2-(4-methylcyclohex-3-en-1-yl)propan-2-ol

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 November to 04 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Objective of study:

toxicokinetics

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was conducted to evaluate the toxicokinetics of the test item Terpineol multiconstituent, and its metabolite alpha-terpineol glucuronide, following daily oral administration (gavage) to male Sprague-Dawley rats for 1, 7 and 14 days.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl CD® (SD) IGS BR

Details on species / strain selection:

The rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study. The Sprague-Dawley strain was selected since background data from previous studies are available at CiToxLAB France. Sprague-Dawley rats are the animal species (and strain) used in the previous toxicological studies performed with the Terpineol multiconstituent.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy.
- Age at study initiation: Approximately 10 weeks
- Weight at study initiation: Mean body weight of 382 g (range: 341-428 g)
- Housing: Animals were housed by three from the same group and subgroup in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm2) containing autoclaved sawdust (SICSA, Alfortville, France).
- Diet: SSNIFF R/M-H pelleted maintenance diet, batch No. 4301997 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly, ad libitum
- Water: Tap water (filtered with a 0.22 μm filter), ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: No contaminants were present in the diet, drinking water or sawdust at levels which could have been expected to interfere with, or prejudice, the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 20 %
- Air changes: Approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: 13 November 2014 To: 04 December 2014

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Preparation procedure: Test item was weighed and mixed with the required quantity of vehicle (corn oil).
The control and test item dose formulations were stirred just before administration and were maintained under continuous magnetic stirring throughout the dosing procedure.
Frequency of preparation: On the days of treatment
Delivery conditions: At room temperature and protected from light

VEHICLE
- Concentration in vehicle: 20, 50, 120 and 150 mg/mL
- Dose volume: 5 mL/kg bw/day

Duration and frequency of treatment / exposure:

1, 7 and 14 days

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Dose / conc.:

750 mg/kg bw/day (actual dose received)

No. of animals per sex per dose / concentration:

6 males/dose

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

Not applicable

Details on study design:

- Dose selection rationale: Dose levels were selected by the Sponsor (no data reported).
- Rationale for animal assignment: During the acclimation period, the required number of animals (72 principal and 18 satellite males) was selected according to body weight and clinical condition. The animals were allocated to groups according to a computerized stratification procedure so that the average body weight of each group was similar.

Details on dosing and sampling:

TOXICOKINETIC STUDY
Tissues and body fluids sampled: blood
Time and frequency of sampling:
- Blood samples for the determination of plasma levels of the test item and its metabolite were taken on all groups: on Day 1; on Day 7 for 750 mg/kg bw/day group only; at the end of the treatment period (Day 14)
- At each occasion, the animals were sampled as follows: 0 (predose), 0.5, 1, 2, 4, 6, 12 and 24 h after test item administration.
- Three animals / subgroup were sampled at each time-point, and each animal was sampled four times during each period.
Blood was collected from the tail vein at each time-point, except for the last sampling before sacrifice for groups 2 to 6 which was performed at the vena cava. Blood was centrifuged at 3000g for 10 minutes under refrigerated conditions (set to maintain 4 °C). The plasma was transferred in two individual tubes, each containing at least 75 µL of plasma and stored at -20 °C until analysis.

Preliminary studies:

Not applicable

Key result

Toxicokinetic parameters:

Tmax: 0.5 h for all doses with the exception of the 100 mg/kg bw/day group at day 1 (Tmax was observed at 1 h)

Key result

Toxicokinetic parameters:

other: mean terminal half-life values

Remarks:

mean terminal half-life values of the metabolite ranged from 8.59 to 20.0 h at 100 and 250 mg/kg bw/day, from 1.70 to 12.4 h at 600 mg/kg bw/day and decreased to 2.91-5.02 h at 750 mg/kg bw/day.

Metabolites identified:

yes

Details on metabolites:

Plasma levels of alpha-terpineol and alpha-terpineol glucuronide:
- No contamination was observed in pre-dose samples collected on Day 1 from all treated and vehicle groups as their respective plasma concentrations of alpha-terpineol and alpha-terpineol glucuronide were either below the Lower Limit of Quantification (LLOQ: 1.00 µg/mL for terpineol) or not detected (S/N<5 for terpineol glucuronide). At all doses and for all sampling time points, terpineol concentration was below the limit of quantification (BLQ < 1.00 µg/mL) in plasma. However, animals were exposed to alpha-terpineol since its metabolite was found in all animals treated. Indeed, alpha-terpineol glucuronide (i.e. the metabolite) was detected in plasma at all times and all doses (except at predose for day 1 groups).
- Since there was no commercial standard available for alpha-terpineol glucuronide, this metabolite could not be quantified. Consequently, only a semi-quantitative analysis was performed and results were given in “counts”.
- Maximum plasma concentration of alpha-terpineol glucuronide was reached at 1h following oral administration of Terpineol Multiconstituent at 100 mg/kg bw/day at day 1, and 0.5 h for all other groups and doses. Moreover, all the concentration-time curves (in a lesser extent at 250 mg/kg bw/day) of the 7-day and 14-day groups revealed 2 peaks: one at 0.5 h and another one at 4 h or 6 h, suggesting possible enterohepatic recycling.

Toxicokinetic calculation:
- Mean toxicokinetic parameters of alpha-terpineol glucuronide are presented in Table 7.1.1/2.
- Since only a semi-quantitative analysis was performed, Cmax could not be estimated. Similarly, AUC values are given for information. The mean terminal half-life values of the metabolite (ln2/ λz values) ranged from 8.59 to 20.0 h at 100 and 250 mg/kg bw/day, from 1.70 to 12.4 h at 600 mg/kg bw/day and decreased to 2.91-5.02 h at 750 mg/kg bw/day.
- The variability in group 600 mg/kg bw/day could be partially explained by the analytical conditions (analysis c.a. 1 year later than other group samples) but also by the interindividual variability in terms of clearance and metabolism. However, the exposure parameters from this group remained consistent with the other groups.
- As the maximal estimated half-life is 20.0 h, one can consider that at day 14 all animals are at the steady state (5 maximal half-lives correspond approximately to 4 days).

Dose and time effects:
- Exposure to alpha-terpineol glucuronide (AUC0-24h) increased almost dose-proportionally in the range of the tested doses at day 1 and at day 14 (Table 7.1.1/3).
- Moreover, alpha-terpineol glucuronide tended to accumulate with the treatment length: 1.58 times at day 7 (750 mg/kg bw/day) and from 1.58 to 1.67 at day 14 for all doses. These results are consistent with the estimated half-lives.

Table 7.1.1/1: Plasma intensity (count) of alpha-terpineol glucuronide following single oral administration of Terpineol Multiconstituent at 100, 250, 600 and 750 mg/kg bw/day in male Sprague-Dawley rats

 

Dose (mg/kg bw/day)	Period	Plasma intensity (count)	Sampling time (hours)
			0.0	0.5	1	2	4	6	12	24
100	Day 1	Mean	0	685932	962493	454732	103359	138857	117639	74994
	Day 14		87010	824787	433410	313717	436783	386066	344019	265749
250	Day 1		0	1921301	1257260	1179185	318406	302592	244639	141430
	Day 14		192822	2076135	1393117	785286	523236	604929	612961	293241
600	Day 1		0	10013615	9300159	2010128	5041774	2567509	427618	89200
	Day 14		55212	3 840 304	1855509	1437818	3048305	1627018	1091329	586481
750	Day 1		0	9399927	7028413	3457456	3001709	2825952	775399	194155
	Day 7		253784	7 570 038	3974550	3834839	3156714	5854943	2220807	440907
	Day 14		30167	10590511	5472195	5952035	8149623	3156401	2923376	54960

 

Table 7.1.1/2: Mean toxicokinetic parameters (median for T_max) of alpha-terpineol glucuronide following oral administration of Terpineol Multiconstituent at 100, 250, 600 and 750 mg/kg bw/day for 1, 7 or 14 days in male Sprague-Dawley rats

Dose (mg/kg bw/day)	Day	R²	N° points λz	λz (h^-1)	t½ (h)	Tmax (h)	AUC0-24h (h*{count})	AUC0-∞(h*{count})	AUC0-∞ Extrap %
100	1	0.995	3	0.0347	20.0	1	3,881,730	-	> 20
	14	0.991	4	0.0807	8.59	0.5	6,336,675	7,425,633	14.7
250	1	0.997	3	0.0427	16.2	0.5	8,312,529	-	> 20
	14	0.883	3	0.0433	16.0	0.5	13,759,810	-	> 20
600	1	0.996	4	0.0408	1.70	0.5	19,537,745	19,756,276	1.11
	14	0.961	3	0.0560	12.4	0.5	30,793,388	-	> 20
750	1	0.980	5	0.138	5.02	0.5	38,287,031	39,692,683	3.54
	7	0.998	3	0.142	4.87	0.5	60,335,093	63,431,344	4.88
	14	0.929	4	0.238	2.91	0.5	63,765,238	63,996,360	0.361

 

Table 7.1.1/3: Dose proportionality assessment of alpha-terpineol glucuronide following oral administration of Terpineol Multiconstituent at 100, 250, 600 and 750 mg/kg bw/day for 1, 7 or 14 days in male Sprague-Dawley rats

Day	Dose (mg/kg bw/day)	AUC0-24h (h*{count})	AUC0-24h/D (count*h/(mg/kg))	Treatment ratio compared with 100 mg/kg bw/day
1	100	3,881,730	38,817	-
	250	8,312,529	33,250	0.9
	750	38,287,031	51,049	1.3
14	100	6,336,675	63,367	-
	250	13,759,810	55,039	0.9
	750	63,765,238	85,020	1.3

Conclusions:

Based on the data obtained, the following conclusions can be made:
Following oral administration of Terpineol multiconstituent to male Sprague-Dawley rats at 100, 250, 600 and 750 mg/kg bw/day, alpha-terpineol concentrations were below the limit of quantification (BLQ < 1.00 µg/mL) for all animals at all sampling times. All animals have detectable alpha-terpineol glucuronide amounts in plasma at all times (except at day 1 predose) for all doses. Rapid oral absorption and metabolism rate were noted with a metabolite Tmax observed at 0.5 h for all doses with the exception of the 100 mg/kg bw/day group at day 1 (Tmax was observed at 1 h). The mean terminal half-life values of the metabolite were highly variable ranging from 1.70 to 20.0 h. Mean concentration versus time profiles of the alpha-terpineol glucuronide suggest either an enterohepatic recycling and/or a saturable absorption. Both on Days 1 and 14, exposure to terpineol glucuronide (AUC0-24h) increased almost dose-proportionally from 100 mg/kg to 600 mg/kg, whilst at 750 mg/kg a more than dose-proportional increase can be observed.

Executive summary:

A toxicokinetic study was conducted to evaluate the toxicokinetics of test item Terpineol multiconstituent, and its metabolite alpha-terpineol glucuronide, following daily oral administration (gavage) to male Sprague-Dawley rats for 1, 7 and 14 days.

 

The toxicokinetics of Terpineol multiconstituent after oral administration on Day 1, 7 (750 mg/kg group only) and 14, to 12 male Sprague-Dawley rats (n=3 per dose group) were characterised using mean plasma concentration vs. time data. The animals received either 100, 250, 600 or 750 mg/kg bw/day of Terpineol multiconstituent by daily gavage administration. Three additional samples from external animals were allocated to the initial groups: 3 blood samples were added to group 100 mg/kg bw/day at 12 h.

Blood samples for the determination of plasma levels of the test item and its metabolite were taken on all groups: on Day 1; on Day 7 for 750 mg/kg bw/day group only; at the end of the treatment period (Day 14). At each occasion, the animals were sampled as follows: 0 (predose), 0.5, 1, 2, 4, 6, 12 and 24 h after test item administration. Three animals / subgroup were sampled at each time-point, and each animal was sampled four times during each period.

Bioanalysis for the determination of Terpineol multicojstituent concentration in plasma was performed using a validated GC-FID method. Conversely, Terpineol multiconstituent glucuronide concentration could not be determined as no commercial standard was available. Consequently, no validated method could be performed and only the peak intensity (in count), obtained by LC-MS, could be reported.

 

The toxicokinetic parameters were estimated using Phoenix WinNonlin^® software v6.4 (Pharsight Corporation, Mountain View, California 94040/USA).

The following toxicokinetic parameters were determined: AUC_0-24h, t_max, λ_z(t½). Parameters were compared in order to evaluate dose-proportionality and time effect.

 

Based on the data obtained, the following conclusions can be made:

- No contamination was observed in pre-dose samples collected on Day 1 from all treated and vehicle groups as their respective plasma concentrations of alpha-terpineol and alpha-terpineol glucuronide were either below the Lower Limit of Quantification (LLOQ: 1.00 µg/mL for alpha-terpineol) or not detected (S/N<5 for alpha-terpineol glucuronide).

- Following oral administration of Terpineol multiconstituent to male Sprague-Dawley rats at 100, 250, 600 and 750 mg/kg bw/day, alpha-terpineol concentrations were below the limit of quantification (BLQ < 1.00 µg/mL) for all animals at all sampling times.

- The male rats were exposed to alpha-terpineol since all animals have detectable alpha-terpineol glucuronide amounts in plasma at all times (except at day 1 predose) for all doses.

- Rapid oral absorption and metabolism rate were noted with a metabolite T_max observed at 0.5 h for all test item doses with the exception of the 100 mg/kg bw/day group at day 1 (T_max at 1 h). The mean terminal half-life values of the metabolite were highly variable ranging from 1.70 to 20.0 h.

- Mean concentration versus time profiles of the alpha-terpineol glucuronide suggest either an enterohepatic recycling and/or a saturable absorption.

- Both on Days 1 and 14, exposure to terpineol glucuronide (AUC_0-24h) increased almost dose-proportionally from 100 mg/kg to 600 mg/kg, whilst at 750 mg/kg a more than dose-proportional increase can be observed.

Reference 2

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well conducted and well described study with minor missing data (details on test substance and animals housing conditions)

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Measurement of UDP-glucuronosyltransferase activities towards monoterpenes including terpineol in Wistar rats, Gunn rats and guinea pigs with or without phenobarbital induction.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Male Wistar and Gunn rats (200 g) were obtained from IFFA-CREDO (Saint-Germain sur l'Abresle, France). They had free access to food (UAR, Villemoisson/Orge) and water.
Male "tricolores" guinea pigs, weighing 350-400 g, were obtained from the Centre d'Elevage d'Animaux de Laboratoire, Ardenay, France.

Route of administration:

intraperitoneal

Vehicle:

physiological saline

Details on exposure:

Each animal was treated by intraperitoneal injection of 0.5 mL of vehicle per 200g of body weight. One group received phenobarbital (80 mg/kg) dissolved in 0.9% NaCl (w/v) daily for 4 days. The corresponding controls received saline solution only.
Another group of Wistar rats received 3-methylcholanthrene in corn oil, as a single injection of 80 mg/kg on the first day and these animals were killed on day 5. Corresponding controls received corn oil only.
A group of four guinea pigs received daily intraperitoneal injections of 20-40 mg of phenobarbital per kg of body weight, for 9 days (total dose: 320 mg/kg of body weight).

Duration and frequency of treatment / exposure:

see details on exposure

Remarks:

Doses / Concentrations:
see details on exposure

No. of animals per sex per dose / concentration:

see details on exposure

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

none

Details on study design:

none

Details on dosing and sampling:

Animals were decapitated 24 h after the last treatment. Liver microsomes were prepared in 0.25 mM sucrose, 1 mM Tris-HCl buffer, pH 7.4, according to the method of Beaufay et al., 1974. Protein was measured by the method of Lowry et al., 1951. UDPGT activities were measured by a modification of the method described by Mulder and van Doorn, 1975. The microsomal suspensions were activated with Triton X-100 such that the ratio of
Triton to protein (w/w) was 0.4. 10 µL of terpineol (0.3 mM final concentration, or as indicated) was used for the reaction. Each run took 10 min, and readings at 340 nm were recorded every 20 s. The specific activity was calculated by linear regression. Each value is given as a mean of four determinations.

Statistics:

none

Preliminary studies:

none

Details on absorption:

none

Details on distribution in tissues:

None

Details on excretion:

none

Metabolites identified:

no

Details on metabolites:

none

Table 1: UDP-glucuronosyltransferase specific activities of terpineol measured in liver microsomes from Wistar rats induced by either phenobarbital or 3-methylcholanthrene

	UDPGT specific activities (nmoles/min/mg)
	Control (NaCl)	Phenobarbital	Control (oil)	3-methylcholanthrene
Terpineol (0.25 mM)	9.0 ± 0.3	13.2 ± 0.4	6.6 ± 0.1	7.8 ± 0.0

Values represent mean ± S.D. of four determinations on pooled microsomes of four animals.

Table 2: Activities of hepatic UDP-glucuronosyltransferases from control and phenobarbital-treated Gunn rats

	Control	Ratio of activity : Gunn/Wistar	Phenobarbital	Ratio of activity : Gunn/Wistar
Terpineol (0.25 mM)	6.4 ± 0.3	0.8	8.8 ± 1.0 (140)	0.7

Values represent the means ± S.D. of four determinations from pooled fractions from four animals, and they are expressed in nmoles/min/mg protein. The numbers in parentheses provide the percentage of induction by phenobarbital. The ratios refer to the similar situation in Wistar rats (from Table 1).

Table 3: Hepatic microsomal UDP-glucuronosyltransferase activities from control and phenobarbital-treated guinea pigs

	Control (A)	Phenobarbital (B)	Ratio B/A
Terpineol (0.175 mM)	8.2 ± 0.2	22.2 ± 0.4	2.8

The values are given as means ± S.D. of four determinations, from pooled fractions from four animals, and are expressed in nmoles/min/mg protein.

Conclusions:

Terpineol is conjugated with UDP-glucuronosyltransferase (UDPGT).

Executive summary:

Four male Wistar rats received intraperitoneal doses of 80 mg/kg phenobarbital in 0.9% NaCl for 4 days. Rats were sacrificed 24 h after the last dose and liver microsomes were prepared. UDPGT activities were measured by a modification of the method of Mulder and Van Doorn (1985) using 10 µL of terpineol (final concentration of terpineol, 0.25 mM). Phenobarbital treatment enhanced the activity of UDP-glucuronosyltransferase toward terpineol by 1.4 times compared to the control values. Another group of rats received a single injection of 80 mg/kg of 3-methylcholanthrene on the first day (control animals received corn oil alone). Animals were sacrificed on day 5. 3-Methylcholanthrene did not enhance UDPGT activity toward terpineol.

In Gunn rats, UDPGT activity toward terpineol was 0.8 fold the activity in Wistar rats and in phenobarbital-treated rats, this activity was 0.7 fold the activity in phenobarbital-treated Wistar rats.

Groups of 4 guinea pigs received intraperitoneal doses of 20-40 mg phenobarbital/kg body weight in 0.9% NaCl for 9 days. Control animals received 0.9% NaCl alone for 9 days. UDP-glucuronosyltransferase (UDPGT) activities were measured using 10 µL of terpineol (final concentration of terpineol, 0.175 mM) as the substrate. Phenobarbital enhanced UDPGT activity towards terpineol by 2.8 times over the control value.

Therefore, it can be concluded that terpineol was conjugated with UDP-glucuronosyltransferase (UDPGT).

Reference 3

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

metabolism

Principles of method if other than guideline:

Study was performed to investigate the inhibitory effects of α-terpineol on liver microsomal enzymes (CYP1A1, CYP1A2 and CYP2B1) involved in the biotransformation of xenobiotic substances.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Oswaldo Cruz Foundation breeding stock
- Housing: animals were housed in standard rat cages
- Diet: pelleted diet for laboratory rats (Nuvital®, Nuvilab, Curitiba, PR, Brazil), ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 1 °C
- Humidity: approximately 70 %
- Photoperiod: 12 h dark / 12 h light

Remarks:

Doses / Concentrations:
Inhibitory effects on PROD activity: 1, 5, 10 and 20 µM
Inhibitory effects on EROD activity: 75 and 150 µM
Inhibitory effects on MROD activity: 9, 38 and 150 µM

Table 7.1.1/1: Inhibitory effects of terpineol on PROD, EROD and MROD activity

 

Substance	Concentration (µM)	Rate of O-dealkylation (pmol resorufin/min per mg protein)	Activity (%)	IC50 (µM)
PROD activity^a
Terpineol	0	2047 ± 134	100	14.8
	1	1466 ± 134*	72
	5	1350 ± 67*	66
	10	1117 ± 67*	55
	20	924 ± 7*	45
EROD activity^b
Substance	Concentration (µM)	Rate of O-dealkylation (nmol resorufin ± min ± mg protein)	Activity (%)	IC50 (µM)
Terpineol	0	10.5 ± 0.6	100	-
	75	9.6 ± 1.3	92
	150	9.5 ± 0.7	91
MROD activity^c
Substance	Concentration (µM)	Rate of O-dealkylation (pmol resorufin ± min ± mg protein)	Activity (%)	IC50 (µM)
Terpineol	0	1918 ± 109	100	-
	9	1942 ± 71	101
	38	1906 ± 137	99
	150	1895 ± 109	99

 

Microsomes were PB-induced (PROD) / β-NF- induced (EROD and MROD) and pentoxyresorufin (a)/ethoxyresorufin (b)/methoxyresorufin (c) concentration was 5 µM. Statistical analysis: ANOVA followed by Student’st-test. * Values different from control values (*P<0.05).

 

Results: Terpineol caused a concentration-related decrease in microsomal PROD activity with an IC50 value of 14.8 µM. Terpineol tested at concentrations up to 150 µM, did not cause any reduction of EROD or MROD activity.

Conclusions:

Terpineol showed inhibitory effect on CYP2B1 activity.

Executive summary:

The effects of terpineol on the activity of pentoxyresorufin-O-depentilase (PROD, a selective marker for CYP2B1) was determined in a pool of liver microsomes prepared from phenobarbital-treated rats, whereas the effect of terpineol on the activities of ethoxyresorufin-O-deethylase (EROD, a marker for CYP1A1) and methoxyresorufin-O-demethylase (MROD, a marker for CYP1A2) were determined in a pool of liver microsomes prepared from β-naphthoflavone-treated rats.

 

Terpineol caused a concentration-related decrease in microsomal PROD (a selective marker for CYP2B1) activity with an IC50 value of 14.8 µM proved to be in vitro inhibitors of PROD reaction. Terpineol tested at concentrations up to 150 µM, did not cause any reduction of EROD or MROD activity. The inhibitory effects on CYP2B1 suggest that terpineol may interfere with the metabolism of xenobiotics which are substrates for this isoenzyme.

Terpineol showed inhibitory effect on CYP2B1 activity.

Reference 4

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 August 2011 to 11 June 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparative metabolism was studied using hepatocytes of different species

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

metabolism

Principles of method if other than guideline:

A study was conducted to compare the in vitro metabolism of test substance between three species (rat, rabbit and human) using hepatocytes. The analytical method used radio-HPLC, followed by HPLC coupled with mass spectrometry (LC-MS) to identify the radiolabelled metabolites generated and to determine metabolite profile.

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP compliance programme (inspected on 01-03 July 2014 / signed on 15 September 2014)

Radiolabelling:

yes

Species:

other: rat, rabbit and human cryopreserved hepatocytes

Strain:

other:

Duration and frequency of treatment / exposure:

Test substance was incubated in triplicate with cryopreserved hepatocytes (1 × 10^6 viable cells) from rat, rabbit and human at test substance concentrations of 1, 10 and 100 μM over incubation times of 0, 1 and 4 h in an orbital shaking water bath (set at 60 rpm and 37 °C). At the end of the requisite incubation period, reactions were terminated by the addition of chilled acetonitrile.

Remarks:

Doses / Concentrations:
1, 10 and 100 μM

No. of animals per sex per dose / concentration:

Incubations were conducted in triplicate, with the exception of the control hepatocytes in the absence of test substance which were incubated singly.

Positive control reference chemical:

Positive control incubations were also conducted with 7-ethoxy[14C]coumarin (7-EC) as substrate, together with control incubations in the absence of hepatocytes.

Details on study design:

PREPARATION OF HEPATOCYTES
Cryopreserved hepatocytes were thawed and decanted directly into warmed 32.4 % (v/v) Percoll (rat and rabbit) or 28.8 % (v/v) Percoll (human) in supplemented Williams' Medium E. Trypan blue exclusion methods were used to determine the cell viability and density.
Incubation components were mixed together to give a total incubation volume of 1.0 mL with final test substance concentrations of 1, 10 and 100 µM:
Foetal calf serum (100 µL); Supplemented Williams' Medium E, Test substance (10 µL of solution in ethanol), Hepatocyte suspension (1 x 10^6 viable cells).

Preliminary incubations were conducted with non-radiolabelled test substance at a nominal concentration of 100 µM.

CONTROLS
On each occasion, control incubations in the absence of hepatocytes were conducted in parallel at each substrate concentration for 4 h only.
Additionally, control incubates were conducted with hepatocytes in the absence of test substance for 4 h only.

Statistics:

None

Preliminary studies:

Preliminary phase results showed that it was not possible to detect any of the unchanged parent test substance using LC-MS. For terpineol, the only metabolite detected by LC-MS was a glucuronide conjugate of terpineol (molecular weight 330).

Metabolites identified:

yes

Details on metabolites:

Metabolite profiling

Metabolite profiling following incubations of [14C]terpineol with rat, rabbit and human hepatocytes showed up to 20 individual regions of radioactivity, assigned T1 to T20 in order of increasing retention time.

Region T20 eluted with a typical retention time of 25.2 min, was consistent with terpineol reference substance, which indicated the identity of this component (although the identity could not be confirmed by LC-MS).
Additional components that were identified by mass spectrometry were regions T10, T11 and T16 which eluted with typical retention times of 9.9, 11.5 and 20.5 min, respectively (glucuronide conjugates of hydroxylated terpineol) and region T18 which eluted with a typical retention time of 22.6 min (glucuronide conjugate of terpineol).

The radioactivity profiles for the rabbit and human hepatocyte incubations tended to be similar, with fewer metabolites observed in the rat profiles.

In rat hepatocytes:
Region T18 was the largest component for each concentration in the rat profiles (mean: between 37.5 and 44.7% of sample radioactivity). Regions T1 and T17 were also major components in the 1 µM profile (mean: 18.6 and 24.6% of sample radioactivity respectively) and 10 µM profile (mean: 19.7 and 27.3% of sample radioactivity respectively), whilst regions T8, T10 and T11 were major components in the 100 µM profiles (mean: 12.4, 10.7 and 11.8% of sample radioactivity respectively).

In rabbit hepatocytes:
The 1 µM rabbit hepatocyte incubations showed three major components: regions T1, T11 and T18 (mean: between 12.2 and 13.2% of sample radioactivity). For the 10 µM rabbit incubations, regions T1 and T11 were the only major components (mean: 11.8 and 18.8% of sample radioactivity, respectively), whilst the only major components in the 100 µM incubations were regions T11 and T20 (mean: 36.8 and 23.3% of sample radioactivity, respectively).

in human hepatocytes:
The 1 µM samples showed three major components, regions T1, T2 and T11 (mean: 28.3, 18.4 and 15.5% of sample radioactivity, respectively). The major regions in the 10 µM incubation samples were also T1, T2 and T11 (respective mean: 24.5, 16.9 and 18.1% of sample radioactivity). The largest region in the 100 µM incubation samples was region T11 (mean: 43.1% of sample radioactivity), with other major regions observed being T2, T13 and T20 (mean: 10.7, 14.1 and 14.0% of sample radioactivity, respectively).

Interspecies comparisons

Incubations were conducted in triplicate and the variability in the percentage of sample radioactivity in each region between replicates was typically within 10 % of the mean result.

 

Table 7.1.1/1: Summary of the mean radioactive concentration range in samples obtained following 0, 1 and 4 h incubations of [14C]terpineol with rat, rabbit and human hepatocytes

 

Incubation time (h)	Mean radioactive concentration range (percentage of nominal)
0	83.4 - 106
1	56.4 - 108
4	38.6 - 145

 

Concentrations of radioactivity following 0 h incubations were generally close to nominal (between 83.4 and 106%). However, concentrations tended to be reduced in the 1 h incubations (between 56.4 to 108% of nominal) and were further reduced in the 4 h incubations (between 38.6 and 145% of nominal). The reduction in concentration was probably related to loss of radiolabelled material due to the volatile nature of the terpineol (and possibly some of its metabolites).

 

Incubation of [¹⁴C]terpineol in the absence of hepatocytes (control)

The only component observed (mean > 99.0 % of sample radioactivity) for each incubation concentration was region T20 (typical retention time 25.2 min) which co-eluted with terpineol reference substance. These results confirmed that [¹⁴C]terpineol was stable under the incubation conditions used in this study.

  

Table 7.1.1/2: Summary of the radioactivity profiles obtained following 4 h incubations of [¹⁴C]terpineol with rat, rabbit and human hepatocytes

 

Region	Typical RT (min)	Mean percentage of sample radioactivity
		Rat			Rabbit			Human
		1 µM	10 µM	100 µM	1 µM	10 µM	100 µM	1 µM	10 µM	100 µM
T1	0.8	18.6	19.7	°	13.2	11.8	-	28.3	24.5	°
T2	1.9	-	-	°	5.3	5.1	7.2	18.4	16.9	10.7
T3	2.5	-	-	°	°	°	°	5.2	7.4	°
T4	3.4	-	-	°	°	°	°	°	°	°
T5	4.9	-	-	°	°	°	°	°	°	°
T6	5.9	-	°	°	°	°	-	°	°	-
T7	6.6	-	°	°	°	°	-	°	°	-
T8	7.9	°	°	12.4	°	5.9	-	°	°	-
T9	8.8	°	°	°	°	5.6	-	°	°	°
T10	9.9	°	°	10.7	6.7	8.3	°	°	°	-
T11	11.5	°	°	11.8	12.3	18.8	36.8	15.5	18.1	43.1
T12	12.9	-	-	°	°	°	°	°	°	°
T13	14.3	-	-	°	°	°	6.6	7.2	8.6	14.1
T14	16.5	-	-	-	-	-	-	-	-	°
T15	18.0	-	-	-	-	-	-	-	-	-
T16	20.5	-	-	-	°	°	°	°	-	°
T17	21.8	24.6	27.3	-	7.4	5.5	°	°	-	-
T18	22.6	44.7	37.5	42.0	12.2	6.4	7.0	°	°	5.2
T19	23.2	-	-	-	-	-	-	-	-	-
T20	25.2	°	°	°	9.6	5.3	23.3	°	°	14

- Not detected or <0.5 % of sample radioactivity

° Minor component: 0.5 to 10 % of sample radioactivity

Values above 5% were reported in the table 7.1.1/2.

Regions T 10, T11 and T16 were identified by LC-MS as glucuronide conjugates of hydroxylated terpineol

Region T18 was identified by LC-MS as a glucuronide conjugate of terpineol

Region T20 (bold text) co-eluted with terpineol reference substance

Conclusions:

Metabolite profiles following hepatocyte incubations of [14C]terpineol showed up to 20 individual regions of radioactivity (assigned T1 to T20). The profiles for the rabbit and human tended to be similar with the main metabolite being the glucuronide conjugates of hydroxylated terpineol (up to 43% of sample radioactivity). Fewer metabolites were observed in the rat profiles with the main metabolite being the glucuronide conjugate of terpineol (T18 up to 45% of the sample radioactivity).

Executive summary:

The in vitro metabolism of [¹⁴C] terpineol was studied following incubation with rat, rabbit and human cryopreserved hepatocytes at concentrations of 1, 10 and 100 µM over incubation times of 0, 1 and 4 h. The initial cell viability of the hepatocytes in suspension was assessed using the trypan blue exclusion test. The cellular integrity of the hepatocytes was assessed by measuring the leakage of lactate dehydrogenase (LDH) activity from the hepatocyte cytosol into the medium in the presence and absence of each test substance over the incubation time course. Positive control incubations were also conducted with 7-ethoxy[¹⁴C]coumarin (7-EC) as substrate, together with control incubations in the absence of hepatocytes. The profile of radioactivity was determined for each sample and representative samples were then analysed by LC-MS to identify the principal metabolites observed (where possible).

 

Concentrations of radioactivity following 0 h incubations were generally close to nominal (between 83.4 and 106 %). However, concentrations of radioactivity reduced with time; this was attributed to loss of radioactivity during the incubation procedures due to the volatility of the test substance and possibly of some metabolites.

 

Radio-HPLC method was used to determine the metabolite profiles generated during incubations of the test substance with rat, rabbit and human hepatocytes. LC-MS was then used to investigate and where possible elucidate the structures of metabolites > 10% of sample radioactivity. The retention time of unchanged test substance was established by co-chromatography which provided a tentative identity.

 

Metabolite profiles following hepatocyte incubations of [¹⁴C]terpineol showed up to 20 individual regions of radioactivity (assigned T1 to T20). Region T20 was consistent with terpineol, although the identity could not be confirmed by LC-MS. The profiles for the rabbit and human tended to be similar, with fewer metabolites observed in the rat profiles. A metabolism pathway was proposed for [¹⁴C]terpineol and the components that were identified by LC-MS were regions T10, T11 and T16 (glucuronide conjugates of hydroxylated terpineol) and region T18 (glucuronide conjugate of terpineol). Other major components (>10 % of sample radioactivity), regions T1, T13 and T17, were observed in some profiles, but the identity of these could not be established.

Reference 5

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1975

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

metabolism

Principles of method if other than guideline:

Urine samples from an infant (human) were analysed to determine the metabolites of α-terpineol.

GLP compliance:

no

Radiolabelling:

no

Species:

other: rat and human

Strain:

Sprague-Dawley

Route of administration:

other: rat (intraperitoneal); human (poisoning)

Remarks:

Doses / Concentrations:
Rat: 100 mg
Human (infant): poisoning case

Control animals:

no

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling (rats): urine samples (0-24 h and 24-48 h)
- Sampling (human): random urine samples were collected from the infant on two occasions and family members (mother, father and sibling).
- Samples were kept frozen (-14 °C) until analyzed.
- Method type(s) for identification: neutral metabolites were isolated from the infant's urine (10 mL) and from rat urine (10 mL) using a DEAE Sephadex procedure. After conversion to derivatives (methylation followed by silylation), the extracted neutral metabolites were identified by gas chromatographic and gas chromatographic-mass spectrometric methods. The acidic metabolites were isolated from urine by the use of a DEAE-Sephadex procedure. After conversion to derivatives (methylation and silylation), the acidic metabolites were identified by GC-MS analyses.
- Other: the structure of the major neutral urinary metabolite excreted by the infant and the rat was confirmed by synthesis from α-terpineol using a procedure described previously (Harvey et al., 1972). The gas chromatographic and mass spectrometric properties of the trimethylsilyl derivatives of the synthetic and urinary metabolite were identical.

Statistics:

none

Metabolites identified:

yes

Details on metabolites:

Gas chromatographic analyses of the neutral fraction isolated from the infant's urine revealed the major metabolite i.e., p-methan-1 ,2, 8-triol ((HO)2-α-terpineol)). Mass spectral analyses indicated that the major metabolite was a triol. The major neutral metabolite excreted by the rat following intraperitoneal injection of α-terpineol was also a triol and was identified by GC-MS as p-menthan-1, 2, 8-triol.

An isomeric triol, present in small amounts in the urine of the infant and the rat, was assigned the structure p-menthan-1, 2, 9-triol on the basis of GC-MS analyses. This triol was a metabolite of p-menth-1-en-9-ol, a minor constituent of pine oil. The glucuronide of α-terpineol (methyl ester trimethylsilyl ether derivative) was identified in the acidic fraction of both the infant's and rat's urine.

These metabolites rapidly disappeared and were not detected in urine collected 12 days after the child was admitted to the hospital when he was alert. However, trace amounts of p-menthan-1, 2, 8-triol were detected in the urine of the father and older sibling; larger amount was found in the maternal urine.

The metabolism of α-terpineol by the infant and the rat was very similar, and probably involved the epoxide-diol pathway. Epoxides were not detected in any of the urine samples collected from the child or from the α-terpineol treated rat. However, the epoxide(s) formed in vivo may have reacted irreversibly with cellular components.

none

Conclusions:

The metabolites identified in the urine of rat and infant was p-methan-1, 2, 8-triol (major metabolite).

Executive summary:

In a metabolism study, random urine samples were collected from the infant on two occasions. The metabolites were identified by gas chromatographic and gas chromatographic-mass spectrometric methods. These metabolites were confirmed by mass spectrometry to be the same metabolites excreted by Sprague-Dawley rats intraperitoneally injected with α-terpineol (100 mg).

 

Gas chromatographic analyses of the neutral fraction isolated from the infant's urine revealed the major metabolite i.e. p-methan-1, 2, 8-triol ((HO)2-α-terpineol)). Mass spectral analyses indicated that the major metabolite was a triol. Similar results were observed in rats administered with α-terpineol. An isomeric triol, present in small amounts in the urine of the infant and the rat was p-menthan-1, 2, 9-triol on the basis of GC-MS analyses, which was the metabolite of p-menth-1-en-9-ol, a minor constituent of pine oil. The glucuronide of α-terpineol (methyl ester trimethylsilyl ether derivative) was identified in the acidic fraction of both the infant's and rat's urine.

 

These metabolites rapidly disappeared and were not detected in urine collected 12 days after the child was admitted to the hospital when he was alert. However, the trace amounts of p-menthan-1, 2, 8-triol were detected in the urine of the father and older sibling; larger amount was found in the maternal urine.

Reference 6

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 January to 22 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD 417 Guideline.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP compliance programme (inspected on 23 August 2011 / signed on 12 December 2011)

Radiolabelling:

yes

Species:

rat

Strain:

other: Sprague Dawley / Crl:CD(SD)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 40-48 days
- Weight at study initiation: 191-234 g
- Housing: During the acclimatisation period animals were housed (in groups of up to five) in polycarbonate cages with a stainless steel mesh lid. After dosing rats were returned to their battery cages (for kinetic and tissue distribution experiments) or housed individually in glass metabolism cages (Metabowls®) which facilitated the separate collection of urine and faeces (excretion/balance experiments).
- Individual metabolism cages: yes
- Diet: food (VRF1 pellets, Special Diets Services Ltd, Witham, Essex, UK), ad libitum
- Water: potable water taken from the public supply, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 55 ± 15 %
- Air changes: approximately 15 per hour
- Photoperiod: 12 h dark / 12 h light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Radiolabelled (in ethanol solution) and non-radiolabelled alpha-terpineol were combined in corn oil to achieve the desired specific activity for the dosed material.

DOSE VOLUME: 5 mL/kg bw

ANALYSIS:
At least duplicate aliquots were taken for liquid scintillation counting (LSC). The actual amount of formulation administered was determined gravimetrically by weighing the dose syringe when loaded and again after administration.
To accurately determine the quantity of radioactivity administered to each animal, further aliquots (1 mL) of dose formulation were similarly weighed into volumetric flasks. The flasks were made up to volume with ethanol (or acetonitrile) and triplicate aliquots of each taken for LSC. The specific activity of the radiodiluted [14C]-alpha-terpineol was determined from these aliquots.

Duration and frequency of treatment / exposure:

single dose

Remarks:

Doses / Concentrations:
75, 250 and 750 mg/kg bw

No. of animals per sex per dose / concentration:

Excretion/tissue distribution: 4 male rats/dose
Plasma/blood cell kinetics: 12 male rats/dose
Tissue distribution: 9 male rats/dose

Control animals:

yes

Positive control reference chemical:

not applicable

Details on dosing and sampling:

EXCRETION/DISTRIBUTION EXPERIMENTS (GROUPS 1-3)
- Each animal was placed in a separate Metabowl®.
- Tissues and body fluids sampled: blood, urine, faeces, cage washes
- Time and frequency of sampling:
Urine: Urine was collected from each animal into receivers cooled in solid carbon dioxide at 0-6, 6-24 h and at 24 h intervals thereafter up to 168 h.
Faeces: Faeces were collected from each animal into receivers cooled in solid carbon dioxide at 0-24 h and at 24 h intervals up to 168 h. The interior of each Metabowl® was washed with water at 24 h intervals and the washings retained for radioactivity measurement.
Blood: Immediately prior to sacrifice, blood samples (6-8 mL) withdrawn by cardiac puncture from the anaesthetised animals (isoflurane). Aliquots were taken for measurement of radioactivity concentration of the whole-blood prior to the remainder being centrifuged to separate the plasma.
- Urine, faeces and cage washes were stored at ≤-15 °C prior to analysis. Blood samples were stored at ca 4 °C.
- Animals were sacrificed by cervical dislocation at 168 h after dosing and the following tissues/organs were removed for analysis: adrenal glands, bone, bone marrow, brain, epididymis, fat (abdominal), gastrointestinal tract (including contents), heart, kidneys, liver, lungs, muscle (skeletal), pituitary, residual carcass, skin, spleen, testes and thyroid. All tissue samples were stored at ≤-15 °C prior to analysis except low weight tissues (adrenal glands, bone marrow, pituitary and thyroid) which were analysed immediately.

PLASMA AND WHOLE-BLOOD KINETICS EXPERIMENTS (GROUPS 4-6)
- Blood samples (ca 0.4 mL) were taken from a tail vein and terminal blood samples were obtained by cardiac puncture.
- Time and frequency of sampling: blood samples were collected in heparinised tubes at the following times from each group (animals in each group were divided into three subgroups of four):
Subgroup 1: Pre-dose, 1, 4, 24 and 96 h
Subgroup 2: 0.25, 2, 6, 48 and 120 h
Subgroup 3: 0.5, 3, 12, 72 and 168 h
Overall sampling schedule: pre-dose, 0.25, 0.5, 1, 2, 3, 4, 6, 12, 24, 48, 72, 96, 120 and 168 h
- Other: plasma was separated from the blood cells by centrifugation. Each subgroup of animals was sacrificed on completion of the specified sampling schedule. Blood samples were stored at ca +4 °C.

TISSUE DISTRIBUTION EXPERIMENTS (GROUPS 7-9)
Sacrifice times for the groups of 3 animals were determined from plasma kinetics experiment (group 4-6). The Sacrifice times were as follows for different parameters:
Tmax: 15 minutes, 1 h and 1 h for 75, 250 and 750 mg/kg bw, respectively
Half Tmax: 90 minutes, 3 h and 6 h for 75, 250 and 750 mg/kg bw, respectively
Latest quantifiable: 24, 24 and 48 h for 75, 250 and 750 mg/kg bw, respectively

Statistics:

no data

Preliminary studies:

Not applicable

Type:

absorption

Results:

Following a single oral dose, alpha-terpineol was well absorbed with 60% excreted in urine and unchanged alpha-tepineol accounted for a max of 18.7% dose in faeces.

Type:

distribution

Results:

Concentrations of radioactivity in tissues were highest in the kidney and liver at each dose level. Overall tissue accumulation after single oral doses was low with only a small proportion of the dose retained in tissues at 168 h (<0.1% dose).

Type:

metabolism

Results:

Unchanged alpha-terpineol represented max 18.7% dose in faeces and 0.1% dose in urine. Four major metabolites in urine (3.4 - 20.7% dose) were identified as glucuronide conjugates of alpha-terpineol or glucuronide conjugates of hydroxy alpha-terpineol.

Type:

excretion

Results:

Following single oral doses of [14C]-alpha-terpineol, most of the radioactivity (>90%) was eliminated in urine and faeces within 48 hours. Excretion was mainly via the urine.

Details on absorption:

The rate of systemic exposure of rats to alpha-terpineol, characterised by Cmax, increased approximately proportionately with increasing dose over the dose range 75 to 750 mg/kg in both plasma and whole blood. The extent of systemic exposure, characterised by AUCall, increased with increasing dose over the dose range 75 to 750 mg/kg; however, these increases were greater than the proportionate dose increment in both plasma and whole blood. Overall, the AUCall values at the highest dose level (750 mg/kg) were ca 2.3-fold higher than those values predicted from a linear relationship.

Details on distribution in tissues:

Tissue distribution (Groups 7 to 9):
- Concentrations of radioactivity in tissues were highest at 90 minutes, 1 h and 1 h after dose administration at 75, 250 and 750 mg/kg bw, respectively. Concentrations of radioactivity in tissues were highest in the kidney and liver at each dose level. Overall tissue accumulation after single oral doses was low with only a small proportion of the dose retained in tissues at 168 h (<0.1% dose). Concentrations in tissues generally declined over time with the exception of animals sacrificed at 15 minutes and 90 minutes, at the low dose level, in which an increase in tissue concentrations was generally observed.
- Tissue/plasma ratios were generally less than one other than for kidney and liver (all sacrifice times) and fat (at the later sacrifice times). Plasma ratios were not calculable at 168 h.

Details on excretion:

Excretion/balance and tissue distribution experiments (Groups 1 to 3):
Following single oral doses of [14C]-alpha-terpineol, most of the radioactivity (>90%) was eliminated in urine and faeces within 48 h. Excretion was mainly via the urine and accounted for 60.13 - 65.28% dose during 0 - 48 h at each dose level. Faecal excretion during 0 - 48 h accounted for 26.00 - 32.63% dose.
During 168 h after administration of test item urinary excretion accounted for 65.85, 66.53 and 61.48% dose at 75, 250 and 750 mg/kg bw, respectively. Most of the urinary radioactivity was excreted during 0-48 h: 64.27, 65.28 and 60.13 % dose at 75, 250 and 750 mg/kg bw, respectively.
Faecal excretion during 0-168 h accounted for 26.78, 27.50 and 33.08% dose at 75, 250 and 750 mg/kg bw, respectively. Most of the faecal radioactivity was excreted during 0-48 h: 26.00, 27.03 and 32.63 % dose at 75, 250 and 750 mg/kg bw, respectively.
Overall excretion of radioactivity was rapid with 90.27, 92.31 and 92.76% dose (75, 250 and 750 mg/kg bw, respectively) excreted during 0-48 h.
Following sacrifice at 168 h after dosing, no significant radioactivity was detected in the carcass. Overall recovery of radioactivity was 93.10, 95.68 and 95.33% dose at 75, 250 and 750 mg/kg bw, respectively.
No radioactivity was recovered in individual tissues at 168 h.

At 75 mg/kg bw, concentrations of radioactivity in tissues at sacrifice were low. The highest concentrations, 0.100 μg equiv/g, were measured in the liver. Concentrations of 0.091 μg equiv/g and 0.063 μg equiv/g were measured in the spleen and kidney respectively. Concentrations ≤0.02 μg equiv/g were measured in the whole-blood, brain, lungs and skin. Concentrations of radioactivity in all other individual tissues were below the limit of detection.

At 250 mg/kg bw, concentrations of radioactivity were highest in the liver (0.295 μg equiv/g) and kidney (0.043 μg equiv/g). No radioactivity was detected in the other individual tissues.

At 750 mg/kg bw, the highest concentrations were measured in the liver (0.811 μg equiv/g) and skin (0.687 μg equiv/g). Concentrations calculated in blood cells were 0. 773 μg equiv/g. Concentrations of 0.113 and 0.322 μg equiv/g were measured in the kidney and whole blood respectively. No radioactivity was detected in the other individual tissues.

Tissue/plasma ratios were generally less than one other than for kidney and liver (all sacrifice times) and fat (at the later sacrifice times). Plasma ratios were not calculable as no radioactivity was detected in plasma.

Toxicokinetic parameters:

Cmax: Plasma: 25.3 µg/mL (75 mg/kg); 84.5 µg/mL (250 mg/kg) and 246 µg/mL (750 mg/kg).

Toxicokinetic parameters:

Cmax: Whole Blood: 14.8 µg/mL (75 mg/kg); 57.5 µg/mL (250 mg/kg) and 167 µg/mL (750 mg/kg).

Toxicokinetic parameters:

AUC: Plasma: 102 µg/mL (75mg/kg); 480 µg/mL (250 mg/kg) and 0.72 µg/mL (250 mg/kg) and 2290 µg/mL (750 mg/kg)

Toxicokinetic parameters:

AUC: Whole blood: 69.4 µg/mL (75 mg/kg); 347 µg/mL (250 mg/kg) and 1650 µg/mL (750 mg/kg).

Metabolites identified:

yes

Details on metabolites:

Metabolites in urine/faeces after single dose (Groups 1 to 3):
- Unchanged alpha-terpineol represented 17.4-18.7% dose at the low and mid dose levels and 11.1% dose at the high dose level in faeces; 0.1 % dose in urine.
- Four major metabolites (U6/7 and U8/9) were observed in urine (3.4-20.7% dose). U9 and U6 were the most significant components of the respective pairs. Proportionally, U6/7 increased with time as U8/9 decreased with time. For each dose level overall, U6 accounted for 12.1 - 20.7% dose, U7 accounted for 3.4 - 4.0% dose, U8 accounted for 11.1 - 18.0% dose and U9 accounted for 15.7 - 19.9% dose. After enzyme treatment, U8 was identified as a glucuronide conjugate of alpha-terpineol. Subsequent analysis by mass spectrometry identified U9 as an isomer of U8 and U6/7 as isomeric forms of glucuronide conjugates of hydroxy alpha-terpineol. There were no major unidentified metabolites.
- U8 and U9 were present at <1% dose at the low and mid dose levels, but increased to 3.8 - 5.8% dose at the high level in faeces.

Metabolites in tissue extracts (Groups 7 to 9):
- Extracts of liver, kidney, plasma and testes were analysed by HPLC. Alpha-terpineol was extensively metabolised in tissues and accounted for ≤ 5% tissue radioactivity in all cases except liver at the low dose level (9.1% dose). At the Tmax timepoint the chromatographic profile of tissue extracts (kidney, liver, plasma and testes) were qualitatively similar to urine. The major metabolites in tissues were glucuronide conjugates of alpha-terpineol and glucuronide conjugates of hydroxy alpha-terpineol.
- U8/9 accounted for 64.4 - 74.4% sample radioactivity for liver, kidney and plasma at the low dose and declined to 24.5 - 39.0% sample radioactivity at the high dose level. U4/5/6/7 increased from 22.3 - 32.6% sample radioactivity at the low dose to 53.1 - 64.6% sample radioactivity at the high dose. A polar component accounted for a maximum of 4.7% sample radioactivity for liver, kidney and plasma at each dose level. A chromatographic profile was only obtained for testes extracts at the mid dose level. alpha-Terpineol accounted for 5.0% sample radioactivity, U4/5/6/7 and U8/9 accounted for 39.2 and 10.0% sample radioactivity respectively. A polar component accounted for 41.9% sample radioactivity; however, this may have been due to matrix effects causing poor retention of radioactivity on the column.
- There were no unidentified components > 5% dose.

Table 7.1.1/2: Excretion of radioactivity during 0 - 168 h after administration

 

Samples	75 mg/kg bw	250 mg/kg bw	750 mg/kg bw
Urine	65.85	66.53	61.48
Cage wash	1.38	1.60	0.65
Faeces	26.78	27.50	33.08
Carcass	0.05	0.05	0.13
GIT	0.03	0.00	0.00
Total	93.10	95.68	95.33

 

 

Plasma and whole blood kinetics (Groups 4 to 6)

 

Table 7.1.1/3: Kinetic parameters

 

Matrix	Cmax (μg/g)	Tmax (h)	AUCall(μg.h/g)	K (1/h)	t1/2(h)
Plasma
75 mg/kg bw	25.3	0.25	102	0.1115	6.2
250 mg/kg bw	84.5	1	480	0.1034	6.7
750 mg/kg bw	246	1	2290	0.1010	6.9
Whole-blood
75 mg/kg bw	14.8	0.25	69.4	0.0494^a	14.0^a
250 mg/kg bw	57.5	1	347	0.0703	9.9
750 mg/kg bw	167	1	1650	0.0943	7.4

^aValue is an estimate as the data did not meet all the acceptance criteria

 

The rate of systemic exposure of rats to alpha-terpineol, characterised by C_max, increased approximately proportionately with increasing dose over the dose range 75 to 750 mg/kg bw in both plasma and whole blood. The extent of systemic exposure, characterised by AUC_all, increased with increasing dose over the dose range 75 to 750 mg/kg bw; however, these increases were greater than the proportionate dose increment in both plasma and whole blood. Overall, the AUC_allvalues at the highest dose level (750 mg/kg bw) were ca 2.3-fold higher than those values predicted from a linear relationship.

The whole blood to plasma ratios, calculated using AUC_all, were 0.68 at the lowest dose level and 0.72 at the 250 and 750 mg/kg bw dose levels. These results suggested that there was relatively little distribution of radioactivity into red blood cells.

The terminal half-life was estimatable for all groups in plasma and was an average of 6.6 h. The terminal half-life for whole blood could not be reliably estimated at the lowest dose level but at the 250 and 750 mg/kg bw dose levels, t_1/2was 9.9 and 7.4 h respectively.

Conclusions:

After single oral doses of 14C-alpha-terpineol (75, 250 and 750 mg/kg), more than 90% of the dose was excreted within 48 h. Excretion was mainly via the urine where 4 major metabolites were identified as glucuronide conjugates of alpha-terpineol or glucuronide conjugates of hydroxy alpha-terpineol. Faecal excretion accounted for 26-33 % dose. Overall, the AUCall values at the highest dose level (750 mg/kg bw) were ca 2.3-fold higher than those values predicted from a linear relationship. The average terminal half-life for all groups in plasma was 6.6 h. Overall tissue accumulation after single oral doses was low (<0.1% dose).

Executive summary:

The absorption, distribution, metabolism and excretion of [isopropyl methyl-¹⁴C]-alpha-terpineol were studied after single oral doses at 75, 250 and 750 mg/kg bw to male rats.

 

After single oral doses of ¹⁴C-alpha-terpineol, more than 90% of the dose was excreted within 48 h. Excretion was mainly via the urine and accounted for 60.13 - 65.28% dose during 0 - 48 h at each dose level. Faecal excretion during 0 - 48 h accounted for 26.00 - 32.63% dose.

 

The rate of systemic exposure of rats to alpha-terpineol, characterised by C_max, increased approximately proportionately with increasing dose over the dose range 75 to 750 mg/kg bw in both plasma and whole blood. The extent of systemic exposure, characterised by AUC_all, increased with increasing dose over the dose range 75 to 750 mg/kg bw; however, these increases were greater than the proportionate dose increment in both plasma and whole blood. Overall, the AUC_all values at the highest dose level (750 mg/kg bw) were ca 2.3-fold higher than those values predicted from a linear relationship. The whole blood to plasma ratios, calculated using AUC_all, were 0.68 at the lowest dose level and 0.72 at the 250 and 750 mg/kg bw dose levels. These results suggested that there was relatively little distribution of radioactivity into red blood cells.

The terminal half-life was estimatable for all groups in plasma and was an average of 6.6 h.

 

Concentrations of radioactivity in tissues were highest in the kidney and liver at each dose level. Overall tissue accumulation after single oral doses was low with only a small proportion of the dose retained in tissues at 168 h (<0.1% dose). Concentrations in tissues generally declined over time with the exception of animals sacrificed at 15 minutes and 90 minutes, at the low dose level, in which an increase in tissue concentrations was generally observed. Tissue/plasma ratios were generally less than one other than for kidney and liver (all sacrifice times) and fat (at the later sacrifice times).

 

Unchanged alpha-terpineol represented a maximum of 18.7% dose in faeces and 0.1% dose in urine. Four major metabolites in urine (3.4 - 20.7% dose) were identified by mass spectrometry as glucuronide conjugates of alpha-terpineol or glucuronide conjugates of hydroxy alpha-terpineol.

 

At the T_max timepoint the chromatographic profile of tissue extracts were qualitatively similar to urine. Major components in tissues were alpha-terpineol, glucuronide conjugates of alpha-terpineol or glucuronide conjugates of hydroxy alpha-terpineol. Alpha-terpineol accounted for a maximum of 9.1% sample radioactivity in the liver at the low dose level. There were no unidentified components > 5% dose.

Reference 7

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well conducted study with missing details such as number of animals used and animals housing conditions.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

metabolism

Principles of method if other than guideline:

Urine metabolites were identified from male rats that were given α-terpineol orally at a daily dose of 600 mg/kg bw for 20 days. Additional animals given the same treatment were sacrificed at different times and the effect of the treatment on the activity of hepatic enzymes was studied.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

other: albino rats IISc., strain

Sex:

male

Details on test animals or test system and environmental conditions:

Male rats weighed 185-225 g. After dosing, they were housed separately in metabolism cages with free access to food and water.

Route of administration:

oral: gavage

Vehicle:

other: methyl cellulose

Details on exposure:

Suspension in 1% methyl cellulose; 2 mL/rat was administered.

Duration and frequency of treatment / exposure:

once daily for 20 days

Remarks:

Doses / Concentrations:
600 mg/kg bw/day

No. of animals per sex per dose / concentration:

no data

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

None

Details on study design:

In this study a group of rats was used to identify the urinary metabolites of α-terpineol and additional animals were used to assess the effect of the treatment on the activity of hepatic enzymes.

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Sampling: urine was collected daily, adjusted to pH 3-4 and extracted twice with equal volume of diethyl ether. The aqueous portion remaining after ether extraction containing conjugated metabolites was then subjected to acid hydrolysis and then extracted with ether. Both the ether extracts were separated into neutral and acid fractions. The acidic fractions were esterified using diazomethane.
- Method type(s) for identification: UV, IR, NMR and visible absorption spectra were recorded and Gas Liquid Chromatography (GLC) analyses were also used. Thin layer chromatography (TLC) (silicagel G) of the metabolites was performed.
- Other: the rats used for studying the effects of α-terpineol on the hepatic microsomal cytochrome P-450 system were dosed as mentioned earlier. The control and test item treated rats were sacrificed after 1, 2, 3, 6 or 9 days of dosing and the liver microsomes were prepared by the method of Lu and Levin (1972). Protein concentrations were determined by the method of Lowry et al. (1951). The concentrations of cytochromes P-450 and b 5 in the liver microsomes were measured by the method of Omura and Sato (1964). The NAD(P)H-cytochrome c reductase activities were determined as reported earlier (Chadha and Madyastha 1982).

Statistics:

none

Preliminary studies:

none

Details on absorption:

none

Details on distribution in tissues:

none

Details on excretion:

α-terpineol is biotransformed into urinary metabolites in the rat.

Metabolites identified:

yes

Details on metabolites:

The neutral fraction (1.1 g) on TLC analysis showed the presence of one major (Rf 0.69) and two minor (Rf 0.63 & 0.35) metabolites. The major metabolite identified was p-methane-1, 2, 8-triol. The neutral fraction (690 mg) from hydrolysed urine on TLC analysis showed a major compound (Rf 0.69) and a few minor metabolites. The minor compounds could not be identified due to paucity of material.

TLC analysis of the esterified acid fraction showed metabolite of a mixture containing oleuropeic acid methyl ester and a compound closely related to it. GLC analysis showed the presence of dihydrooleuropeic acid (IV) and oleuropeic acid (III) methyl esters in the mixture. The presence of these two compounds was confirmed by GC-mass spectral analysis.

The methylated acid fraction from the hydrolysed urine upon TLC analysis showed a major (Rf 0.44) and a few minor metabolites. GLC analysis showed the presence of dihydrooleuropeic acid (IV) and oleuropeic acid (III) methyl esters in the mixture.

Biotransformation of α-terpineol to p-methane 1, 2, 8-triol (II) was reported earlier (Horning et al., 1976). The present study clearly suggests that allylic methyl oxidation and the reduction of the 1,2-double bond are the major routes for the metabolism of α-terpineol in rat. Although allylic oxidation of C-1 methyl seems to be the major pathway, the alcohol p-menth-1-ene-7,8-diol (V) could not be isolated from the urine of rats. Probably this compound does not get accumulated and is readily oxidized further to oleuropeic acid (III). The present studies have clearly demonstrated that the endocyclic double bond in oleuropeic acid (III) readily gets reduced to yield dihydrooleuropeic acid (IV). It is reasonable to assume that the allylic methyl group (C-7) gets oxidized prior to the reduction of the 1, 2-double bond.

Oral administration of alpha-terpineol to rats increased the levels of liver microsomal cytochrome P-450 by 72, 104, 90, 54 and 52% after 1, 2, 3, 6 and 9 days of treatment, respectively. A moderate increase in the levels of liver microsomal NADPH-cytochrome c reductase was also observed during the first three-days of repeated treatment. The effect on cytochrome b5 and NADH-cytochrome c reductase was not significant. These results suggest that the oxidation is mediated by CYP450.

Conclusions:

Allylic methyl oxidation and reduction of the 1, 2-double bond are the major routes for the metabolism of α-terpineol in rat.

Executive summary:

In a metabolism study, male rats administered with α-terpineol through oral gavage at 600 mg/kg bw, once daily for 20 days and urine samples were collected daily. UV, IR, NMR and visible absorption spectra were recorded. Gas Liquid Chromatography (GLC) analyses and Thin Layer Chromatography (TLC) analyses (silicagel G) were performed.

The present study clearly suggests that allylic methyl oxidation and reduction of the 1, 2 -double bond are the major routes for the metabolism of α-terpineol in rat. Results of analyses showed the presence of major metabolites, p-methane-1, 2, 8-triol and mixture of dihydrooleuropeic acid (IV) and oleuropeic acid (III) methyl esters.

Administration of α-terpineol to rat increases the liver microsomal cytochrome P-450 system to a significant extent.

Reference 8

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1969

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Enzyme induction was studied

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

metabolism

Principles of method if other than guideline:

Rats were pretreated for 3 days with terpineol administered by intraperitoneal injection or by admixture with their food, and enzyme activities and cytochrome P-450 were determined.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Route of administration:

other: oral: feed and intraperitoneal

Vehicle:

not specified

Duration and frequency of treatment / exposure:

3 days

Control animals:

no

Pretreatment with terpineol produced smaller increases of about 25% in the activities of all three enzymes (biphenyl 4-hydroxylase, glucuronyl transferase and 4-nitrobenzoate reductase) and cytochrome P-450.

Terpineol is very lipid-soluble, which is metabolized by reduction and conjugation with glucuronic acid, and it would appear that, with this class of substances, at least a compound induces those enzymes that are involved in its own metabolism.

Conclusions:

Pretreatment with terpineol produced smaller increases of about 25% in the activities of all three enzymes (biphenyl 4-hydroxylase, glucuronyl transferase and 4-nitrobenzoate reductase) and cytochrome P-450.

Executive summary:

Rats were pretreated for 3 days with terpineol administered by intraperitoneal injection or by admixture with their food. Biphenyl 4-hydroxylase, glucuronyl transferase, 4-nitrobenzoate reductase activities and cytochrome P-450 were determined in the liver homogenates.

 

Pretreatment with terpineol produced smaller increases of about 25% in the activities of all three enzymes (biphenyl 4-hydroxylase, glucuronyl transferase and 4-nitrobenzoate reductase) and cytochrome P-450.

Reference 9

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:

Alpha-terpineol is one of the main constituents of multiconstituent substance TERPINEOL MULTICONSTITUENT. Therefore, data on alpha-terpineol can be used for extrapolation to TERPINEOL MULTICONSTITUENT.

Reason / purpose for cross-reference:

read-across source

Preliminary studies:

none

Details on absorption:

none

Details on distribution in tissues:

none

Details on excretion:

alpha-Terpineol is biotransformed into urinary metabolites in the rat.

Metabolites identified:

yes

Details on metabolites:

Oxidation of the allylic methyl group yielded the corresponding carboxylic acid which, to a small extent, was reduced to yield the corresponding saturated carboxylic acid.

Conclusions:

The oxidation of alpha-terpineol is mediated by CYP450.

Executive summary:

Urine metabolites were identified from male rats that were given alpha-terpineol orally at a daily dose of 600 mg/kg bw for 20 days. Additional animals given the same treatment were sacrificed at different times and the effect of the treatment on the activity of hepatic enzymes was studied.

Allylic methyl oxidation of alpha-terpineol is the major route for its biotransformation in rat. Oxidation of the allylic methyl group yielded the corresponding carboxylic acid, which to a small extent, was reduced to yield the corresponding saturated carboxylic acid. On the TLC analysis

the major compound was identified as alpha-terpineol. The major compound in the neutral fraction (690 mg) from hydrolysed urine was also identified as alpha-terpineol.

Oral administration of alpha-terpineol to rats increased the levels of liver microsomal cytochrome P-450. A moderate increase in the levels of liver microsomal NADPH-cytochrome c reductase was also observed during the first three-days of repeated treatment. The effect on cytochrome b5 and NADH-cytochrome c reductase was not significant. These results suggest that the oxidation is mediated by CYP450.

Reference 10

Endpoint:

dermal absorption 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:

Alpha-terpineol is one of the main constituents of multiconstituent substance TERPINEOL MULTICONSTITUENT. Therefore, data on alpha-terpineol can be used for extrapolation to TERPINEOL MULTICONSTITUENT.

Reason / purpose for cross-reference:

read-across source

Signs and symptoms of toxicity:

not specified

Dermal irritation:

yes

Absorption in different matrices:

See other information on results

Total recovery:

See other information on results

Table 1: The Flux, Skin Deposition, and Enhancement Ratio of (+)-Catechin across Skin after in Vitro Permeation for 24 h Duration

	Flux (nmol/cm2/h)	ERFlux a)	Skin deposition (nmol/mg)	ERDeposition b)
Control	0.58 ± 0.18	-	0.48 ± 0.22	-
Alpha -Terpineol	217.50 ± 13.55	375.00	5.08 ± 0.55	10.58

a) The enhancement ratio (ERFlux) was the (+)-catechin flux with terpene treatment/(+)-catechin flux of control group.b) The enhancement ratio (ERDeposition) was the (+)-catechin deposition in skin with terpene treatment/(+)-catechin deposition in skin of control group. All the values of terpenes (flux and skin deposition) were significantly higher than the control (p<0.05). Each value represents the mean±S.D. (n=4).

Table 2: The Flux (nmol/cm2/h) of (+)-Catechin, (-)-Epicatechin, EGCG, and Theophylline across Skin after in Vitro Permeation for 24 h Duration

	(+)-catechin	(-)-Epicatechin	EGCG	Theophylline
Control	0.58 ± 0.18	0.62 ± 0.12	0	11.38 ± 5.68
Alpha -Terpineol	217.50 ± 13.55	214.19 ± 31.05	33.11 ± 6.53	233.673 ± 30.56

All the flux values of terpenes were significantly higher than the control (p<0.05). Each value represents the mean±S.D. (n=4).

Table 3: Skin/Vehicle Partition Coefficient of (+)-Catechin and Theophylline by Treating Skin in 3% Terpenes

	(+)-catechin	Theophylline
Control	2.51 ± 0.36	0.23 ± 0.18
Alpha -Terpineol	9.43 ± 3.30	0.84 ± 0.09

All the partition coefficient values of terpenes were significantly higher than the control (p<0.05). Each value represents the mean±S.D. (n=4)

Table 4: In Vivo TEWL and Erythema of Rat Skin after Treatment of 3% Terpenes for 24 h

	ΔTEWL (g/m2/h)	Δa* (arbitrary unit)
Control	2.66 ± 0.65	-1.06 ± 0.73
Alpha -Terpineol	4.82 ± 1.21a)	1.95 ± 0.22a)

a) The value significantly higher than the control (p<0.05). Each value represents the mean ± S.D. (n=6)

Table 5: Skin Deposition (nmol/mg) of (+)-Catechin, (-)-Epicatechin, EGCG, and Theophylline after in Vivo Application for 6 h Duration

	Control	Alpha-terpineol
(+)-catechin	0.11 ± 0.03	0.46 ± 0.16
(-)-Epicatechin	0.26 ± 0.09	0.59 ± 0.18
EGCG	0.003 ± 0.001	0.55 ± 0.14
Theophylline	0.11 ± 0.08	0.37 ± 0.13

All the partition coefficient values of alpha-terpineol were significantly higher than the control (p<0.05). Each value represents the mean±S.D. (n=6).

Conclusions:

alpha-Terpineol was found to be the best enhancer for catechins and theophylline. The high enhancement by alpha terpineol was due to macroscopic perturbation of the stratum corneum (SC) and the biological reaction in viable skin as evaluated by transepidermal water loss (TEWL) and colorimetry.

Executive summary:

Using in vitro and in vivo techniques, terpenes were evaluated as enhancers to improve the skin permeation of therapeutically active agents derived from tea, including tea catechins and theophylline. The in vitro permeation was determined by Franz cells. The skin deposition and subcutaneous amounts of drugs sampled in vivo were evaluated by microdialysis. In vivo, terpenes promoted the skin uptake but not the subsequent subcutaneous concentration of (-)-epigallocatechin gallate (EGCG). Both increased skin/vehicle partitioning and lipid bilayer disruption of the stratum corneum (SC) contributed to the enhancing mechanisms of terpenes for topically applied tea catechins and theophylline based on the experimental results from the partition coefficient and transepidermal water loss (TEWL). alpha-Terpineol was found to be the best enhancer for catechins and theophylline. The high enhancement by alpha terpineol was due to macroscopic perturbation of the SC and the biological reaction in viable skin, as evaluated by TEWL and colorimetry.

Description of key information

Metabolite profiles following hepatocyte incubations of [14C]terpineol showed up to 20 individual regions of radioactivity (assigned T1 to T20).

The profiles for the rabbit and human tended to be similar with the main metabolite being the glucuronide conjugates of hydroxylated terpineol (up to 43% of sample radioactivity). Fewer metabolites were observed in the rat profiles with the main metabolite being the glucuronide conjugate of terpineol (T18 up to 45% of the sample radioactivity).
After single oral doses of 14C-alpha-terpineol (75, 250 and 750 mg/kg), more than 90 % of the dose was excreted within 48 h. Excretion was mainly via the urine where 4 major metabolites were identified as glucuronide conjugates of alpha-terpineol or glucuronide conjugates of hydroxy alpha-terpineol. Faecal excretion accounted for 26-33 % dose. Overall, the AUCall values at the highest dose level (750 mg/kg bw) were ca 2.3-fold higher than those values predicted from a linear relationship. The average terminal half-life for all groups in plasma was 6.6 h. Concentrations of radioactivity in tissues were highest in the kidney and liver at each dose level. Overall tissue accumulation after single oral doses was low (<0.1% dose). Several studies also suggest that terpineol is conjugated by UDP-glucuronosyltransferases in several species and excreted in urine.

A toxicokinetic study was conducted to evaluate the toxicokinetics of test item Terpineol multiconstituent, and its metabolite alpha-terpineol glucuronide, following daily oral administration (gavage) to male Sprague-Dawley rats for 1, 7 and 14 days. The toxicokinetics of Terpineol multiconstituent after oral administration on Day 1, 7(750 mg/kg group only) and 14, to 12 male Sprague-Dawley rats (n=3 per dose group) were characterised using mean plasma concentration vs. time data. The animals received either 100, 250, 600 or 750 mg/kg bw/day of Terpineol multiconstituent by daily gavage.

Based on the data obtained, the following conclusions can be made:

- Following oral administration of Terpineol multiconstituent to male Sprague-Dawley rats at 100, 250, 600 and 750 mg/kg bw/day up to 14 days, alpha-terpineol concentrations were below the limit of quantification (BLQ < 1.00 µg/mL) for all animals at all sampling times.

- The male rats were exposed to alpha-terpineol since all animals have detectable alpha-terpineol glucuronide amounts in plasma at all times (except at day 1 predose) for all doses.

- Rapid oral absorption and metabolism rate were noted with a metabolite T_maxobserved at 0.5 h for all test item doses with the exception of the 100 mg/kg bw/day group at day 1 (T_maxat 1 h). The mean terminal half-life values of the metabolite were highly variable ranging from 1.70 to 20.0 h.

- Mean concentration versus time profiles of the alpha-terpineol glucuronide suggest either an enterohepatic recycling and/or a saturable absorption.

- Both on Days 1 and 14, exposure to terpineol glucuronide (AUC_0-24h) increased almost dose-proportionally from 100 mg/kg to 600 mg/kg, whilst at 750 mg/kg a more than dose-proportional increase can be observed.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Absorption:

The absorption, distribution, metabolism and excretion of [isopropyl methyl-¹⁴C]-alpha-terpineol were studied after single oral doses at 75, 250 and 750 mg/kg bw to male rats. Following a single oral dose, alpha-terpineol was well absorbed with 60% excreted in urine and unchanged alpha-terpineol accounted for a maximum of 18.7% dose in faeces. (Knight, 2013)

In an inhalation experiment on mice with alpha-terpineol (exposure for 1 h to 50-108 mg/m3), serum concentrations of alpha-terpineol of 6.9 ± 1 ng/mL were measured (Jirovetz et al., 1992; cited in Bhatia et al., 2008b), showing absorption by this route of exposure.

The skin absorption of terpineol was studied in 5 male mice over a 2 h period. A 2.2 cm² shaved area on the abdominal skin was used. Eserine (0.23%) was used as an indicator and the test material was used as a carrier for the Eserine. The latency period between application to the skin and the appearance of the eserine effect in the periodically stimulated masticatory muscles was used as a measure of the absorption rate. Absorption of terpineol was very rapid (33 min). (Meyer and Meyer, 1959, cited in Bathia et al., 2008a)

Terpineol multiconstituent is well known as an enhancer of dermal absorption of various chemicals such as rhodamine B (Meyer, 1965, cited in Bhatia et al., 2008a) and alpha-terpineol was also found to enhance dermal absorption of several catechins from tea and theophylline (Fang et al., 2007). The high enhancement by alpha-terpineol is thought to be due to macroscopic perturbation of the stratum corneum and the biological (inflammatory) reaction in viable skin.

Distribution:

The absorption, distribution, metabolism and excretion of [isopropyl methyl-¹⁴C]-alpha-terpineol were studied after single oral doses at 75, 250 and 750 mg/kg bw to male rats. The whole blood to plasma ratios, calculated using AUC_all, were 0.68 at the lowest dose level and 0.72 at the 250 and 750 mg/kg bw dose levels. These results suggested that there was relatively little distribution of radioactivity into red blood cells. Concentrations of radioactivity in tissues were highest in the kidney and liver at each dose level. Overall tissue accumulation after single oral doses was low with only a small proportion of the dose retained in tissues at 168 h (<0.1 % dose). (Knight, 2013)

Metabolism and excretion:

In a key in vitro study, the metabolism of [¹⁴C] terpineol was studied following incubation with rat, rabbit and human cryopreserved hepatocytes at concentrations of 1, 10 and 100 µM over incubation times of 0, 1 and 4 h. Metabolite profiles following hepatocyte incubations of [14C]terpineol showed up to 20 individual regions of radioactivity (assigned T1 to T20). The profiles for the rabbit and human tended to be similar with the main metabolite being the glucuronide conjugates of hydroxylated terpineol (up to 43% of sample radioactivity). Fewer metabolites were observed in the rat profiles with the main metabolite being the glucuronide conjugate of terpineol (T18 up to 45% of the sample radioactivity) (Harrison, 2014).

The absorption, distribution, metabolism and excretion of [isopropyl methyl-¹⁴C]-alpha-terpineol were studied after single oral doses at 75, 250 and 750 mg/kg bw to male rats. Excretion was mainly via the urine where 4 major metabolites were identified as glucuronide conjugates of alpha-terpineol or glucuronide conjugates of hydroxy alpha-terpineol. Faecal excretion accounted for 26-33 % dose. Overall, the AUCall values at the highest dose level (750 mg/kg bw) were ca 2.3-fold higher than those values predicted from a linear relationship. The average terminal half-life for all groups in plasma was 6.6 h. Unchanged alpha-terpineol represented a maximum of 18.7 % dose in faeces and 0.1 % dose in urine. Four major metabolites in urine (3.4 - 20.7 % dose) were identified by mass spectrometry as glucuronide conjugates of alpha-terpineol or glucuronide conjugates of hydroxy alpha-terpineol. (Knight, 2013)

Several studies also suggest that terpineol is conjugated by UDP-glucuromosyltransferases in several species and excreted in urine.

In male albino IISc strain rats given alpha-terpineol orally at a daily dose of 600 mg/kg bw for 20 days, oxidation of the allylic methyl group was the major metabolic pathway and was observed to yield the corresponding carboxylic acid which was hydrogenated to a small extent to yield the corresponding saturated carboxylic acid. On the TLC analysis the major compound was identified as alpha-terpineol. The major compound in the neutral fraction from hydrolysed urine was also identified as alpha-terpineol. alpha-Terpineol also increased the liver microsomal P450 content and the activity of NADPH-cytochrome c reductase suggesting that the oxidation is mediated by CYP450 (Madyastha et al., 1988).

Following oral administration of Terpineol multiconstituent to male Sprague-Dawley rats at 100, 250, 600 and 750 mg/kg bw/day up to 14 days, alpha-terpineol concentrations were below the limit of quantification (BLQ < 1.00 µg/mL) for all animals at all sampling times. The male rats were exposed to alpha-terpineol since all animals have detectable alpha-terpineol glucuronide amounts in plasma at all times (except at day 1 predose) for all doses. Rapid oral absorption and metabolism rate were noted with a metabolite T_maxobserved at 0.5 h for all test item doses with the exception of the 100 mg/kg bw/day group at day 1 (T_maxat 1 h). The mean terminal half-life values of the metabolite were highly variable ranging from 1.70 to 20.0 h. Mean concentration versus time profiles of the alpha-terpineol glucuronide suggest either an enterohepatic recycling and/or a saturable absorption. Both on Days 1 and 14, exposure to terpineol glucuronide (AUC_0-24h) increased almost dose-proportionally from 100 mg/kg to 600 mg/kg, whilst at 750 mg/kg a more than dose-proportional increase can be observed.

In an in vitro study using human cell lines, the glucuronidation of various chemicals by embryonic kidney 293 cells expressing UDP-glucuronosyltransferase 1.4 protein was investigated. The rate of glucuronidation of 0.5 mM alpha-terpineol by this human embryonic model was 20 pmol/min/mg protein after 0.5 to 2-h incubation (Green and Tephly, 1996; cited in Bhatia et al., 2008b).

In an in vivo metabolism study various terpenes including alpha-terpineol were fed to sheep and their products were analyzed. When 10 g of alpha-terpineol was fed to groups of 2-6 sheep, presence of a conjugated glucuronide was observed in the urine (Wright, 1945; cited in Bhatia et al., 2008b).

Urine samples from male Sprague-Dawley rats administered a single intraperitoneal injection of alpha-terpineol or pine oil at 100 mg were collected at 0–24 and 24–48 h. The metabolites were observed to be p-menthan-1,2,8-triol and the glucuronide conjugate of alpha-terpineol. Neutral metabolites were also isolated from the rat urine (Hill et al., 1975; cited in Bhatia et al., 2008b).

References:

Reviews not included in 7.1.1 Basic toxicokinetics endpoint

Bhatia et al., 2008a, Fragrance material review on terpineol, Food and Chemical Toxicology 46:275-279

Bhatia et al., 2008b, Fragrance material review on alpha terpineol, Food and Chemical Toxicology 46:280-285