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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
Thymol was dissolved in propylene glycol (1-2 ml) and given 6 male albino rats per stomach tube at a dose level of 1 mmol/kg bw (ca. 150 mg/kg bw). Similar animals receiving solvent only served as controls. Urine samples were collected at -10°C at 24 hours intervals and anaylsed by chromatography after conjugate hydrolysis.
GLP compliance:
not specified
Radiolabelling:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Mollegaards Breeding Centre Ltd., Ejby, Denmark
- Weight at study initiation: 250 - 350 g
- Diet (e.g. ad libitum): ad libitum. They were switched from a standard pellet diet to a purified diet two days before dosing and during the experiments in order to reduce the number of normal chromatographic peaks in the urine extracts.
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test compounds was dissolved in propylene glycol (1-2 mL) and given by stomach tube at a dose level of 1 mmol/kg.
Duration and frequency of treatment / exposure:
Single application
Dose / conc.:
150.22 mg/kg bw/day
Remarks:
1 mmol = 150.22 mg/kg bw
No. of animals per sex per dose / concentration:
6
Control animals:
yes, concurrent vehicle
Details on dosing and sampling:
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: 24 h intervals
- From how many animals (samples pooled or not) : 6 animals, not pooled
- Method type(s) for identification: GLC-MS

TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable): conjugate hydrolysis using a glucuronidase + sulphatase preparation

ANALYTICAL METHOD
- Complete description including: The capillary GLC columns employed were prepared in using SE-54 (bonded phase), OV-1701 (bonded phase), Pluronic 64 and Emulphor. TMS-derivatives were chromatographed using 20 m SE-54 column (0.32 mm x 20 m) at 100°C for 2 min., then increasing at 8°C/min to 260°C. Methyl ester-derivatives chromatographed using OV-1701 column (0.29 mm x 30 m) at 100°C, then increasing at 8°C/min to 250°C. Mass spectra recorded from > m/z 80 (TMS-derivatives) or > m/z 50 (methyl ester-derivatives).
Type:
metabolism
Results:
Oxidation of the methyl and isopropyl groups occurred. This resulted in the formation of derivatives of benzyl alcohol and 2-phenylpropanol and their corresponding carboxylic acids. In contrast, ring hydroxylation of the two phenols was a minor reaction.
Details on excretion:
The urinary excretion of metabolites were rapid. Only very small amounts were excreted after 24 h.
Metabolites identified:
yes
Details on metabolites:
In the 24 h rat urine the following metabolites were identified:
2.5-Dihydroxy-p-cymene, 2-(2-hydroxy-4-methylphenyl)propan-1-ol, 5-Hxdroxymethyl)-2-(1-methylethyl)phenol, 2-(4-Hydroxymethyl-2-hydroxyphenyl)propan-1-ol, 2-(2-Hydroxy-4-methylphenyl)propionic acid, 3-Hydroxy-4-(1-methylethyl)benzoic acid (please refer to attached figure)

The urinary excretion of metabolites were rapid. Only very small amounts were excreted after 24 h. Although large quantities of thymol was excreted unchanged (or as their glucuronide and sulfate conjugates), extensive oxidation of the methyl and isopropyl groups also occurred. This resulted in the formation of derivatives of benzyl alcohol and 2-phenylpropanol and their corresponding carboxylic acids. In contrast, ring hydroxylation of the two phenols was a minor reaction.

The following metabolites were identified: 2.5-Dihydroxy-p-cymene, 2-(2-hydroxy-4-methylphenyl)propan-1-ol, 5-Hxdroxymethyl)-2-(1-methylethyl)phenol, 2-(4-Hydroxymethyl-2-hydroxyphenyl)propan-1-ol, 2-(2-Hydroxy-4-methylphenyl)propionic acid, 3-Hydroxy-4-(1-methylethyl)benzoic acid (please refer to attached figure).

Conclusions:
The urinary excretion of metabolites were rapid. Only very small amounts were excreted after 24 h. Although large quantities of thymol was excreted unchanged (or as their glucuronide and sulfate conjugates), extensive oxidation of the methyl and isopropyl groups also occurred. This resulted in the formation of derivatives of benzyl alcohol and 2-phenylpropanol and their corresponding carboxylic acids. In contrast, ring hydroxylation of the two phenols was a minor reaction.
Executive summary:

Thymol was dissolved in propylene glycol (1-2 mL) and given 6 male albino rats per stomach tube at a dose level of 1 mmol/kg bw (ca. 150 mg/kg bw). Similar animals receiving solvent only served as controls. Urine samples were collected at -10°C at 24 hours intervals and analysed by chromatography after conjugate hydrolysis.

The urinary excretion of metabolites were rapid. Only very small amounts were excreted after 24 h. Although large quantities of thymol was excreted unchanged (or as their glucuronide and sulfate conjugates), extensive oxidation of the methyl and isopropyl groups also occurred. This resulted in the formation of derivatives of benzyl alcohol and 2-phenylpropanol and their corresponding carboxylic acids. In contrast, ring hydroxylation of the two phenols was a minor reaction. The following metabolites were identified: 2.5-Dihydroxy-p-cymene, 2-(2-hydroxy-4-methylphenyl)propan-1-ol, 5-Hxdroxymethyl)-2-(1-methylethyl)phenol, 2-(4-Hydroxymethyl-2-hydroxyphenyl)propan-1-ol, 2-(2-Hydroxy-4-methylphenyl)propionic acid, 3-Hydroxy-4-(1-methylethyl)benzoic acid.

Endpoint:
basic toxicokinetics in vivo
Type of information:
other: expert statement
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The toxicokinetic profile of thymol (CAS 89-83-8) was assessed based on existing studies on toxicokinetics and toxicity and the physical-chemical properties of the substance.
Objective of study:
absorption
distribution
excretion
toxicokinetics
Qualifier:
no guideline available
Principles of method if other than guideline:
The toxicokinetic profile of thymol (CAS 89-83-8) was assessed based on existing studies on toxicokinetics and toxicity and the physical-chemical properties of the substance.
GLP compliance:
no
Type:
absorption
Results:
An absorption of 100 % is assumed for the oral, dermal and inhalation route as worst-case.
Type:
distribution
Results:
Thymol is expected to be widely distributed in the body via blood circulation. It may diffuse through the aqueous pores of cell membranes. Based on the low log P (< 3) no accumulation in fatty tissue or stratum corneum is expected.
Type:
excretion
Results:
The main excretion route is urinary excretion.
Details on absorption:
Oral absorption
To determine the systemic availability of thymol a clinical trial was carried out in 12 healthy volunteers. Each subject received a single dose of a Bronchipret® TP tablet, which is equivalent to 1.08 mg thymol. Thymol was absorbed quickly. Considerable plasma concentrations of thymol sulfate could already be detected after 20 minutes. This fast absorption indicates that thymol is mainly absorbed in the upper part of the gut (Kohlert et al. 2002). Also, based on the physical-chemical properties a high absorption rate via the gastro-intestinal tract is expected for thymol. In fact, absorption by passive diffusion is favoured for substances with moderate log P values (between -1 and 4). With a log P of 3.3, thymol is likely to follow this route of absorption. Additionally, based on the low molecular weight (< 200 g/mol) and due to the its solubility in water (980 mg/L), thymol may also be absorbed by passing through aqueous pores or being carried through the epithelial barrier by the bulk passage of water.
Taken together, based on the available studies in combination with the physical-chemical properties an oral absorption rate of 100 % is assumed for thymol.

Dermal absorption
In a study according to OECD Guideline 404 thymol was shown to have skin corrosive effects. Therefore, the substance is expected to destroy the barrier function of the skin. Moreover, the physical-chemical properties of thymol indicate the potential for dermal absorption. Absorption in the stratum corneum is favoured for substances with a molecular weight below 100 g/mol, but it is also possible for substances with a molecular weight of below 500 g/mol. To cross the lipid-rich stratum corneum a certain degree of lipophilicity is required. Log P values between 1 and 4 favour dermal absorption (values between 2 and 3 are optimal). Thymol has a molecular weight of 150 g/mol and a log P of 3.3. Thus, thymol is expected to be able to be absorbed by the stratum corneum. To partition from the stratum corneum into the viable part of the epidermis, a substance must be sufficiently soluble in water (>1 mg/L). Having a water-solubility of 980 mg/L thymol is able to partition from the stratum corneum into the lower epidermis and thus, be taken up by the systemic circulation.
Overall, based on the physical-chemical properties of thymol dermal absorption is possible. What is more, as a corrosive substance thymol is able to impede the barrier function of the skin enhancing dermal absorption. Thus, a dermal absorption rate of 100 % is assumed.

Respiratory absorption
The substance is solid particulate. The particle size distribution of the test item was assessed by the sponsor in an in-house non-GLP test via laser diffraction with a Malvern device. The procedure is similar to ISO 13320. It was observed, that the particles have the following sizes:
d10: 234.527 µm
d50: 574.311 µm
d90: 1095.964 µm
In humans, particles with aerodynamic diameters below 100 μm have the potential to be inhaled. Particles with aerodynamic diameters below 50 μm may reach the thoracic region and those below 15 μm the alveolar region of the respiratory tract. Thus, based on the measured particle size distribution of thymol (d10 = 234.5 µm), only a minor percentage of the particles, if any, will be available for inhalation. However, respiratory exposure cannot fully be excluded. Due to the vapour pressure of 0.022 hPa at 25°C and a boiling point of 231.8°C, thymol has a low volatility and thus it is unlikely to be available as a vapour. If absorbed, thymol is favourable for absorption directly across the respiratory tract epithelium by passive diffusion based on its moderate logP value of 3.3.
Based on the available data, it can be concluded that inhalatory exposure may be possible. As worst-case, 100 % inhalation absorption is assumed.
Details on distribution in tissues:
Due to the low molecular weight (150 g/mol) and the rather high water solubility (980 mg/L), thymol is expected to be widely distributed in the body via blood circulation. It may diffuse through the aqueous pores of cell membranes. Based on the low log P (< 3) no accumulation in fatty tissue or stratum corneum is expected.
Details on excretion:
The rather low molecular weight (150 g/mol) and high water solubility of thymol favours urinary excretion. This is also confirmed by the available studies. In rats urinary excretion of metabolites were rapid. Only very small amounts were excreted after 24 h (Austgulen et al. 1986). In humans the terminal elimination phase set in after 10 to 12 hours, and thymol could be detected up to an average of 38 hours. Elimination half-life was determined to be 10.2 ± 1.4 h (mean ± SD). In urine, the elimination of thymol conjugates was detectable for the first 24-hour interval, with most being eliminated after 6 hours. The combined amount of both thymol sulfate and glucuronide excreted in 24-hour urine was 16.2% ± 4.5% of intake. The renal clearance was calculated to be 0.271 ± 0.7 L/h.
Metabolites identified:
yes
Details on metabolites:
It was proven, that human phase I metabolism leads to a hydroxylation of the aromatic ring as well as of the isopropyl side chain. Hydroxylation of the isopropyl group results in the formation of the rather unstable p-cymene-3,8-diol and the corresponding dehydration product p-cymene-3-ol-8-ene which could be clearly detected in human urine samples. Furthermore, the aromatic hydroxylation products p-cymene-2,5-diol, its oxidation product p-cymene-2,5-dione and p-cymene-2,3-diol were also unambiguously identified (Thalhammer et al. 2011). As phase II metabolites glucuronide and sulphate conjugates were identified in the urine of human and rats after oral thymol administration (Kohlert et al. 2002, Austgulen et al. 1986).
Conclusions:
The absorption rate of thymol is assumed to be 100 % via the oral, dermal and inhalation route. The substance is expected to be distributed widely through the body via blood circulation. No accumulation is expected. The main excretion route is urinary excretion.
Executive summary:

Toxicokinetic Statement for Thymol (CAS 89-83-8)

General

The toxicokinetic profile of thymol (CAS 89-83-8) was assessed based on existing studies on toxicokinetics and toxicity and the physical-chemical properties of the substance.

Substance identity

Table 1: Physical-chemical properties of thymol

 

Thymol (CAS 89-83-8)

Structural formula

C10H14O

Structure

Molecular Weight [g/mol]

150

Physical state

solid

Water Solubility (20°C)

980 mg/L

Log P

3.3

Vapour Pressure (25°C)

0.022 hPa

 

 

Oral absorption

To determine the systemic availability of thymol a clinical trial was carried out in 12 healthy volunteers. Each subject received a single dose of a Bronchipret® TP tablet, which is equivalent to 1.08 mg thymol. Thymol was absorbed quickly. Considerable plasma concentrations of thymol sulfate could already be detected after 20 minutes. This fast absorption indicates that thymol is mainly absorbed in the upper part of the gut (Kohlert et al. 2002). Also, based on the physical-chemical properties a high absorption rate via the gastro-intestinal tract is expected for thymol. In fact, absorption by passive diffusion is favoured for substances with moderate log P values (between -1 and 4). With a log P of 3.3, thymol is likely to follow this route of absorption. Additionally, based on the low molecular weight (< 200 g/mol) and due to the its solubility in water (980 mg/L), thymol may also be absorbed by passing through aqueous pores or being carried through the epithelial barrier by the bulk passage of water.

Taken together, based on the available studies in combination with the physical-chemical properties an oral absorption rate of 100 % is assumed for thymol. 

 

Dermal absorption

In a study according to OECD Guideline 404 thymol was shown to have skin corrosive effects. Therefore, the substance is expected to destroy the barrier function of the skin. Moreover, the physical-chemical properties of thymol indicate the potential for dermal absorption. Absorption in the stratum corneum is favoured for substances with a molecular weight below 100 g/mol, but it is also possible for substances with a molecular weight of below 500 g/mol. To cross the lipid-rich stratum corneum a certain degree of lipophilicity is required. Log P values between 1 and 4 favour dermal absorption (values between 2 and 3 are optimal). Thymol has a molecular weight of 150 g/mol and a log P of 3.3. Thus, thymol is expected to be able to be absorbed by the stratum corneum. To partition from the stratum corneum into the viable part of the epidermis, a substance must be sufficiently soluble in water (>1 mg/L). Having a water-solubility of 980 mg/L thymol is able to partition from the stratum corneum into the lower epidermis and thus, be taken up by the systemic circulation.

Overall, based on the physical-chemical properties of thymol dermal absorption is possible. What is more, as a corrosive substance thymol is able to impede the barrier function of the skin enhancing dermal absorption. Thus, a dermal absorption rate of 100 % is assumed.

Respiratory absorption

The substance is solid particulate. The particle size distribution of the test item was assessed by the sponsor in an in-house non-GLP test via laser diffraction with a Malvern device. The procedure is similar to ISO 13320. It was observed, that the particles have the following sizes:

d10: 234.527 µm

d50: 574.311 µm

d90: 1095.964 µm

In humans, particles with aerodynamic diameters below 100 μm have the potential to be inhaled. Particles with aerodynamic diameters below 50 μm may reach the thoracic region and those below 15 μm the alveolar region of the respiratory tract. Thus, based on the measured particle size distribution of thymol (d10 = 234.5 µm), only a minor percentage of the particles, if any, will be available for inhalation. However, respiratory exposure cannot fully be excluded. Due to the vapour pressure of 0.022 hPa at 25°C and a boiling point of 231.8°C, thymol has a low volatility and thus it is unlikely to be available as a vapour. If absorbed, thymol is favourable for absorption directly across the respiratory tract epithelium by passive diffusion based on its moderate logP value of 3.3.

Based on the available data, it can be concluded that inhalatory exposure may be possible. As worst-case, 100 % inhalation absorption is assumed.

 

Distribution and Accumulation

Due to the low molecular weight (150 g/mol) and the rather high water solubility (980 mg/L), thymol is expected to be widely distributed in the body via blood circulation. It may diffuse through the aqueous pores of cell membranes. Based on the low log P (< 3) no accumulation in fatty tissue or stratum corneum is expected.

Metabolism

It was proven, that human phase I metabolism leads to a hydroxylation of the aromatic ring as well as of the isopropyl side chain. Hydroxylation of the isopropyl group results in the formation of the rather unstable p-cymene-3,8-diol and the corresponding dehydration product p-cymene-3-ol-8-ene which could be clearly detected in human urine samples. Furthermore, the aromatic hydroxylation products p-cymene-2,5-diol, its oxidation product p-cymene-2,5-dione and p-cymene-2,3-diol were also unambiguously identified (Thalhamer et al. 2011). As phase II metabolites glucuronide and sulphate conjugates were identified in the urine of human and rats after oral thymol administration (Kohlert et al. 2002, Austgulen et al. 1986).

 

Elimination

The rather low molecular weight (150 g/mol) and high water solubility of thymol favours urinary excretion. This is also confirmed by the available studies. In rats urinary excretion of metabolites were rapid. Only very small amounts were excreted after 24 h (Austgulen et al. 1986). In humans the terminal elimination phase set in after 10 to 12 hours, and thymol could be detected up to an average of 38 hours. Elimination half-life was determined to be 10.2 ± 1.4 h (mean ± SD). In urine, the elimination of thymol conjugates was detectable for the first 24-hour interval, with most being eliminated after 6 hours. The combined amount of both thymol sulfate and glucuronide excreted in 24-hour urine was 16.2% ± 4.5% of intake. The renal clearance was calculated to be 0.271 ± 0.7 L/h.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
absorption
bioaccessibility (or bioavailability)
distribution
excretion
metabolism
toxicokinetics
Qualifier:
no guideline followed
Principles of method if other than guideline:
Twelve healthy male volunteers were recruited for the study after complete clinical examination. Routine blood and urine laboratory tests were performed. Mean age was 29.5 ± 6.74 years (mean ± SD), and mean body mass index was 24.6 ± 2.0 kg/m² (mean ± SD). Subjects were not allowed to use any medicine during the study.
Each subject received a single dose of Bronchipret® TP tablets containing 60 mg of primrose dry extract (6.0-7.0:1; extracted by ethanol 47% (v/v)) and 160 mg of thyme dry extract (5.9-10.0:1, extracted by ethanol 50% (m/m)), which was batch-specific equivalent to 1.08 mg of thymol. The subjects were fasted at the time they received the medication. They stayed in the clinic for the first 15 hours of the study and returned to the clinic for regular blood sampling visits. Prestudies indicated that long sampling times would be necessary to cover the elimination phase completely. Therefore, a sampling period of 72 hours was chosen.

Collection of Blood and Urine Samples
Venous blood samples (9 mL per bloodsample) were collected into EDTA tubes once before subjects were administered the medication and 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 24, 31, 38, 48, 55, 62, and 72 hours after administration. Blood was centrifuged for 10 minutes at 4000 × g. The supernatant plasma was transferred into reaction cups in aliquots of 0.5 mL, and 20 µL acetic acid 0.58 M were added to each aliquot for stabilization. The plasma was stored at -20°C until analysis. Urine was collected in plastic bottles in intervals of 0 to 3, 3 to 6, 6 to 9, 9 to 14, 14 to 24, 24 to 31, 38 to 48, 48 to 55, 55 to 62, and 62 to 72 hours. An aliquot of 50 ml of each sample was mixed with 0.1 g ascorbic acid as antioxidant and stored at -20°C until analysis.
GLP compliance:
no
Specific details on test material used for the study:
Bronchipret® TP tablet, which is equivalent to 1.08 mg thymol.
Radiolabelling:
no
Species:
human
Strain:
other: not applicable
Details on species / strain selection:
All subjects signed an informedconsent form according to good clinical practice (GCP) guidelines. The protocol was approved by the ethics committee of the University of Erlangen, Germany.
Sex:
male
Details on test animals or test system and environmental conditions:
During the entire time, the subjects were on an essential oil-free diet, and they were not allowed to apply any cosmetics (e.g., toothpaste, aftershave, etc.) containing essential oil compounds to avoid interference with any other essential oil compounds. Therefore, the subjects were handed out a list with food and cosmetics they were not allowed to eat and apply during the time of the study period.
Route of administration:
oral: capsule
Vehicle:
other: Bronchipret® TP tablets containing 60 mg of primrose dry extract (6.0-7.0:1; extracted by ethanol 47% (v/v)) and 160 mg of thyme dry extract (5.9-10.0:1, extracted by ethanol 50% (m/m)), which was batch-specific equivalent to 1.08 mg of thymol.
Details on exposure:
Each subject received a single dose of Bronchipret® TP tablets containing 60 mg of primrose dry extract (6.0-7.0:1; extracted by ethanol 47% (v/v)) and 160 mg of thyme dry extract (5.9-10.0:1, extracted by ethanol 50% (m/m)), which was batch-specific equivalent to 1.08 mg of thymol.
Duration and frequency of treatment / exposure:
single dose
Dose / conc.:
1.08 other: mg of thymol
No. of animals per sex per dose / concentration:
12
Control animals:
not specified
Details on study design:
please refer to the field 'Principles of method if other than guideline'
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, plasma
- Time and frequency of sampling:
plasma: 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 24, 31, 38, 48, 55, 62, and 72 hours after administration
urine: in intervals of 0 to 3, 3 to 6, 6 to 9, 9 to 14, 14 to 24, 24 to 31, 38 to 48, 48 to 55, 55 to 62, and 62 to 72 hours.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, plasma
- Time and frequency of sampling:
plasma: 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 24, 31, 38, 48, 55, 62, and 72 hours after administration
urine: in intervals of 0 to 3, 3 to 6, 6 to 9, 9 to 14, 14 to 24, 24 to 31, 38 to 48, 48 to 55, 55 to 62, and 62 to 72 hours.
- From how many subjects: 12 men, not pooled
- Method type(s) for identification: headspace solid-phase microextraction (HS-SPME) prior to gas chromatographic analysis
- Limits of detection and quantification: The lower limit of quantification for plasma analysis was 8.1 ng/mL and for urine 10.9 ng/mL.
- Other (validation of method): At the limit of quantification (LOQ), within-day precision of the plasma and urine assay was below 19 % and 12 % (coefficient of variation [CV]), respectively, and below 5.6% (plasma) and 7.2% (urine) at higher concentrations.
Accuracy was below 9 % for plasma and below 13 % for urine. Stability of both plasma and urine samples was given over 12 weeks at –20°C.

TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable): enzymatic hydrolysis of conjugated thymol

ANALYTICAL METHOD
- Complete description including: For the determination of total concentration of thymol, thawed plasma samples were analyzed after enzymatic hydrolysis of conjugated thymol by headspace solid-phase microextraction (HS-SPME) prior to gas chromatographic analysis. For urine analysis,only 20 µL of acetic acid 0.58 M compared to 50 µ used in the plasma methodwere necessary to adjust to pH 5 because ascorbic acid was added before the samples were frozen. In addition, adaptation of the SPME extraction time to 25 minutes insteadof 35 minutes for plasma analysis was performed, which is due to the different matrix. Plasma samples were analyzed in 12 runs and the urine samples in 5 runs. Each run was checked for linearity by a separate calibration curve. Precision and accuracy were controlled by running six external quality control samples (spiked blank plasma and urine samples with reference compounds) covering the whole range.
The identification of phase II metabolites and the determination of free thymol in both plasma and urine were carried out exemplarily in 2 subjects each. For identification of the phase II metabolites, LC-MS/MS measurements were performed. Prior to LC-MS/MS (ionization mode: ESI negative), the following sample preparation procedures were applied. After addition of 550 µL acetonitrile to 550 µL plasma, the solution was vigorously shaken for 20 seconds and centrifuged at 7800 × g for 4 minutes. The solvent was removed under nitrogen stream at 40°C. The residue was dissolved in 5 µL acetonitrile and 45 µL 2 mM ammonium acetate by ultrasonication. After centrifugation at 7800 × g for 4 minutes, the supernatant was used for HPLC analysis.
For urine sample preparation, 4 mL urine were acidified with 8 µl formic acid; 14 mL ethylacetate were added, and the samples were shaken for 2 minutes. For separation of the organic andaqueous phase, the samples were centrifuged at 7800 × g for 4 minutes, and the supernatant was evaporated under nitrogen stream at 40°C. The residues were dissolved in 12 µL acetonitrile and 108 µL 2 mm ammonium acetate by means of ultrasonication and used for HPLC analysis.
Determination of free thymol in plasma was carried out by HPLC. To 400 µL of plasma (pH 5), 50 µL internal stock solution (o-Cresol, 4 µg/mL), 50 µL water, and 500 µL methanol were added, vortexed, and centrifuged for 10 minutes at 7800 × g. The supernatant was used for HPLC analysis.
Determination of free thymol in urine was carried out by GC-MS. n-Heptane (250 µL) was added to 500 µLof urine (pH 5) and vortexed for 20 minutes at 350 rpm. The supernatant was used for GC-MS analysis.
Statistics:
Pharmacokinetic data were determined by non compartmental analysis using Microsoft Excel based on the equations given by Gibaldi and Perrier. The maximum observed plasma concentration Cmax and the time to reach Cmax (tmax) were determined directly from the data. The AUC (0 → clast) was calculated using the linear trapezoidal rule. The apparent terminal elimination half-life was determined by linear regression of the terminal phase of the semilogarithmic plasma concentration
versus time profiles. The mean absorption time (MAT) was calculated from 1/ka, which was obtained from ke and tmax by the Excel Solver function from tmax =
ln(ka/ke)/(ka – ke).
Preliminary studies:
Prestudies indicated that long sampling times wouldbe necessary to cover the elimination phase completely.
Type:
absorption
Results:
Thymol was absorbed quickly. Considerable plasma concentrations of thymol sulfate could already be detected after 20 minutes. This fast absorption indicates that thymol is mainly absorbed in the upper part of the gut.
Type:
distribution
Results:
Thymol is distributed in the plasma as the metabolite thymol sulfate. The volume of distribution (Vdss/f) of 14.7 L indicates that thymol sulfate stays mainly in the extracellular space.
Type:
metabolism
Results:
Thymol sulfate but not the glucuronide was identified in human plasma. Thymol sulfate and thymol glucuronide were found in the urine.
Type:
excretion
Results:
Terminal elimination phase began about 10 hours after administration and lasting up to an average of 38 hours. Elimination half-life was 10.2 ± 1.4 h (mean ± SD). The renal clearance was calculated to be 0.271 ± 0.7 L/h.
Details on absorption:
Thymol was rapidly absorbed. Thymol sulfate could be detected in plasma 20 minutes after application. This fast absorption indicates that thymol is mainly absorbed in the upper part of the gut.
Details on distribution in tissues:
Maximum plasma levels of 93.1 ± 24.5 ng/ml (mean ± SD) were reached after 1.97 ± 0.77 hours (mean ± SD). The plasma concentration versus time profile was biphasic, subdivided into a distribution phase and a slow terminal elimination phase beginning at about 10 hours after administration and lasting up to an average of 38 hours.
Observation:
not determined
Details on excretion:
Thymol plasma concentrations - after enzymatic hydrolysis of thymol sulfate - showed a biphasic profile. The terminal elimination phase set in after 10 to 12 hours, and thymol could be detected up to an average of 38 hours. Elimination half-life was determined to be 10.2 ± 1.4 h (mean ± SD). In urine, the elimination of thymol conjugates was detectable for the first 24-hour interval, with most being eliminated after 6 hours. The combined amount of both thymol sulfate and glucuronide excreted in 24-hour urine was 16.2% ± 4.5% of intake. The renal clearance was calculated to be 0.271 ± 0.7 L/h.

Although plasma levels were detectable up to an average of 38 hours, the renal elimination of thymol conjugates was completed within 24 hours. This discrepancy might be due to the fact that very small amounts of renally eliminated thymol conjugates could not be quantified any more after 24 hours (< LOQ). The fact that thymol sulfate is eliminated slowly can also be deduced from the small clearance (Cltot/f) of 1.2 L/h. The renal clearance of 0.271 L/h indicates high protein binding and/or reabsorption in the kidney, respectively.
The volume of distribution (Vdss/f) of 14.7 L indicates that thymol sulfate stays mainly in the extracellular space. The bioavailability of thymol sulfate after administration of thymol is at least 16%, because 16% of the dose administered was excreted into the urine as thymol conjugates. However, since Vdss/f is only 14.7 L, a much larger number for f is likely.
Toxicokinetic parameters:
half-life 1st: 10.2 ± 1.4 h
Toxicokinetic parameters:
Cmax: 93.11 ± 24.47 ng/mL
Toxicokinetic parameters:
Tmax: 1.97 ± 0.77 h
Toxicokinetic parameters:
AUC: 837.3 ± 278.5 ng h/mL
Toxicokinetic parameters:
other: MRT(abs): 12.6 ± 2.1 h
Remarks:
mean residence time after extravascular administration
Toxicokinetic parameters:
other: MAT: 0.53 ± 0.04 h
Remarks:
mean absorption time
Toxicokinetic parameters:
other: CL(lot)/f: 1.2 ± 0.3 L/h
Remarks:
total body clearance with respect to unknown bioavailability f
Toxicokinetic parameters:
other: Vdss/f: 14.7 ± 5.1 L
Remarks:
volume of distribution at steady state with respect to unknown bioavailability f
Toxicokinetic parameters:
other: Vd(area)/f: 17.7 ± 5.6 L
Remarks:
volume of distribution during the elimination phase with respect to unknown bioavailability f
Metabolites identified:
yes
Details on metabolites:
Thymol was present in human plasma only as thymol sulfate. In urine it was present as thymol sulfate and thymol glucuronide.
Bioaccessibility (or Bioavailability) testing results:
The bioavailability of thymol sulfate after administration of thymol is at least 16%, because 16% of the dose administered was excreted into the urine as thymol conjugates. However, since Vdss/f is only 14.7 L, a much larger number for f is likely.

Free thymol could not be detected in human plasma. By means of LC-MS/MS analysis, only thymol sulfate but not the glucuronide was identified in human plasma. No free thymol was found in urine either. Instead, two phase II conjugates were identified by LC-NS/MS as thymol sulfate and glucuronide. The ratio of peak areas of thymol sulfate and thymol glucuronide was constant over the different urine fractions. Thymol was rapidly absorbed. Thymol sulfate could be detected in plasma 20 minutes after application. Maximum plasma levels of 93.1 ± 24.5 ng/mL were reached after 1.97 ± 0.77 hours. The plasma concentration was biphasic, subdivided into a distribution phase and a slow terminal elimination phase beginning at about 10 hours after administration and lasting up to an average of 38 hours. Elimination half-life was calculated to be 10.2 ± 1.4 hours (mean ± SD). In urine, the elimination of thymol conjugates was detectable for the first 24-hour interval, with most being eliminated after 6 hours. The combined amount of both thymol sulfate and glucuronide excreted in 24-hour urine was 16.2% ± 4.5% of intake. The renal clearance was calculated to be 0.271 ± 0.7 L h–1.

Conclusions:
Thymol was rapidly absorbed. Thymol sulfate could be detected in plasma 20 minutes after application. Maximum plasma levels of 93.1 ± 24.5 ng/mL were reached after 1.97 ± 0.77 hours. The plasma concentration was biphasic, subdivided into a distribution phase and a slow terminal elimination phase beginning at about 10 hours after administration and lasting up to an average of 38 hours. Elimination half-life was calculated to be 10.2 ± 1.4 hours (mean ± SD).
Executive summary:

To determine the systemic availability and the pharmacokinetics of thymol after oral application to humans, a clinical trial was carried out in 12 healthy volunteers. Each subject received a single dose of a Bronchipret® TP tablet, which is equivalent to 1.08 mg thymol. No thymol could be detected in plasma or urine. However, the metabolites thymol sulfate and thymol glucuronide were found in urine and identified by LC-MS/MS. Plasma and urine samples were analyzed after enzymatic hydrolysis of the metabolites by headspace solid-phase microextraction prior to GC analysis and flame ionization detection. Thymol sulfate, but not thymol glucuronide, was detectable in plasma. Peak plasma concentrations were 93.1 ± 24.5 ng/mL and were reached after 2.0 ± 0.8 hours. The mean terminal elimination half-life was 10.2 hours. Thymol sulfate was detectable up to 41 hours after administration. Urinary excretion could be followed over 24 hours. The amount of both thymol sulfate and glucuronide excreted in 24-hour urine was 16.2% ± 4.5% of the dose.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
metabolism
Qualifier:
no guideline followed
Radiolabelling:
no
Species:
other: human
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST SUBJECTS
- All probands explicitly declared their agreement of taking part in these studies and providing urine samples
- Age at study initiation: average age was 33.8 years (s = 8.1)
- Weight at study initiation: average body mass index was 25.6 (s = 4.1)
- Diet: All the probands just lived on rice or potatoes and mineral water, avoiding all contact to herbs, oils, dietary supplements or cosmetic products which might include terpenoid components for 72 h (48 h before and 24 h after test item administration).
- Health status: healthy
Route of administration:
oral: drinking water
Vehicle:
ethanol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
50 mg thymol (dissolved in 1 mL ethanol and 200 mL H2O)
Duration and frequency of treatment / exposure:
single dose
Dose / conc.:
50 other: mg thymol
No. of animals per sex per dose / concentration:
3
Control animals:
no
Details on dosing and sampling:
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: during 24 h after test item administration all the urine of the probands was collected
- From how many animals: 6 probands, not pooled
- Method type(s) for identification: headspace sorptive extraction (HSSE) method in combination with thermal desorption gas chromatography coupled to a mass spectrometer (TD–GC/MS)

TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable): enzymatic hydrolysis of glucuronated or sulphated phase II metabolites of thymol and of the respective phase I metabolites prior to analysis.

ANALYTICAL METHOD
- Complete description including: Thymol (0.1 g) was dissolved in 1 L 18 MΩ H2O giving the standard stock solution (100 µg/ml) which was then further diluted giving solutions of different concentrations (10/25/50/100/250/500/1000/2500/5000/10,000 ng/mL) which were used in the HSSE–TD–GC/MS quantitation experiments. 1000 µL of these standard solutions were transferred in a 20 ml crimp cap headspace vial, followed by addition of 1000 µL H2O. Then the twister was placed in the glass insert and put into the twister headspace vial which was closed and heated to 60°C for 20 min. After cooling to room temperature (4 min) the twister was placed into the thermodesorption glass tube and analysed by TD–GC/MS. This procedure was then used to examine the influence of HSSE extraction time and extraction temperature.
Type:
metabolism
Results:
Preferred hydroxylation of the aromatic system of thymol resultingin the formation of p-cymene-2,5-diol and its oxidized form (p-cymene-2,5-dione) as the main products and a functionalization of the iso-propyl side chain was observed.
Metabolites identified:
yes
Details on metabolites:
It was proven, that human metabolism leads to a hydroxylation of the aromatic ring as well as of the isopropyl side chain. Hydroxylation of the iso-propyl group results in the formation of the rather unstable p-cymene-3,8-diol and the corresponding dehydration product p-cymene-3-ol-8-ene which could be clearly detected in human urine samples. Furthermore, the aromatic hydroxylation products p-cymene-2,5-diol, its oxidation product p-cymene-2,5-dione and p-cymene-2,3-diol were also unambiguously identified by comparison with synthesized reference compounds.
Conclusions:
It was clearly shown that human metabolism leads to a preferred hydroxylation of the aromatic system of thymol resulting in the formation of p-cymene-2,5-diol and its corresponding oxidized form (p-cymene-2,5-dione) as the main products. It was also possible to detected small quantities of the p-cymene-2,3-diol upon prolonged extraction times. In addition also a functionalization of the iso-propyl side chain was observed. Although we just detected the elimination product p-cymene-3-ol-8-ene, it can be rationalized that this compound might originate from the p-cymene-3,8-diol upon H2O-cleavage. Furthermore, it was shown that some of the derivatives which were found in rat metabolism could not be detected in human samples and vice versa.
Executive summary:

Six healthy probands (three male and three female) were administered a single dose of 50 mg thymol and their urine was collected for 24 h. The identification of thymol and several phase I metabolites in human urine was performed using a novel highly sensitive headspace sorptive extraction (HSSE) method in combination with thermal desorption gas chromatography coupled to a mass spectrometer (TD–GC/MS). Combined with an enzymatic hydrolysis of glucuronated or sulphated phase II metabolites of thymol and of the respective phase I metabolites prior to analysis, even trace quantities of hitherto not detected thymol phase I metabolites could be identified in urine samples of test persons after oral administration of 50 mg thymol. It was proven, that human metabolism leads to a hydroxylation of the aromatic ring as well as of the isopropyl side chain. Hydroxylation of the iso-propyl group results in the formation of the rather unstable p-cymene-3,8-diol and the corresponding dehydration product p-cymene-3-ol-8-ene which could be clearly detected in human urine samples. Furthermore, the aromatic hydroxylation products p-cymene-2,5-diol, its oxidation product p-cymene-2,5-dione and p-cymene-2,3-diol were also unambiguously identified by comparison with synthesized reference compounds.

Description of key information

The absorption rate of thymol is assumed to be 100 % via the oral, dermal and inhalation route. The substance is expected to be distributed widely through the body via blood circulation. No accumulation is expected. The main excretion route is urinary excretion.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

To determine the systemic availability and the pharmacokinetics of thymol after oral application to humans, a clinical trial was carried out in 12 healthy volunteers. Each subject received a single dose of a Bronchipret® TP tablet, which is equivalent to 1.08 mg thymol. No thymol could be detected in plasma or urine. However, the metabolites thymol sulfate and thymol glucuronide were found in urine and identified by LC-MS/MS. Plasma and urine samples were analyzed after enzymatic hydrolysis of the metabolites. Thymol sulfate, but not thymol glucuronide, was detectable in plasma. Peak plasma concentrations were 93.1 ± 24.5 ng/mL and were reached after 2.0 ± 0.8 hours. The mean terminal elimination half-life was 10.2 hours (Kohlert 2002).

In another trial male Wistar rats received 1 mmol/kg bw (150 mg/kg bw) thymol by stomach tube.The urinary excretion was very rapid. Only very small amounts were excreted after 24 hrs. Although large quantities of thymol were excreted unchanged (or as their glucuronide and sulphate conjugates) extensive oxidation of the methyl and isopropyl group also occurred. This resulted in the formation of derivatives of benzyl alcohol and 2-phenylpropanol and their corresponding carboxylic acid. Ring hydroxylation was a minor reaction. Based on these study results no bioaccumulation potential is expected (Austgulen 1986).