative technique: According to accepted method- Membrane integrity check: Yes, by assessing permeation of tritiated water- Storage conditions: -20 degrees CelsiusPRINCIPLES OF ASSAY- Diffusion cell: Franz-type- Receptor fluid: PBS- Solubility of test substance in receptor fluid: 1 mg/ml- Static system: Yes, containing diffusion cell donor and receptor chamber- Test temperature: 37.0 +/- 0.5 degrees Celsius (skin surface: 32 +/- 1 degrees Celsius)- Occlusion: Open and closed- Reference substance(s): Benzoic acid (3.99 mg/ml in 50/50 (v/v) ethanol/water)
Signs and symptoms of toxicity:
not examined
Remarks:
in vitro study
Dermal irritation:
not examined
Remarks:
in vitro study
Absorption in different matrices:
See field "Remarks on results including tables and figures"
Total recovery:
- Total recovery (mean +/- SD): Unoccluded: 8.01 +/- 2.1%Occluded: 36.3 +/- 10.1%Control: no radiolabel contamination was detected in any of the compartmentsOverall evaporative loss: ~97%Permeation benzoic acid: 89.6 +/- 3.0%, indicating a valid test system- Recovery of applied dose acceptable: No, recovery should be 100 +/- 10%- Results adjusted for incomplete recovery of the applied dose: No, but evaporative loss is determined
Key result
Time point:
24 h
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 12.7 %
Remarks on result:
other: Occluded conditions (excl. epidermis)
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 0.7 %
Remarks on result:
other: 0.5 h
Remarks:
Open conditions (excl. epidermis)
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 1.3 %
Remarks on result:
other: 1 h
Remarks:
Open conditions (excl. epidermis)
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 2.7 %
Remarks on result:
other: 6 h
Remarks:
Open conditions (excl. epidermis)
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 2.9 %
Remarks on result:
other: 12 h
Remarks:
Open conditions (excl. epidermis)
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 3 %
Remarks on result:
other: 24 h
Remarks:
Open conditions (excl. epidermis)
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 1.3 %
Remarks on result:
other: 0.5 h
Remarks:
Occluded conditions (excl. epidermis)
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 3.4 %
Remarks on result:
other: 1 h
Remarks:
Occluded conditions (excl. epidermis)
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 10.1 %
Remarks on result:
other: 6 h
Remarks:
Occluded conditions (excl. epidermis)
Dose:
40.2 mg/ml
Parameter:
percentage
Absorption:
ca. 11.7 %
Remarks on result:
other: 12 h
Remarks:
Occluded conditions (excl. epidermis)
Conversion factor human vs. animal skin:
Not applicable

Distribution of linalool and percentage absorption in different compartments (see Appendix 2):

Mean (+/- SD) distribution of linalool in all compartments (ug/cm2) (after 24 hours) 24h wipe Donor chamber Strip 1* Strips 2-3 Strips 4-6 Strips 7-10 Epidermis Filter paper Receptor phase
Unoccluded conditions Mean 5.41 2.16 0.399 0.553 0.295 0.172 0.65 0.609 5.95
  SD 1.06 0.9 0.256 0.443 0.164 0.097 0.448 0.191 3.17
Occluded conditions Mean 6.32 36.7 0.317 0.312 0.227 0.161 1.02 2.39 25.6
  SD 1.83 18.1 0.232 0.204 0.13 0.098 0.51 0.71 7.5

* Skin membranes were stripped 10 times and the strips were grouped

Absorption (%) of linalool in all compartments (after 24 hours) 24h wipe Donor chamber Strip 1* Strips 2-3 Strips 4-6 Strips 7-10 Epidermis Filter paper Receptor phase
Unoccluded conditions Mean 2.67 1.07 0.198 0.274 0.146 0.085 0.321 0.301 2.95
  SD 0.52 0.44 0.128 0.221 0.082 0.049 0.223 0.095 1.58
Occluded conditions Mean 3.14 18.2 0.158 0.155 0.113 0.08 0.506 1.19 12.7
  SD 0.91 9.0 0.116 0.101 0.065 0.049 0.253 0.35 3.7

* Skin membranes were stripped 10 times and the strips were grouped

Conclusions:
Under the conditions of this test, linalool was found to penetrate through human skin. Occlusion of the test system resulted in a higher absorption rate after 24 hours (3.0% after open application versus 12.7% after occlusion). However, a large portion of the applied dose evaporated within 24 hours.
Executive summary:

This study was conducted to determine the skin penetrating potential of linalool. An in vitro diffusion cell test system was used (methods equivalent or similar to OECD guideline 428). Prepared human skin membranes were exposed open and occluded to 40.2 mg/ml (201 ug/cm2, 1.2 cm2 cell) radiolabelled linalool (in 70/30% ethanol/water) for 24 hours and the amount of test substance left on the skin (by wipe), in the donor chamber, stratum corneum (tape strips), epidermis, filter paper and receptor chamber were determined by liquid scintillation counting. One control skin membrane was used (unoccluded) where only the vehicle was added. Evaporation loss was also determined in an additional experiment. A reference substance (benzoic acid) was used to test the validity of the test sytem.

Linalool was found to penetrate through skin and after 24 hours 3.0% of the applied dose was recoveredin receptor fluid in the open test system, versus 12.7% in the receptor fluid in the occluded test system. It was found that about 97% of the applied linalool dose evaporated within 24 hours, resulting in a very low total recovery in the open test system. Benzoic acid was found to penetrate rapidly through skin, total recovery was 89.6%. The test system is therefore shown to be valid.

Under the conditions of this test, linalool was found to penetrate through human skin. Occlusion of the test system resulted in a higher absorption rate after 24 hours (3.0% versus 12.7%). However, a large portion (97%)of the applied dose evaporated within 24 hours.

Description of key information

Toxicokinetics ethyllinalyl acetate (ELAC)

 

Background

As part of an OECD 422 28-day repeated dose study (Thiel A and van Oetelaar D (2018b)), the toxicokinetics (TK) of Ethyllinalyl Acetate and its metabolite Ethyllinalool were evaluated. Wistar Han rats received daily oral gavage administration of the test substance at dose levels of 40, 200 and 1000 mg/kg for a minimum of 28 days. On day 10 blood samples were taken and parameters that were calculated included tlast, tmax, Cmax, AUClast, dose effect, sex effect and exposure to metabolite versus parent. Further information regarding the absorption, distribution and excretion, via inhalation and via the dermal route on ethyllinalyl acetate is limited. Therefore, some aspects of this toxicokinetic assessment are read-across substance linalool.

The substance linalool is widely used as flavour and fragrance ingredient. Considerable information on toxicokinetic behaviour, metabolism and dermal absorption is available. The studies addressing this subject are available in the REACH dossier. A study concerning the absorption, distribution and excretion of linalool after inhalation is available. Dermal absorption of linalool was studied in one in vivo and an in vitro study. A prediction of the metabolic pathway of ethyllinalyl acetate was also performed with the program METEOR and the results are included as attachment.

 

Information from physchem and Toxicokinetics/toxicity studies for ethyllinalyl acetate

Physical/chemical parameters

Log Kow, water solubility, vapour pressure, and molecular weight, as well as parameters like hydrolysis can provide useful information regarding the behaviour of a substance in the body. Ethyllinalyl acetate has a log Kow of 4.4 and a water solubility of 2.64 mg/l. The vapour pressure is 8.77 Pa, the molecular weight is 210.32 and the half-life for hydrolysis is 4.764 year.

 

 Absorption

The high log Kow and relatively low water solubility generally do not favour oral or inhalation absorption, but a lipophilic compound may be taken up nevertheless by micellular solubilisation. The low molecular weight and the fact that the substance does not hydrolize quickly supports that absorption can take place. Based on the previous, it is likely that ethyllinalyl acetate is absorbed in the human body via the oral and inhalation route, which is in fact supported by the finding of systemic effects in the oral 28-day repeated dose toxicity study by Thiel A and van Oetelaar D (2018a). Furthermore, the toxicokinetic evaluation which was performed as part of this study showed that:

-       Ethyllinalyl Acetate was absorbed from the gastrointestinal track and was metabolized to Ethyllinalool.

-       The peak blood concentration, Cmax, of test item was reached at 2-4 h.

-       The time point of last measurable blood concentration (Tlast) ranged from 8 to 24 h post-dose and increased with increasing dose levels.

-       The apparent terminal half-life of the test item for females at 1000 mg/kg and was 3.2 h.

-       Cmax of the metabolite was 4 h post-dose.

-       Tlast of the metabolite ranged from 4 to 24 h post-dose and increased with increasing dose levels.

-       In males, both Cmax and systemic exposure of the test item expressed as AUClast increased less than dose-proportionally. In females, Cmax increased dose-proportionally and AUClast increased more than dose-proportionally.

-       Sex differences in Cmax and AUClast were observed at 1000 mg/kg for the test item and its metabolite. Cmax and AUClast were about 5-fold higher in females than in males.

-       Systemic exposure to the metabolite, in terms of Cmax and AUClast, was lower than that to the unchanged test item. In males at 200 and 1000 mg/kg systemic exposure to the metabolite was about 10 times lower. In females at 1000 mg/kg exposure to the metabolite was about three times lower.

 

For inhalation exposure, inhalation is expected due to the technical function of the substance (fragrance), despite of the low vapour pressure. Given the Log Kow, the rate of penetration through skin may be limited by the rate of transfer between the stratum corneum and the epidermis, but uptake into the stratum corneum will be high which would not lead to systemic availability. Other phys-chem parameters like water solubility, molecular weight also does not favour penetration through skin. This is supported by the low dermal absorption rates found in studies for the read-across substance linalool and the lack of systemic effects found in a dermal repeated dose toxicity study (not included in this dossier).

 

Distribution

As the substance can be considered lipophilic, accumulation in adipose tissue would be favourable when the substance is absorbed systemically. The intracellular concentration will be higher than the extracellular concentration. Given the low water solubility, wide distribution throughout the body is not expected. This is supported by the available toxicokinetic evaluation data for Ethyllinalyl Acetate, which shows that the blood concentrations of Ethyllinalyl acetate increased slowly. The peak blood concentration in rat, Cmax, was reached at 2 to 4 hours after dosing, and the Cmax/D ranged from 0.314-1.62 in males and females.

 

Metabolism

Based on physchem paramaters it is difficult to predict metabolism. An in silico analysis was therefore performed using Meteor software from Lhasa (Meteor Nexus 3.0.0). The following mammalian metabolism steps were predicted:

(i)          hydrolysis of the ester bond to Ethyllinalool and acetate,

(ii)         hydroxylation either at the allylic position or at terminal methyl groups,

(iii)        epoxidation of the mono-substituted double bond followed by either GSH-addition or hydrolysis,

(iv)        oxidation of the formed primary alcohols to carboxylic acid,

(v)         glucuronidation of the carboxylic acid,

(vi)        oxidation of secondary alcohol to corresponding ketone.

A detailed overview of the results is provided in the attachment to this summary.

 

The formation and kinetics of the metabolite ethyllinalool was examined in the oral 28-day repeated dose toxicity study. The exposure to the metabolite Ethyllinalool, in terms of Cmax and AUClast, was lower compared with Ethyllinalyl acetate. In males, at 200 and 1000 mg/kg Ethyllinalyl acetate, the exposure of the metabolite Ethyllinalool was approximately a factor 10 lower compared to Ethyllinalyl acetate. In females, at 1000 mg/kg Ethyllinalyl acetate, the exposure of the metabolite Ethyllinalool, was approximately a factor 3 lower compared to Ethyllinalyl acetate. Indicating that not all Ethyllinalyl acetate is metabolised to Ethyllinalyl.

Furthermore, in the oral 28-day repeated dose toxicity study, treatment with Ethyllinalyl Acetate resulted in increased CYP content, increased microsomal protein and/or increased EROD, BROD, PROD, as well as glucuronidation activities. In combination with observations of increased liver weights, these findings are indicative of adaptive response due to metabolic enzyme induction.

 

Excretion

Based on the molecular weight of ethyllinalyl acetate the substance is favourable for excretion via the urine. The renal effects observed in the OECD422 study also suggest involvement in the excretory process. This further supported by the available data for the read-across substance linalool. Exfoliation is likely after dermal exposure as ethyllinalyl acetate is expected to not penetrate further based on its high lipophilicity.

 

Studies on toxicokinetics for read-across substance linalool

The oral absorption of linalool in rats was rapid after an oral dose of 500 mg/kg bw linalool (radiolabelled). Within 2 days after treatment, radioactivity was excreted via urine (approx. 60%), expired air (approx. 23%), and faeces (approx. 15%) indicating that at least 85% of the applied dose was absorbed. A separate experiment showed that considerable enterohepatic circulation is possible, and that bilary conjugates and non-polar ether extractable metabolites are formed and excreted via faeces. Overall, this may indicate that radioactivity was completely absorbed. Three percent of the dose was distributed in tissues after 72 h of dosing; linalool was detected in liver (0.5%), gut (0.6%), skin (0.8%) and skeletal muscle (1.2%). Other organs including kidneys contained insignificant residual radioactivity. Metabolites detected in urine and bile indicated that linalool is largely excreted in the form of glucuronic acid conjugates. (Parke et al., 1974a).

 

After exposure to linalool by inhalation, linalool could be detected in blood of exposed mice (7-9 ng/mL serum). (Jirovetz et al., 1991) Other reports showed that after inhalation of linalool at 3.21 mg/L for 30, 60, and 90 min, plasma levels were around 1, 2.5, and 3 ng/mL plasma. (Buchbauer et al., 1991).

 

To determine dermal absorption, two in vitro studies were performed that were both similar to guideline OECD 428. In a study in which human skin was exposed to radiolabelled linalool for 24 hours, both open and occluded, in a solution of ethanol/water (70:30).12.7%in receptor fluid, excl. epidermiswhen exposure was occluded, open exposure resulted in an3.0%in receptor fluid, excl. epidermis). When linalool was applied open, approximately 97% of the applied dose evaporated within 24 hours. (Green, 2007).

 

A second in vitro dermal absorption study studied the absorption of linalool in human skin after 4 hours of (occluded) exposure. Linalool easily penetrated into the skin, however it was not detected in the acceptor fluid. The dermal absorption of linalool was determined to be 0.17%. (Cal and Sznitowska, 2003).

 

An in vivo dermal absorption study confirms the limited dermal absorption of linalool. In this study the dermal absorption of linalool from lavender oil was studied in one male human subject. The applied dose was about 7 mg linalool. Results showed that linalool present in plasma peaks approximately after 20 min (ca. 120 ng/mL). Plasma levels return to almost background after 90 min indicating rapid elimination from plasma. (Jäger et al., 1991).

 

In four studies, metabolism of linalool was studied, mainly by investigating enzyme induction upon exposure with linalool. Increased activity of cytochrome P450, cytochrome b5, biphenyl-4-hydroxylase and 4-methylumbelliferone glucuronyltransferase were observed in rats that were exposed to linalool for 64 days at a dose level of 500 mg/kg bw/d. (Parke et al., 1974b)

In another in vivo study in which rats (600 mg/kg bw/d) were exposed to linalool for six days, increased P450-activity was observed up to three days of dosing, whereas activities had returned to normal after six days of dosing. In the same study, the metabolites 8-hydroxy linalool and 8-carboxy linalool were identified in urine (after acidic hydrolysis) after 20 days of exposure, indicating that 8-hydroxylation seems to be the major Phase I metabolism pathway. In addition, other minor metabolites could not be characterised. (Chadha and Madhava Madyastha, 1984).

The metabolism of linalool by human skin P450 enzymes CYP2C19 and CYP2D6 was studied in another in vitro study. Linalool was metabolized to (R/S)-furanoid-linalool oxide, (R/S)-pyranoid-linalool oxide and (cis/trans)-8-hydroxylinalool, dependent on time, linalool, and enzyme concentration. (Meesters et al., 2007).

Phase II metabolism was also studied using an in vitro experiment. It was shown that when the phase II enzyme UDP-glucuronosyltransferase (UDPGT) was induced by phenobarbital, UDPGT specific activity towards linalool was higher than when UDPGT was induced by 3-methylcholanthrene. These experiments indicate that linalool is conjugated with glucuronic acid. (Boutin, J.A. et al., 1985).

 

It can be concluded that linalool is rapidly absorbed after oral administration (at least 85%). After 72h, 97% of the administered radioactivity were excreted. 3% of the dose was detected in tissues (liver, gut, skin and skeletal muscle) 72 h after dosing. Linalool was excreted mainly via urine (60%), exhaled air (23%) and faeces (15%) and is subject to enterohepatic recirculation The dermal absorption of linalool is low: 0.17% after 4 hours exposure (occluded). Experiments show that most of the dermally applied linalool evaporates from skin.

 

Information from public literature for read-across substance linalool. The most reliable references from literature were included in the dossier and summarized in the paragraph above. Additionally, an OECD SIDS assessment is available in which the toxicokinetics of linalool is discussed. In the document the rapid oral absorption of linalool is confirmed, as well as the excretion in urine, faeces, and via the expired air. The conclusion of the OECD SIDS assessment is that the relatively rapid overall excretion of linalool and its metabolites suggests no long-term hazard. The studies of Parke (1974) with linalool suggest that its metabolites are harmless. A large doses of linalool may be metabolised in the rat by conjugation and excretion in urine and bile, while a substantial resorption may enter intermediary metabolism up to formation of carbon dioxide that is excreted by pulmonary route. The rapid excretion of linalool and its metabolites suggests no long-term hazard from tissue accumulation on chronic concentrations normally encountered in foods. However, enterohepatic re-circulation might have the effect of prolonging the metabolic load on the liver over a relatively short period.

 

Conclusions

In conclusion, ethyllinalyl acetate is expected to be absorbed after oral exposure and is excreted mainly and rapidly via urine and faeces. Overall, the assessment indicates no potential for bioaccumulation (limited distribution in the body is expected). The data indicate further, that ethyllinalyl acetate is expected to be metabolized to harmless metabolites. Dermal absorption after exposure is considered to be low and not expected to contribute substantially to the toxicity of the substance. As a worst-case 12.7% absorption can be used for hazard assessment.

 

References

Boutin, J.A., Thomassin, J., Siest, G., Cartier, A. (1985). Heterogeneity of hepatic microsomal UDP-glucuronosyltransferase activities. Conjugation of phenolic and monoterpenoid aglycones in control and induced rats and guinea pigs. Biochemical Pharmacology, 34, 113: 2235-2249.

 

Cal., K., Sznitowska, M. (2003). Cutaneous absorption and elimination of three acyclic terpenes. Journal of Controlled Release, 92: 369-376.

 

Chadha, A., Madhava Madyastha, K. (1984). Metabolism of geraniol and linalool in the rat and effects on liver and lung microsomal enzymes. Xenobiotica, 1984, 14, 5: 365-374.

 

Green, D.M. (2007). In vitro human skin penetration of fragrance material linalool under both in-use and occluded conditions from an ethanol/water vehicle. Research Institute for Fragrance Materials (RIFM), report nr. R03/13a/05.

 

Jäger, W., Buchbauer, G., Jirovetz, L., Fritzer, M. (1991). Percutaneous absorption of lavender oil from a massage oil. Journal of the Society of Cosmetic Chemists, 43: 49-54.

 

Jirovetz, L., Jäger, W., Burchbauer, G., Nikiforev, A., Raverdino, V. (1991). Investigations of animal blood samples after fragrance drug inhalation by gas chromatography/mass spectrometry with chemical ionization and selected ion monitoring. Biological Mass Spectrometry, 20: 801-803.

 

Meesters, R.J.W., Duisken, M., Hollender, J. (2007). Study on the cutochrome P450-mediated oxidative metabolism of the terpene alcohol linalool: Indication of biological epoxidation. Xenobiotica, 37 (6):604-617.

 

OECD SIDS (2002). Linalool. UNEP Publications.

 

Parke, D.V., Quddusur Rahman, K.H.M., Walker, R. (1974a). The absorption, distribution and excretion of linalool in the rat. Biochemical Society Transactions 2: 612-615.

 

Parke, D.V., Quddusur Rahman, K.H.M., Walker, R. (1974b). Effect of linalool on hepatic drug-metabolizing enzymes in the rat. Biochemical Society Transactions 2: 615-618.

 

Remus, T. (2017). Summary of Toxicological Data for REACH Dossier (10 – 100 t) for Ethyllinalyl acetate. Draft internal document, version 1. DSM Nutritional Products.

 

Thiel A and van Oetelaar D (2018a) 14-day dose range finding study for a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of Ethyllinalyl Acetate by oral gavage in rats, Study performed at CRL Den Bosch, The Netherlands. DSM Internal Document RDR 00057531

Thiel A and van Oetelaar D (2018b) Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of Ethyllinalyl Acetate by oral gavage in rats, Study performed at CRL Den Bosch, The Netherlands. DSM Internal Document RDR 00057532

 

 

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
12.7
Absorption rate - inhalation (%):
100

Additional information