(1S,5S)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

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:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

other: elimination kinetics

Principles of method if other than guideline:

Metabolism and elimination kinetics of alpha-pinene in 4 volunteers

GLP compliance:

no

Specific details on test material used for the study:

(1S,5S)-(−)-α-pinene (enantiomeric reference for αPN; ≥99.0 %),

Species:

other: human

Sex:

male/female

Details on test animals or test system and environmental conditions:

Four healthy human volunteers (3 men and 1 woman), mean age 33 ± 11 years, mean body weight (80 ± 8 kg)

Route of administration:

oral: capsule

Details on exposure:

Volunteers were orally exposed to 9.0 ± 0.4 mg (66 ± 3 μmol,M = 136.23 mg mmol−1) αPN via spiked gelatin capsules.
The volunteers ingested the capsules in the morning on an empty stomach After the exposure, they fasted for 1 h. During the remaining time of the experiment, they were allowed to eat and drink normally.

Dose / conc.:

9 other: mg

Remarks:

9.0 ± 0.4 mg (66 ± 3 μmol,M = 136.23 mg mmol−1)

No. of animals per sex per dose / concentration:

4 healthy human volunteers (3 men and 1 woman)

Control animals:

no

Positive control reference chemical:

no

Details on study design:

The volunteers ingested the capsules in the morning on an empty stomach

Details on dosing and sampling:

The volunteers ingested the capsules in the morning directly after the collection of one preexposure urine sample (n = 4 volunteers) and one blood sample (n = 2 volunteers).

- Tissues and body fluids sampled: Urine and blood
- Time and frequency of sampling: Urine samples were collected by every volunteer throughout 24 h as often as possible. For every urine sample, the sampling time and the excretion volume were recorded. Blood samples were drawn from two volunteers every hour until 5 h after exposure.
- Storage: Urine samples were stored frozen under nitrogen protective gas at −20 °C until analysis. Blood samples: after aliquotation of 2 mL for headspace analysis, the remaining volumes were stored frozen at −20 °C until analysis.
- Method type(s) for identification: Gas chromatographic–mass spectrometric analyses (HS–GC–MS and GC-PCI-MS/MS); alpha pinene in blood was analysed by HS-GC-MS. alpha pinene metabolites in blood and urine was analysed by GS-PCI-MS/MS.
- Urine samples were analysed according to the procedure of Schmidt et al. (2015).

Statistics:

none

Details on absorption:

Maximum blood concentrations (cmax) were reached in the samples drawn 1 and 3 h after exposure (tmax), respectively.

Details on distribution in tissues:

Noticeable amounts of αPN or related volatile αPN metabolites obviously seem to reach the lungs since all volunteers reported a characteristic flavour of the exhaled air.

Details on excretion:

Renal excretion of cVER in the pre-exposure samples was 22 ± 11 μg L−1. After oral administration renal elimination rates of cVER increased distinctly within the first 1–3 h after exposure to its maximum of 1600 ± 650 μg L−1 and declined afterwards to the base levels within the 24-h observation period. At its maximum urinary concentrations, cVER was present in conjugated forms by 28–76 %. Renal excretion kinetics of cVER showed an elimination half-life of about 1.5 ± 0.2 h. The cumulative excretion within 24 h after exposure correlated with about 5.6 ± 0.5 % of the applied dose.
Renal excretion of tVER in the pre-exposure samples was 14 ± 7.5 μg L−1 . After oral administration renal elimination rates of tVER increased distinctly within the first 1–3 h after exposure to its maximum of 1900 ± 1700 μg L−1and declined afterwards to the base levels within the 24-h observation period . At its maximum urinary concentrations, tVER was present in conjugated forms by 4–68 %. Renal excretion kinetics of tVER showed an elimination half-life of about 1.6 ± 0.2 h. The cumulative excretion within 24 h after exposure correlated with about 4.1 ± 0.4 % of the applied dose
Renal excretion of MYR in the pre-exposure samples was 7.3 ± 1.4 μg L−1. After oral administration renal elimination rates of MYR, increased distinctly within the first 1–3 h after exposure to its maximum of 690 ± 530 μg L−1 and declined afterwards to the base levels within the 24-h observation period . At its maximum urinary concentrations, MYR was present in conjugated forms by 99–100 %. Renal excretion kinetics of MYR showed an elimination half-life of about 1.6 ± 0.2 h. The cumulative excretion within 24 h after exposure correlated with about 1.5 ± 0.2 % of the applied dose
Renal excretion of MYRA in the pre-exposure samples was 16 ± 6.3 μg L−1. After oral administration renal elimination rates of MYRA increased distinctly within the first 1–3 h after exposure to its maximum of 3200 ± 2700 μg L−1and declined afterwards to the base levels within the 24-h observation period. At its maximum urinary concentrations, MYRA was present in conjugated forms by 99–100 %. Renal excretion kinetics of MYRA showed an elimination half-life of about 1.4 ± 0.1 h. The cumulative excretion within 24 h after exposure correlated with about 6.7 ± 0.3 % of the applied dose.

Elimination of the 3 ununknown αPN metabolites (αPN-M1, αPN-M2, and αPN-M3) . accounted for about 1 % (αPN-M1), 10 % (αPN-M2), and 2 % (αPN-M3) of the orally applied dose.

The cumulative excretion graphs suggest completeness ofelimination about 10 h after exposure.

Toxicokinetic parameters:

half-life 1st: 1.5 ± 0.2 h

Remarks:

mean renal elimination half-live of cVER

Toxicokinetic parameters:

half-life 1st: 1.6 ± 0.2 h

Remarks:

mean renal elimination half-live of tVER

Toxicokinetic parameters:

half-life 1st: 1.6 ± 0.2 h

Remarks:

mean renal elimination half-live of MYR

Toxicokinetic parameters:

half-life 1st: 1.4 ± 0.1 h

Remarks:

mean renal elimination half-live of MYRA

Metabolites identified:

yes

Details on metabolites:

In blood: detection of cVER (cis- verbenol) , tVER (trans-verbenol) , and MYR (myrtenol) over the entire blood sampling period of 1–5 h.

In urine: detction of: cVER, tVER, MYRA (myrtenic acid), and MYR and three peaks of unknown αPN metabolites (αPN-M1, αPN-M2, and αPN-M3) . cVER account for 5.6 ± 0.5 % of the applied oral αPN dose, tVER for 4.1 ± 0.4 %, MYR for 1.5 ± 0.2 % and MYRA for 6.7 ± 0.3 %. It was estimated that the newly identified metabolites accounted for about 1 % (αPN-M1), 10 % (αPN-M2), and 2 % (αPN-M3) of the orally applied dose. The quantified metabolites in urine only accounted for about 22 % of the orally applied dose

It was supposed that αPN-M1 is MYRA-4-OH and αPN-M2 is identical with MYRA. αPN-M3 may be an isomer of saturated MYRA, i.e. DHMYRA.

None of the volunteers reported any adverse health effects due to the supplementation with 10 mg of αPN. Though, all volunteers mentioned a characteristic aromatic smell of the exhaled breath which occurred about 1 h after oral exposure and subsequently vanished within 2–3 h after exposure. It was assume that respiratory elimination of unmetabolized αPN was a substantial elimination pathway, as well, which may have contributed to a large extend to the 78 % of the oral dose which was not recovered in form of renal metabolites. This is in accordance with the volunteers’ reports on olfactory perception during the exposure experiments. However, the amount of αPN exhaled was not quantified.

The synchronous and steep metabolite time courses observed in blood and urine may be explained by simultaneous and competing reactions during first-pass metabolism. This first-pass effect, which rapidly yields in polar phase I and II metabolites, is presumably responsible for the low blood concentrations as well as the short elimination half-lives in the first elimination phases. The second, slower elimination phases observed for all metabolites are most probably caused by release of low amounts of αPN or its metabolites from tissue compartments. However, the cumulative elimination indicate that the second phase only plays a minor role to the cumulatively eliminated amount. At the elimination peak, MYR and MYRA are almost entirely conjugated to glucuronic acid (or sulphate), whereas cVER and tVER are only conjugated up to 76 %. This effect may be due to steric hindrance of the secondary hydroxyl groups caused by the bicyclic backbone or due to instability of the conjugates.

Table1: Characteristics of the blood kinetics ofαPN metabolites after oral exposure to 9.0±0.4 mg (66±2.8 µmol)αPN (mean val- ues±range;n=2 volunteers)

Metabolite	cmax(µgL^−1)	cmax(nM)	tmax(h)	t1/2(h)
cVER	1.4±0.7	9.3±4.8	1–3	0.8a
tVER	4.0±2.1	26±14	1–3	1.0a
MYR	1.7±0.4	11±2.4	1–3	1.7a

a  Elimination not completed within 5 h observation period

Table 2:  Characteristics of the renal αPN metabolite elimination kinetics after oral exposure to 9.0±0.4 mg (66±2.8 µmol)αPN (mean values±SD;n=4 volunteers)

Metabolites	RE,max(µg h−1)	tmax(h)	t1/2(h)	kel(h−1)	AUC0    tf(µmol)	Share of oral dose(%)
cVER	170±97	1.6±0.9	1.5±0.2	0.461	3.7±0.3	5.6± 0.5
T VER	120±64	1.6±0.9	1.6±0.2	0.436	2.7±0.3	4.1±0.4
MYR	48±30	1.6±0.9	1.6±0.2	0.440	1.0±0.1	1.5±0.2
MYRA	230±130	1.6±0.9	1.4±0.1	0.483	4.4±0.1	6.7±0.3

R_E,maxmaximum renal excretion;t_maxtime to reach maximum renal excretion;t_1/2elimination half-life;AUC_0→tfarea under the renal excretion vs. time curve (from time 0 to final sampling timetf);V_totalsummarized excreted urine volume

Conclusions:

The present study demonstrates in details the human αPN metabolism after oral uptake and the elimination kinetics of four relevant metabolites (cVER, tVER, MYRA, and MYR ). Two unknown human metabolites, whose predicted structures [4-hydroxymyrtenic acid (MYRA-4-OH) and dihydromyrtenic acid (DHMYRA)] were identified. It is demonstrated that human metabolism of αPN proceeds fast and the body is almost entirely cleared from the metabolites 10 h after exposure.

Executive summary:

In a metabolism study, four healthy human volunteers were orally exposed to a single dose of 9 mg of (1S,5S)-(−)-α- pinene (αPN) via spiked gelatin capsules. Each volunteer gave one urine sample before administration and subsequently collected each urine sample within 24 h after administration. Blood samples were collected directly after administration of the capsule and every hour until 5 hours exposure for 2 volunteers. The concentration of the αPN metabolites were determined using a very specific and sensitive GC-PCI-MS/MS procedure. Concentration of αPN was analysed in blood by HS-GC-MS procedure.

 

αPN metabolites cVER (cis- verbenol) , tVER (trans-verbenol) , and MYR (myrtenol) were detected in blood samples over the entire blood sampling period of 1–5 h, unmetabolised αPN were below the limit of detection.

The metabolite concentrations showed synchronous time courses even though the levels were low and varied between the two volunteers. Metabolite blood levels were low. The non-detection of αPN in blood after low oral doses in contrast to the detectable metabolite levels indicates a fast and approximately entire pre-systemic metabolism such as hepatic or intestinal firstpass metabolism.

 

αPN metabolites were detected in urine in considerably higher amounts in contrast to blood levels. The low blood concentrations compared to the high urinary levels, thus, indicate a fast transfer from blood to urine and a rapid renal elimination.

 

In addition to the known and established αPN metabolites cVER and tVER, the relevance of MYR and MYRA as products of the human in vivo metabolism of αPN was confirmed. Two unknown human metabolites were identified and these structures could be predicted as 4-hydroxymyrtenic acid (MYRA-4-OH) and dihydromyrtenic acid (DHMYRA).

 

Human in vivo metabolism of αPN is similarly dominated by extensive oxidation reactions on the methyl side-chains yielding in carboxylic acid structures. Nonetheless, only 22% of the applied dose was quantified as metabolite. Thus, further metabolites and the share of αPN eliminated unchanged via lungs remain unclear.

 

Human metabolism of αPN proceeds fast and the body is almost entirely cleared from the metabolites 10 h after exposure

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well described study giving reliable information on several terpenes in vivo metabolism in rabbit.

Reason / purpose for cross-reference:

read-across: supporting information

Objective of study:

metabolism

Principles of method if other than guideline:

Albino rabbits were orally administered test item and urine was collected for 3 days for identification of urinary metabolites.

GLP compliance:

no

Radiolabelling:

no

Species:

rabbit

Strain:

other: albino (Japanese White)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Miyamoto Jikken Dobutsu, Hiroshima, Japan
- Weight at study initiation: 2-3 kg
- Fasting period before study: for 2 days before experiment
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): Oriental RC-4, ad libitum
- Water (e.g. ad libitum): ad libitum

Route of administration:

oral: gavage

Vehicle:

other: 100 mL water containing 0.1 g Tween 80

Details on exposure:

Rabbits were administered 20 mL solution through stomach tube followed by 20 mL water, corresponding to 400-700 mg/kg bw.

Duration and frequency of treatment / exposure:

Once

Remarks:

Doses / Concentrations:
400-700 mg/kg bw

No. of animals per sex per dose / concentration:

6

Control animals:

no

Positive control reference chemical:

None

Details on study design:

None

Details on dosing and sampling:

The urine was collected daily for 3 days after drug administration and stored at 0-5°C until time of analysis.

Extraction of urinary metabolites:
The urine was adjusted to pH 4.7 with acetate buffer and incubated with beta-glucuronidase-arylsulfatase (3 mL/1000 mL of the fresh urine) at 37°C for 48 h, followed by continuous ether extraction for 48 h. The ether extracts were washed with 5% NaHCO3 and 5% NaOH to remove the acidic and phenolic fractions, respectively, and dried (magnesium sulfate). Ether was evaporated under reduced pressure to give neutral metabolites. The neutral metabolites were chromatographed on a column containing 100 g of silicic acid (200 mesh). Elution was started with n-hexane, and n-hexane-ethyl acetate mixtures (95:5, 90:10, 85:15, 70:30, and 50:50) were used as subsequent eluents. The acidic metabolites were recovered from the sodium bicarbonate layer by acidification with 5% HCl, followed by ether extraction. The ether extracts were esterified with diazomethane in ether or with dimethyl sulfate in the presence of potassium carbonate in anhydrous acetone. These esters of the acidic metabolites also were chromatographed in the same manner as the neutral metabolites.

Identification of urinary metabolites:
Purification by silicic acid gave pure metabolites. When necessary, metabolites were isolated by preparative TLC or GLC. Structure determination or identification was based on spectral data and chemical transformations.

Statistics:

None

Preliminary studies:

None

Details on absorption:

None

Details on distribution in tissues:

None

Details on excretion:

None

Metabolites identified:

yes

Details on metabolites:

The main urinary metabolite from (+)-, (-)-, and (+/-)-alpha-pinenes was (-)-trans-verbenol.

None

Conclusions:

The main urinary metabolite from (+)-, (-)-, and (+/-)-alpha-pinenes was (-)-trans-verbenol.

Executive summary:

The biotransformation of (+)-, (-)-, and (+/-)-alpha-pinene was studied in albino rabbits orally administered 400-700 mg/kg bw of the respective alpha-pinenes in water with 0.1% Tween 80. Urine was collected daily for 3 days and urinary metabolites were identified. In this study, the main urinary metabolite from (+)-, (-)-, and (+/-)-alpha-pinenes was (-)-trans-verbenol.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well described and well conducted in vivo study performed in humans, giving valuable data about absorption and excretion of several terpenes after inhalation exposure.

Reason / purpose for cross-reference:

read-across: supporting information

Objective of study:

absorption

excretion

Principles of method if other than guideline:

Absorption and excretion rates of alpha-pinene, beta-pinene and 3-carene after inhalation exposure to turpentine vapour in human volunteers.

GLP compliance:

no

Specific details on test material used for the study:

Name of test material (as cited in study report): turpentine
Source: Swedish turpentine, Alcro Beckers

Radiolabelling:

no

Species:

human

Sex:

male

Details on test animals or test system and environmental conditions:

Eight healthy men, not occupationally exposed to solvents, with a mean (range) age of 28 (18-37) years and weight of 75 (62-83) kg participated in the study. All were nonsmokers and they were instructed to refrain from alcohol and activities that could cause exposure to organic solvents during the preceding two days and throughout the experiment.

Route of administration:

inhalation: vapour

Vehicle:

unchanged (no vehicle)

Details on exposure:

The subjects were exposed to turpentine for two hours (450 mg/m3 (75 ppm)) during light physical exercise (50 W on a bicycle ergometer). The
exposure was conducted in an exposure chamber. The volume of the chamber was 12 m3. The air was changed 10 times/h. To prevent the solvent from leaking from the chamber into the surroundings the inlet flow was 115 m3/h and the outlet flow was 135 m3/h. The solvent was injected into the inflowing air stream by means of a high-performance liquid chromatography pump (Gilson 302). The mean temperature in the chamber was 21°C and varied by less than 2.5°C during a two hour exposure to turpentine and between different days.

Duration and frequency of treatment / exposure:

2 h, once

Remarks:

Doses / Concentrations:
450 mg/m3 (75 ppm)

No. of animals per sex per dose / concentration:

8

Control animals:

no

Positive control reference chemical:

None

Details on study design:

A low concentration of 3-carene, 10 mg/m3, was used as a control. The control concentration was chosen as low as possible so that no effect was to be expected, but the subjects could still smell the solvent.
The workload 50 W was chosen to imitate the physical activity generally found in light industries.

Details on dosing and sampling:

The solvent concentration in the air of the chamber was continuously monitored at atmospheric pressure by an infrared spectrophotometer (Miran 80) equipped with a 20 m thermostatically controlled (45°C) gas cell. Measurements of monoterpenes in the air of the chamber were made at 3.39 µm with 3.6 µm as the reference wavelength. The concentration of monoterpenes varied by less than 2% during the two hour exposure of turpentine and less than 3% between different days.

Air sampling: at four regular time intervals during the exposure exhaled air was individually collected in polyester laminated aluminium foil bags (about 20 L) for about four minutes each. Exhaled air samples were also collected after exposure (5, 15, 25, 85, 150, and 230 minutes after the end of exposure). The volume of the expired air was measured with a balanced spirometer and the concentrations of monoterpenes were analysed on a gas chromatograph (Carlo Erba, Fractovap 2350) equipped with a flame ionisation detector and a polar column (Supelcowax 10, 30 m, 0-75 mm inner diameter).

Blood sampling: arterial capillary blood (200 µL) was collected from a prewarmed fingertip at selected intervals during the exposure and up to 21 h after end of exposure, for measurement of monoterpenes in blood. Blood sampling during exposure was taken by the subject holding out one hand through a closable hole in the wall. Blood samples were collected in heparinised capillary tubes (2 x 100 µL) and transferred to gas tight head space vials (22-4 mL) which were capped immediately with Teflon lined membranes. The samples were kept for 20 minutes at 37°C before analysis.
The headspace of the blood samples was analysed on a gas chromatograph (Perkin Elmer 8700) equipped with a head space autosampler (Perkin Elmer HS-101), a polar column (Supelcowax 10, 30 m, 0-75 mm inner diameter), and a flame ionisation detector. The concentration of monoterpenes in the blood was measured by comparison with individual standard curves prepared in the same concentration range by adding monoterpenes to blood samples taken before the exposure.

Statistics:

None

Preliminary studies:

None

Details on absorption:

The relative net uptakes of the total inhaled alpha-pinene, beta-pinene, and 3-carene averaged 62%, 66%, and 68% respectively (% of total inhaled).

Details on distribution in tissues:

None

Details on excretion:

Between 2% and 8% of the net uptake was excreted unchanged in the expired air after the end of exposure. The mean blood clearance 21 h after exposure (CL21h) of alpha-pinene, beta-pinene and 3-carene, were 0.8, 0.5, and 0.4 L/kg/h, respectively. The mean half lives (t1/2) of the last phase of alpha-pinene, beta-pinene, and 3-carene averaged 32, 25, and 42 h, respectively.

Metabolites identified:

not measured

Details on metabolites:

None

The mean total concentration of the monoterpenes in the air of the chamber from all the turpentine exposures was 449 mg/m3, with a relative SD of 1-4%, based on air concentrations from four different occasions at each exposure.

If the assumption is made that the turpentine consists of alpha-pinene, beta-pinene, and 3-carene, the proportions of the vapour were about 54%, 11%, and 35%, respectively.

Table 1: Mean (SD) experimental results ofsome physiological and toxicokinetic vaiables from two hour inhalation exposure to 450 mg/m3 of turpentine during physical exercise at a workload of 50 W (also, results from previous single exposure to alpha-pinene and 3-carene are presented)

	Alpha-pinene			Beta-pinene   Exposure to turpentine	3-carene
	Exposure to turpentine	Exposure to alpha-pinene	Exposure to alpha-pinene		Exposure to turpentine	Exposure to 3-carene	Exposure to 3-carene
Air concentration (mg/m3)	242 (3.3)	455 (5.3)	225 (5.1)	49 (0.67)	157 (2.1)	451 (5.8)	228 (5.8)
Net uptake (% of total inhaled)	62 (5.3)	58 (5.3)	60 (3.9)	66 (5.8)	68 (7.5)	71 (4.7)	70 (4.4)
Respiratory elimination after exposure (% of net uptake)	3.8 (1.1)	7.7 (3.1)	5.7 (5.7)	5.0 (5.8)	2.4 (1.5) *	4.8 (1.2)	1.9 (1.5)
Concentration at steady state (30 min before end of exposure) (µmol/L)	9.0 (0.75)	19 (3.6)	9.6 (1.9)	2.6 (0.29)	9.9 (0.89)	25 (0.92)	12 (1.4)

* P<0.05, Student's t test v. pure 3-carene (450 mg/m3)

Table 2: mean (SD) apparent CLs and t1/2s of alpha-pinene, beta-pinene, and 3-carene from two hour inhalation exposure to 450 mg/m3 of alpha-pinene, 3-carene or turpentine during physical exercise at a workload of 50 W

	Alpha-pinene		Beta-pinene   Exposure to turpentine	3-carene
	Exposure to turpentine	Exposure to alpha-pinene		Exposure to turpentine	Exposure to 3-carene
CL4h (L/kg/h)	1.6 (0.3)	1.4 (0.31)	0.8 (0.3)	1.0 (0.2)	0.3 (0.1)
CL21h (L/kg/h)	0.8 (0.09) §	1.1 (0.2)	0.5 (0.3) §	0.4 (0.1) §	0.9 (0.3)
t1/2 terminal (h)	32(18)	12 (4)	25 (18)	42 (23)	30 (22)

§ four subjects due to analytical problems resulting in problems calculating AUC

Conclusions:

The mean relative uptakes of alpha-pinene, beta-pinene, and 3-carene were 62%, 66%, and 68% respectively, of the amount inhaled. Between 2% and 5 % of the net uptake was excreted unchanged in the expired air after the end of exposure. The mean blood clearance 21 h after exposure (CL21h) of alpha-pinene, beta-pinene and 3-carene, were 0.8, 0.5, and 0.4 L/kg/h, respectively. The mean half lives (t1/2) of the last phase of alpha-pinene, beta-pinene, and 3-carene averaged 32, 25, and 42 h, respectively.

Executive summary:

In this study, eight male volunteers were exposed to 450 mg/m3 turpentine by inhalation (2 h with physical exercise workload of 50 W) in an exposure chamber and the extent of alpha-pinene, beta-pinene and 3-carene inhaled as well as their excretion rates from blood were investigated.

The mean relative uptakes of alpha-pinene, beta-pinene, and 3-carene were 62%, 66%, and 68% respectively, of the amount inhaled. Between 2% and 5 % of the net uptake was excreted unchanged in the expired air after the end of exposure.

The mean blood clearance 21 h after exposure (CL21h) of alpha-pinene, beta-pinene and 3-carene, were 0.8, 0.5, and 0.4 L/kg/h, respectively. The mean half lives (t1/2) of the last phase of alpha-pinene, beta-pinene, and 3-carene averaged 32, 25, and 42 h, respectively.

The last phase of the t1/2s tended to be longer after exposure to turpentine than monoterpenes. The total blood clearance CL21h of 3-carene found in this study was lower, and CL4h of 3-carene was significantly lower than the values obtained from similar exposure to pure 3-carene.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Results from publication with well described details

Reason / purpose for cross-reference:

read-across: supporting information

Objective of study:

toxicokinetics

Principles of method if other than guideline:

The blood and urine monoterpene concentrations were continuously monitored from a patient attempting suicide by ingestion of 400-500 mL pine oil.

GLP compliance:

no

Specific details on test material used for the study:

Name of test material (as cited in study report): pine oil
Composition of test material, percentage of components: 57% alpha-pinene, 8% beta-pinene, 26% carene, 6% limonene and 3% other hydrocarbons

Radiolabelling:

no

Species:

human

Sex:

male

Details on test animals or test system and environmental conditions:

A 49 year old male (height 185 cm, body weight 85 kg)

Route of administration:

oral: unspecified

Vehicle:

unchanged (no vehicle)

Duration and frequency of treatment / exposure:

Once

Remarks:

Doses / Concentrations:
400-500 mL of pine oil

No. of animals per sex per dose / concentration:

1

Control animals:

no

Preliminary studies:

None

Details on absorption:

See other information on results

Details on distribution in tissues:

See other information on results

Details on excretion:

See other information on results

Metabolites identified:

yes

Details on metabolites:

See other information on results

Clinical signs: psychomotric excitation, headache, erythem of mouth and larynx, a flush of the face, ataxia, and a spontaneous hyperventilation. With a latence of 10 h after ingestion the consciousness of the patient was impaired and the circulatory parameters became instable although a hypovolemy could be excluded. Three weeks later the patient left the clinic without any bodily complaints.

Examination results: the circulatory parameters and the laboratory data were in the normal range. The EEG recorded the second day revealed a decelerated activity. No epileptogenic activities could be detected. The patient had a retrograde amnesia for the period of somnolence and sopor. At this time a leukocytosis (21000/mm3), a slight raise of the transaminases, and a reduction of the pseudocholinesterase (1446 U/L) were observed. The renal functions were not affected except a transient oliguria which was due to the drop of the blood pressure.

Alpha-pinene was the monoterpene with the highest concentration in blood. The high affinity of the monoterpenes to lipophilic body compartments may be partly responsible for the comparatively low alpha-pinene blood level. The metabolites of the monoterpenes could be detected solely in urine whereas in blood only the original monoterpenes were identified. The main metabolite is bornylacetate. Ten days after ingestion a total of 100 mg bornylacetate was excreted with the urine. The renal excretion of metabolized monoterpenes reaches its peak level 5 days after ingestion. This indicates that the resorbed portion of the monoterpenes is slowly metabolized and then excreted via kidneys. The main metabolic pathways are hydratation, hydroxylation, rearrangement, and acetylation.

Conclusions:

The data suggest that monoterpenes are poorly absorbed in the gastrointestinal tract. The absorbed portion of the hydrocarbons accumulates in the lipophilic body compartments and is slowly metabolized and then excreted by the kidneys. The main metabolic pathways are hydratation, hydroxylation, rearrangement, and acetylation. Five metabolites were identified.

Executive summary:

A patient attempting suicide ingested 400-500 mL pine oil and was admitted to the clinic. Since more than the potentially lethal dose had been ingested hemoperfusions with activated charcoal and amberlite and a hemodialysis were performed. Clinical signs observed were: psychomotric excitation, head ache, erythem of mouth and larynx, a flush of the face, ataxia, and a spontaneous hyperventilation. With a latence of 10 h after ingestion the consciousness of the patient was impaired and the circulatory parameters became instable although a hypovolemy could be excluded. Three weeks later the patient left the clinic without any bodily complaints. The composition of the ingested pine oil was determined by gaschromatography/mass spectrometry. Four monoterpenes were identified in the pine oil ingested: 57% alpha-pinene, 8% beta-pinene, 26% carene, 6% limonene and 3% other hydrocarbons. The blood and urine monoterpene concentrations were continuously monitored. The data suggest that monoterpenes are poorly absorbed in the gastrointestinal tract. The absorbed portion of the hydrocarbons accumulates in the lipophilic body compartments and is slowly metabolized and then excreted by the kidneys. The main metabolic pathways are hydratation, hydroxylation, rearrangement, and acetylation. Five metabolites were identified.

Reference 5

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1967

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Old but well conducted and well described study.

Reason / purpose for cross-reference:

read-across: supporting information

Objective of study:

distribution

Principles of method if other than guideline:

Turpentine content of tissues after chronic exposure of adult male rats to commercial turpentine.

GLP compliance:

no

Specific details on test material used for the study:

Name of test material (as cited in study report): turpentine
Composition: 95% of alpha-pinene

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

No food was offered during the exposures while water was given ad libitum. The animals were kept under an artificial day-light cycle of 12 h for 2 weeks before and during the exposures.

Route of administration:

inhalation: vapour

Vehicle:

unchanged (no vehicle)

Duration and frequency of treatment / exposure:

6 h /day, 5 days/ weeks for 8 weeks

Remarks:

Doses / Concentrations:
300 ppm (12 µmol/L)

No. of animals per sex per dose / concentration:

40

Control animals:

yes, concurrent no treatment

Positive control reference chemical:

None

Details on dosing and sampling:

5 rats were killed by decapitation 1, 4, 5, 6, 7 and 8 weeks after the beginning of the experiment while ten rats were killed at the second week. Brain and perinephric fat samples were taken after killing.
The amount of alpha-pinene was analyzed gas-chromatographically after extraction with dimethylformamide (DMFA).

Statistics:

None

Preliminary studies:

None

Details on absorption:

None

Details on distribution in tissues:

Alpha-pinene was found in the perinephric fat and brain. The brain pinene content remained similar throughout the experiment and it was about 10% of that in fat.

Details on excretion:

None

Metabolites identified:

not measured

Details on metabolites:

None

Table 1: mean alpha-pinene content in brain and perinephric fat

Weeks of exposure	Alpha-pinene in brain (mmol/g)	Alpha-pinene in perinephric fat (mmol/g)
1	26±6	365±85
2	28±10	353±110
4	27±7	307±76
5	21±4	322±72
6	23±4	371±65
7	-	-
8	-	-

Conclusions:

Chronic exposure of adult male rats to commercial turpentine resulted in an accumulation of the solvent in perinephric fat and brain.

Executive summary:

Male wistar rats were exposed to 300 ppm of turpentine by inhalation 6 h/day, 5days/week for 8 weeks. 5 rats were killed by decapitation 1, 4, 5, 6, 7 and 8 weeks after the beginning of the experiment while ten rats were killed at the second week. Brain and perinephric fat samples were taken after killing. The amount of alpha-pinene was analyzed gas-chromatographically after extraction with dimethylformamide (DMFA). After chronic exposure of adult male rats, commercial turpentine was found in perinephric fat and brain. The brain pinene content remained similar throughout the experiment and it was about 10% of that in fat.

Reference 6

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Recent well described and well conducted study.

Reason / purpose for cross-reference:

read-across: supporting information

Principles of method if other than guideline:

Skin absorption and elimination kinetics were studied using human skin from the region of thorax of 40-50-years old Caucasian women, mounted on flow-through Teflon diffusion cells. 500 mg alpha-pinene was applied and after 1 to 4-h exposure, the content in the stratum corneum layers (separated by a tape-stripping method) and in the epidermis/dermis was determined using GC. Similarly, the elimination kinetics in the skin were analysed during 4 h following 1 h absorption.

GLP compliance:

no

Radiolabelling:

no

Species:

human

Sex:

female

Details on test animals or test system and environmental conditions:

Human cadaver skin was obtained from the region of thorax of 40-50-years old Caucasian women. The subjects did not have skin diseases. Before the experiment, the skin was stored frozen at -20 °C.

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Duration of exposure:

1, 2 or 4 h

Doses:

500 mg on 0.65 cm²

No. of animals per group:

4 for each time point

Control animals:

no

Details on study design:

No data

Details on in vitro test system (if applicable):

Diffusion cell: flow-through Teflon diffusion cell (Crown Glass, USA)
The diffusion area of the skin was 0.65 cm². The donor compartment was occluded with Parafilm (Sigma-Aldrich, Steinheim, Germany), and the system was maintained at temperature 37 ± 0.5 °C. An isotonic pH 7.3 phosphate buffer, 10 mL, preserved with 0.005% sodium azide (Fluka, Buchs, Switzerland) was recirculated beneath the skin with a constant rate 10 mL/h. The two-phase acceptor fluid protected against evaporation was used and sink conditions were ensured for all steps of the study. It was obtained by addition of 5 mL of methylene chloride to the vial served as reservoir of the buffer. The skin was only in contact with the aqueous phase. The experiment was terminated by removing terpenes from the skin surface and very short rinsing with methanol. The stratum corneum layers were separated by a tapestripping method, using 21 fragments of an adhesive
tape (3M Medica Pharma, St. Paul, USA). Collected samples were divided into three fractions (SC I-III). Each fraction, as well as the remaining viable epidermis with dermis (ED) was extracted with methanol (HPLC-grade, P.O.Ch., Gliwice, Poland).
In the elimination studies, the terpenes were applied only for 1 h and next, after removing terpenes from the donor chamber as described above, the skin was left in the chambers for 1, 2, 3 or 4 h. The acceptor medium was replaced by a fresh portion and its circulation was maintained. After the specified time, the skin was rinsed, removed and separated as described above. The terpenes in the extracts were analysed by GC with the detection limit 0.5 mg/mL.

Signs and symptoms of toxicity:

not specified

Dermal irritation:

not specified

Absorption in different matrices:

The dermal penetration of pure terpenes was studied during 4 h. The terpenes were present on the skin in infinite doses and the system was protected against evaporation. During that time no terpenes were detected in the acceptor fluid but extensive accumulation in the skin tissue occurred (see table 1).
Analysis of stratum corneum (SC) collected with an adhesive tape and merged into three groups demonstrates cumulation of terpenes in the outer (SC I), middle (SC II) and inner (SC III) layers. The results demonstrate rapid penetration of terpenes not only to the SC I layers but also to viable epidermis and dermis. The distance-dependent decreasing gradient of concentration for all terpenes is observed, although the concentrations were not normalized in respect of the collected SC mass. A steady-state concentration of terpenes in the SC can be assumed as soon as after 1 h. Maximum concentration in the SC was achieved as soon as after 1 h and did not further increase in the course of the study.
All studied terpenes are absorbed in high amounts in the viable epidermis with dermis (ED), however penetration into this layers is time-dependent process, constantly increasing during 4 h.

Total recovery:

No data

Conversion factor human vs. animal skin:

None

Table 1: Absorption of alpha-pinene (mg/cm2) into human skin layers (mean ± S.D., n = 4)

Skin layer	1-h exposure	2-h exposure	4-h exposure
SC I	4.3 ± 0.8	3.4 ± 0.4	7.7 ± 4.3
SC II	4.0 ± 0.5	3.2 ± 0.6	6.5 ± 4.1
SC III	3.0 ± 0.4	3.4 ± 0.8	6.0 ± 4.0
SC total	11.3 ± 4.7	10.0 ± 2.1	18.2 ± 12.1
ED	66.4 ± 16.8	147.2 ± 25.9	313.7 ± 38.3
Skin total	77.7 ± 14.5	157.2 ± 25.8	331.9 ± 34.7

Table 2: Elimination of alpha-pinene (mg/cm2) from human skin layers following 1 h absorption (t = 0) (mean ± S.D., n = 4)

	Time after 1-h exposure
Skin layer	0	1	2	3	4
SC I	4.3 ± 0.8	3.2 ± 1.9	2.0 ± 1.8	0.7 ± 0.6	0.6 ± 0.7
SC II	4.0 ± 0.5	0.9 ± 1.0	0	0	0
SC III	3.0 ± 0.4	0.2 ± 0.2	0	0	0
SC total	11.3 ± 4.7	4.3 ± 3.0	2.0 ± 1.8	0.7 ± 0.6	0.6 ± 0.7
ED	66.4 ± 16.8	45.7 ± 13.0	35.9 ± 8.2	37.3 ± 3.6	33.9 ± 5.7
Skin total	77.7 ± 14.5	50.0 ± 10.1	37.9 ± 6.8	38.0 ± 3.3	34.5 ± 5.1

Conclusions:

Alpha-pinene absorption into the different skin layers is rapid (steady-state concentrations in the skin obtained after 1-h exposure) but do not permeate through the skin to the acceptor medium due to large accumulation into the skin tissue.

Executive summary:

Skin absorption and elimination kinetics were studied using human skin from the region of thorax of 40-50-years old Caucasian women, mounted on flow-through Teflon diffusion cells. Alpha-pinene (500 mg) was applied onto the human skin (0.65 cm²), and after 1 to 4-h exposure, the content in the stratum corneum layers (separated by a tape-stripping method) and in the epidermis/dermis was determined using GC. Similarly, the elimination kinetics in the skin were analysed during 4 h following 1 h absorption. Quadruplicates were used for each time point.

The results demonstrate rapid penetration of terpenes not only to the first stratum corneum layers but also to viable epidermis and dermis (steady-state concentrations assumed to be obtained at 1-h exposure). However, alpha-pinene did not permeate across the skin to the acceptor medium due to large cumulation in the skin tissue. Two mechanisms of elimination process of terpenes from the SC are suggested: evaporation and slightly progressive penetration from inner layer into dermis.

Description of key information

Although (-)-alpha-pinene can be found in fat tissues, bioaccumulation is not expected to occur since the substance is efficiently metabolised

to yield oxygenated metabolites that are subsequently conjugated with glucuronic acid and excreted mainly in the urine.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Alpha-pinene toxicokinetics following turpentine or pine oil exposure:

Eight male volunteers were exposed to 450 mg/m3 turpentine oil by inhalation (2 h, 50 W) in an exposure chamber. The mean relative uptakes of alpha-pinene was 62% of the amount inhaled. Between 2% and 5% of the net uptake was excreted unchanged in the expired air after the end of exposure. The mean blood clearance 21 h after exposure (CL21h) of alpha-pinene was 0.8 L/kg/h. The mean half-lives (t1/2) of the last phase of alpha-pinene averaged 32 h. (Filipsson et al., 1996)

Since turpentine oil is a lipophilic substance, it can potentially accumulate in fatty tissues. In rats, the highest concentrations of inhaled turpentine oil (mainly composed of alpha-pinene) were found in the spleen, kidneys, brain, and perinephric fat (Savolainen et al., 1978).

The blood and urine monoterpene concentrations were continuously monitored from a patient attempting suicide by ingestion of 400-500 mL pine oil. The data suggest that monoterpenes, including alpha-pinene, are poorly reabsorbed in the gastrointestinal tract. The resorbed portion of the hydrocarbons cumulates in the lipophilic body compartments and is slowly metabolized and then excreted by the kidneys. The main metabolic pathways are hydration, hydroxylation, rearrangement, and acetylation (Koppel, 1981).

Alpha-pinene toxicokinetics following alpha-pinene exposure:

The biotransformation of (+)-, (-)-, and (+/-)-alpha-pinene was studied in albino rabbits orally administered 400-700 mg/kg bw of the respective alpha-pinenes in water with 0.1% Tween 80. Urine was collected daily for 3 days and urinary metabolites were identified. In this study, the main urinary metabolite from (+)-, (-)-, and (+/-)-alpha-pinenes was (-)-trans-verbenol. (Ishida, 1981)

Eight healthy males were exposed to 10, 225, or 450 mg/m3 (+)-alpha-pinene or 450 mg/m3 (-)-alpha-pinene for 2 h in an inhalation chamber while performing light work (50 watts). Average pulmonary uptake of (+)-alpha-pinene and (-)-alpha-pinene amounted to 59% of the exposure concentration. Absolute uptake increased linearly with concentration. Mean blood concentration at the end of exposure were linearly related to inhaled concentration. The terminal t1/2 of alpha-pinene from the blood was 695 min for (+)-alpha-pinene and 555 min for (-)-alpha-pinene. Cumulative urinary excretion of unchanged alpha-pinene amounted to less than 0.001% of each dose. Respiratory elimination of (+)-alpha-pinene and (-)-alpha-pinene was 7.7 and 7.5% of total uptake, respectively (Falk et al., 1990; cited in HSDB 2009a).

Similarly, the renal elimination of verbenols after experimental exposure to (+)-alpha-pinene and (-)-alpha-pinene was studied in humans following exposure to 10, 225 and 450 mg/m3 terpene in an exposure chamber. The pulmonary uptake was about 60%. About 8% was eliminated unchanged in exhaled air. Depending on the exposure level, about 1%-4% of the total uptake was eliminated as cis- and trans-verbenol. Most of the verbenols were eliminated within 20 h after a 2-h exposure. The renal excretion of unchanged alpha-pinene was less than 0.001%. (Levin et al., 1992, cited in HSDB 2009a)

In healthy volunteers orally exposed to (-)-alpha-pinene, metabolites cVER (cis- verbenol), tVER (trans-verbenol) and MYR (myrtenol) were detected in blood samples over the entire blood sampling period of 1–5 h; unmetabolised alpha-pinene was below the limit of detection. In addition to the known and established alpha-pinene metabolites cVER and tVER, the relevance of MYR and MYRA as products of the human in vivo metabolism of alpha-pinene was confirmed. Two unknown human metabolites were identified as 4-hydroxymyrtenic acid and dihydromyrtenic acid. Human in vivo metabolism of alpha-pinene is dominated by extensive oxidation reactions on the methyl side-chains yielding in carboxylic acid structures. Human metabolism of alpha-pinene proceeds fast and the body is almost entirely cleared from the metabolites 10 h after exposure. (Schmidt, 2015)

 

Dermal absorption:

Skin absorption and elimination kinetics were studied using human skin from the thorax region mounted on flow-through diffusion cells. Results demonstrate rapid penetration of alpha-pinene not only to the first stratum corneum layers but also to viable epidermis and dermis. Alpha-pinene did not permeate across the skin to the acceptor medium due to large cumulation in the skin tissue (Cal et al., 2006). However, following immersion of young pigs and one human subject for 30 minutes in baths containing 150 mL of a pine-oil mixture, alpha-pinene was detected in exhaled air within 20 minutes reaching maximum levels 50-75 minutes after start of the bath and remained detectable after 1 day. (Opdyke, 1979, cited in HSDB 2009)

 

In conclusion, although (-)-alpha-pinene can be found in fat tissues, bioaccumulation is not expected to occur since the substance is efficiently metabolised to yield oxygenated metabolites that are subsequently conjugated with glucuronic acid and excreted mainly in the urine.

 

HSDB (Hazardous Substances Data Bank). 2009a. Alpha-pinene. HSDB No. 720. Produced by the National Library of Medicine (NLM), Bethesda, M.D. Last Revision Date: 26 June 2009