Reaction mass of 7,7-dimethyl-2-methylidenebicyclo[2.2.1]heptane and (1R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane and (1S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane and (1S)-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:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

No data on test method. No GLP.

Objective of study:

metabolism

Principles of method if other than guideline:

The metabolism of Camphene was studied on rabbits. The test substance was administered by gavage, with olive oil as vehicle.
No more data provided on the method.

GLP compliance:

no

Radiolabelling:

no

Species:

rabbit

Strain:

not specified

Sex:

not specified

Route of administration:

oral: gavage

Vehicle:

olive oil

Duration and frequency of treatment / exposure:

No data

No. of animals per sex per dose / concentration:

No data

Control animals:

not specified

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled (delete / add / specify): urine (as fresh as possible)

Metabolites identified:

yes

Details on metabolites:

Camphene was the only studied terpene in rabbits that produced glucuronic acid as a metabolite in the urine.

Camphene was the only studied terpene in rabbits that produced glucuronic acid as a metabolite in the urine.

Conclusions:

Camphene was the only studied terpene in rabbits that produced glucuronic acid as a metabolite in the urine.

Executive summary:

The metabolism of camphene was studied on rabbits. The test substance was administered by gavage, with olive oil as vehicle. Camphene was the only studied terpene in rabbits that produced glucuronic acid as a metabolite in the urine.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Albino rabbits were orally administered with test item and urine was collected for 3 days for identification of urinary metabolites.
- Short description of test conditions: see below
- Parameters analysed / observed: Determination and 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
- Age at study initiation: no data
- Weight at study initiation: 2-3 kg.
- Housing: individual stainless steel metabolism cages
- 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:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in water (100 ml) containing 0.1 g Tween 80 and were homogenized well.

Duration and frequency of treatment / exposure:

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

Remarks:

400-700 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

6

Control animals:

no

Positive control reference chemical:

None

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled (delete / add / specify): urine
- Time and frequency of sampling: The urine was collected daily for 3 days after drug administration and stored at 0-5ºC until time of analysis.
- From how many animals: 6
- Method type(s) for identification: GLC-MS, TLC, NMR, IR
- Other:
Extraction and fractionation of urinary metabolites: The urine was centrifuged to remove feces and hairs at 0ºC, and the supernate was used for the experiments. The urine was adjusted to pH 4.76 with acetate buffer and incubated with β-glucuronidase-arylsulfatase (3 ml/1000 ml of the fresh urine) at 37ºC for 48 hr, followed by continuous ether extraction for 48 hr. The ether extracts were washed with 5% NaHC03 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% HCI, 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

Metabolites identified:

yes

Details on metabolites:

The main urinary metabolite from (+)-, (-)-, and (+/-)-alpha-pinenes was (-)-trans-verbenol.
As minor metabolites of (-) alpha-pinene, two allylic products, myrtenol and myrtenic acid, were obtained.

Conclusions:

The main urinary metabolite from (-) alpha-pinene was (-)-trans-verbenol.

Executive summary:

The biotransformation of (-)-alpha-pinene was studied in albino rabbits orally administered 400-700 mg/kg bw of test item 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 (-)-alpha-pinene was (-)-trans-verbenol.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Test method was not according to any guideline. No GLP.

Objective of study:

excretion

Principles of method if other than guideline:

Evaluation of the elimination of injected camphene through respiration.

GLP compliance:

no

Radiolabelling:

not specified

Species:

other: human

Sex:

not specified

Route of administration:

intravenous

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
0.05 mL camphene in 2.5 mL 1,2-propanediol
IV injection (a single dose)
Volume: 3 mL

Duration and frequency of treatment / exposure:

A single dose

Dose / conc.:

0.001 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

One human subject (no data on sex)

Control animals:

no

Details on excretion:

After 30 minutes 90% of injected camphene was detected in the respiratory air.

Metabolites identified:

not measured

After 30 minutes 90% of injected camphene was detected in the respiratory air.

Conclusions:

After intravenous administration, camphene is mostly excreted through the respiratory air.

Executive summary:

To evaluate the elimination of injected camphene through respiration, a single dose of 0.6 µg/kg bw was intravenous administered to a human subject. After 30 minutes 90% of injected camphene was detected in the respiratory air.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

excretion

metabolism

Principles of method if other than guideline:

- Principle of test: human in vivo metabolism and the elimination kinetics of alpha-pinene
- Short description of test conditions: see description below
- Parameters analysed / observed: Metabolites and excretion rates

GLP compliance:

not specified

Radiolabelling:

no

Species:

other: human

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
- Age at study initiation: 33±11 years
- Weight at study initiation: 80±8 kg

Route of administration:

oral: capsule

Vehicle:

not specified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Oral exposure of the volunteers was achieved using standardised custom-made gelatin capsules.

Duration and frequency of treatment / exposure:

single oral dose

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

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- 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.
- Other:
METABOLITE CHARACTERISATION STUDIES
- 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.
- 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 and alpha pinene metabolites in blood and urine were analysed by GS-PCI-MS/MS
- Limits of detection and quantification: LOD and LOQ were determined to be 0.2 and 0.5 μg L−1.

Type:

metabolism

Results:

Metabolites identified: cis-verbenol (cVER), trans-verbenol (tVER), myrtenol (MYR), myrtenic acid (MYRA) and two unknown human metabolites, 4-hydroxymyrtenic acid (MYRA-4-OH) and dihydromyrtenic acid (DHMYRA).

Type:

excretion

Results:

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

Details on absorption:

Blood concentrations of unmetabolised alpha pinene were below the limit of detection (<4 μg L−1) over the entire blood sampling period of 1–5 h. In contrast, the metabolites cis-verbenol (cVER), trans-verbenol (tVER), and myrtenol (MYR) were detectable in almost all of the blood samples. Solely cVER was below the LOD in pre-exposure blood (<0.2 μg L−1). After exposure, the mean metabolite concentrations showed synchronous time courses even though the levels were low and varied between the two volunteers. Maximum blood concentrations (cmax) were reached in the samples drawn 1 and 3 h after exposure (tmax), respectively. After cmax, the blood levels decreased towards the base level; however, they did not reach it within the observation period.

Details on distribution in tissues:

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. Thus, alpha pinene or related volatile metabolites obviously seem to reach the lungs.

Details on excretion:

The pre-exposure urine samples showed background levels of 22 ± 11 μg L−1 (cVER), 14 ± 7.5 μg L−1 (tVER), 7.3 ± 1.4 μg L−1 (MYR) and 16 ± 6.3 μg L−1 (MYRA).
The metabolite concentrations in urine and hence calculated renal elimination rates (RE) increased distinctly within the first 1–3 h after exposure to their maxima of 1600 ± 650 μg L−1 cVER, 1900 ± 1700 μg L−1, tVER, 690 ± 530 μg L−1, MYR, and 3200 ± 2700 μg L−1 myrtenic acid (MYRA) (mean values ± SD; n = 4).
Afterwards, they rapidly declined to the base levels within the 24-h observation period.
At their maximum urinary concentrations, the metabolites were present in conjugated forms by 28–76 % (cVER), 4–68 % (tVER), 99–100 % (MYR), and 99–100 % (MYRA).
Assuming a first-order elimination kinetic, the mean elimination half-lives (t1/2) of the first, rapid elimination phases were 1.5 ± 0.2 h for cVER, 1.6 ± 0.2 h for tVER, 1.6 ± 0.2 h for MYR, and 1.4 ± 0.1 h for MYRA.
Following the first elimination phase, additional slower eliminations can be observed for all metabolites about 7 h after exposure.
The cumulative excretion graphs suggest completeness of elimination about 10 h after exposure.
Accordingly, the molar excreted total amounts of the recovered renal metabolites accounted for 5.6 ± 0.5 % (cVER), 4.1 ± 0.4 % (tVER), 1.5 ± 0.2 % (MYR), and 6.7 ± 0.3 % (MYRA) of the applied oral alpha pinene dose (66 ± 2.8 μmol).
Elimination of the other 2 metabolites detected accounted for about 1 % (αPN-M1) and 2 % (αPN-M3) of the orally applied dose.

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 1.5 ± 0.2 h

Remarks:

mean renal elimination of cVER

Key result

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: 1.6 ± 0.2 h

Remarks:

mean renal elimination of tVER

Key result

Test no.:

#3

Toxicokinetic parameters:

half-life 1st: 1.6 ± 0.2 h

Remarks:

mean renal elimination of MYR

Key result

Test no.:

#4

Toxicokinetic parameters:

half-life 1st: 1.4 ± 0.1 h

Remarks:

mean renal elimination of MYRA

Metabolites identified:

yes

Details on metabolites:

Previously known metabolites of alpha pinene were detected: cis-verbenol (cVER), trans-verbenol (tVER), and myrtenol (MYR).
Three novel metabolites (αPN-M1–αPN-M3), of which one (αPN-M2) conforms to myrtenic acid (MYRA), were identified.
αPN-M1 was identified as 4-hydroxymyrtenic acid (MYRA-4-OH) and αPN-M3 was identified to be an isomer of saturated MYRA, i.e. dihydromyrtenic acid (DHMYRA), which hints to a reductive branch of the metabolism pathway.

None of the volunteers reported any adverse health effects due to the supplementation with 10 mg of test item. 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 alpha pinene 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 alpha pinene exhaled was not quantified.

Table1: Characteristics of the blood kinetics of alpha pinene metabolites after oral exposure to 9.0±0.4 mg (66±2.8 µmol) alpha pinene (mean values ± 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	0.8 a
tVER	4.0±2.1	26±14	1–3	1.0 a
MYR	1.7±0.4	11±2.4	1–3	1.7 a

a Elimination not completed within 5 h observation period

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

Metabolites	RE, max (µg h−1)	Tmax (h)	T½ (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→tf area under the renal excretion vs. time curve (from time 0 to final sampling timetf); V_totalsummarized excreted urine volume

Conclusions:

The study on the human alpha pinene metabolism after oral uptake identified four relevant metabolites: cis-verbenol (cVER), trans-verbenol (tVER), myrtenol (MYR) and myrtenic acid (MYRA). 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 alpha pinene 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 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 alpha pinene metabolites was determined using a very specific and sensitive GC-PCI-MS/MS procedure. Concentration of alpha pinene was analysed in blood by HS-GC-MS procedure.

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 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. The non-detection of alpha pinene 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 first pass metabolism.

Alpha pinene 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 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 and these structures could be predicted as 4-hydroxymyrtenic acid (MYRA-4-OH) and dihydromyrtenic acid (DHMYRA).

Human in vivo metabolism of alpha pinene is consequently 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 alpha pinene eliminated unchanged via lungs remain unclear.

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

Reference 5

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:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance d-camphene which shares the same functional groups with the main components of the multi-constituent substance reaction mass of fenchene and laevo camphene and dextro camphene and laevo alpha pinene also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Metabolites identified:

yes

Details on metabolites:

Based on read across from the analogue camphene, the reaction mass is expected to produced glucuronic acid as a metabolite in the urine.

Conclusions:

Based on read across from the analogue camphene, the reaction mass is expected to produced glucuronic acid as a metabolite in the urine.

Executive summary:

The metabolism of camphene was studied on rabbits. The test substance was administered by gavage, with olive oil as vehicle. Camphene was the only studied terpene in rabbits that produced glucuronic acid as a metabolite in the urine. Based on these results, the read-across approach was applied and the reaction mass is expected to produced glucuronic acid as a metabolite in the urine.

Reference 6

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:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance L-alpha pinene which shares the same functional groups with the main components of the multi-constituent substance reaction mass of fenchene and laevo camphene and dextro camphene and laevo alpha pinene also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Metabolites identified:

yes

Details on metabolites:

Based on read across from the analogue L-alpha pinene, the following results were predicted for the reaction mass:
The main urinary metabolite is (-)-trans-verbenol and
As minor metabolites two allylic products, myrtenol and myrtenic acid, are obtained

Conclusions:

Based on read across from the analogue L-alpha pinene, the main urinary metabolite for the reaction mass is predicted to be (-)-trans-verbenol.

Executive summary:

The biotransformation of (-)-alpha-pinene was studied in albino rabbits orally administered 400-700 mg/kg bw of test item 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 (-)-alpha-pinene was (-)-trans-verbenol. Based on these results, the read-across approach was applied and the main urinary metabolite for the reaction mass is predicted to be (-)-trans-verbenol.

Reference 7

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:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance camphene which shares the same functional groups with the main components of the multi-constituent substance reaction mass of fenchene and laevo camphene and dextro camphene and laevo alpha pinene also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Details on excretion:

Based on read across from the analogue camphene, the reaction mass is expected to be mostly excreted through the respiratory air.

Metabolites identified:

not measured

Conclusions:

Based on read across from the analogue camphene, the reaction mass is expected to be mostly excreted through the respiratory air after intravenous administration.

Executive summary:

To evaluate the elimination of injected camphene through respiration, a single dose of 0.6 µg/kg bw was intravenous administered to a human subject. After 30 minutes 90% of injected camphene was detected in the respiratory air. Based on these results, the read-across approach was applied and the reaction mass is expected to be mostly excreted through the respiratory air after intravenous administration.

Reference 8

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:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance L-alpha pinene which shares the same functional groups with the main components of the multi-constituent substance reaction mass of fenchene and laevo camphene and dextro camphene and laevo alpha pinene also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Type:

metabolism

Results:

Metabolites identified: cis-verbenol (cVER), trans-verbenol (tVER), myrtenol (MYR), myrtenic acid (MYRA) and two unknown human metabolites, 4-hydroxymyrtenic acid (MYRA-4-OH) and dihydromyrtenic acid (DHMYRA).

Type:

excretion

Results:

Based on read across from the analogue L-alpha pinene, human metabolism of the reaction mass is expected to proceed fast and the metabolites leave the body 10 h after exposure.

Details on absorption:

Based on read across from the analogue L-alpha pinene, maximum blood concentrations for the reaction mass are predicted up to 3 h after exposure.

Details on distribution in tissues:

Based on read across from the analogue L-alpha pinene, the reaction mass or related volatile metabolites are predicted to reach the lungs.

Details on excretion:

Based on read across from the analogue L-alpha pinene, human metabolism of the reaction mass is expected to proceed fast and the metabolites leave the body 10 h after exposure.

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 1.5 ± 0.2 h

Remarks:

mean renal elimination of cVER

Key result

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: 1.6 ± 0.2 h

Remarks:

mean renal elimination of tVER

Key result

Test no.:

#3

Toxicokinetic parameters:

half-life 1st: 1.6 ± 0.2 h

Remarks:

mean renal elimination of MYR

Key result

Test no.:

#4

Toxicokinetic parameters:

half-life 1st: 1.4 ± 0.1 h

Remarks:

mean renal elimination of MYRA

Metabolites identified:

yes

Details on metabolites:

Based on read across from the analogue L-alpha pinene, the following results were predicted for the reaction mass:
Previously known metabolites of alpha pinene were detected: cis-verbenol (cVER), trans-verbenol (tVER), and myrtenol (MYR).
Three novel metabolites (αPN-M1–αPN-M3), of which one (αPN-M2) conforms to myrtenic acid (MYRA), were identified.
αPN-M1 was identified as 4-hydroxymyrtenic acid (MYRA-4-OH) and αPN-M3 was identified to be an isomer of saturated MYRA, i.e. dihydromyrtenic acid (DHMYRA), which hints to a reductive branch of the metabolism pathway.

Conclusions:

Based on read across from the analogue L-alpha pinene, human metabolism of the reaction mass is expected to proceed fast and the metabolites leave the body 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 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 alpha pinene metabolites was determined using a very specific and sensitive GC-PCI-MS/MS procedure. Concentration of alpha pinene was analysed in blood by HS-GC-MS procedure.

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 and these structures could be predicted as 4-hydroxymyrtenic acid (MYRA-4-OH) and dihydromyrtenic acid (DHMYRA).

Human in vivo metabolism of alpha pinene is consequently 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 alpha pinene eliminated unchanged via lungs remain unclear.

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

Based on these results, the read-across approach was applied and the human metabolism of the reaction mass is expected to proceed fast and the metabolites leave the body 10 h after exposure.

Reference 9

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

No data on test method. No data on GLP.

Principles of method if other than guideline:

No data on test method.

GLP compliance:

not specified

Radiolabelling:

not specified

Species:

other: no data

Strain:

not specified

Sex:

not specified

Type of coverage:

not specified

Vehicle:

not specified

Duration of exposure:

No data

Doses:

No data

No. of animals per group:

No data

Camphene is readily absorbed following a topical application.

Conclusions:

Camphene is readily absorbed following a topical application.

Executive summary:

Camphene is readily absorbed following a topical application.

Reference 10

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Test method is not according to any guideline. No GLP.

Principles of method if other than guideline:

The permeability of human skin to terpenes was studied by Römmelt et al., who demonstrated percutaneous absorption from 30-min full baths (Kneipp) containing pine oil additives by measuring camphene and other terpene components in the expired air of one human subject. The results of dermal absorption were calculated with respect to the values obtained from the elimination of camphene through the lungs after intravenous administration.

GLP compliance:

no

Radiolabelling:

not specified

Species:

other: Human

Sex:

not specified

Details on test animals or test system and environmental conditions:

In all experiments, the water and the bath temperature were kept constant.
The subject breathed through a breathing mask with double valve.

Type of coverage:

open

Vehicle:

water

Duration of exposure:

30 minutes

Doses:

150 mL pine bath oil in 450 L water
The exact dose was controlled by a pressure reducer in the stock tank and a diving bell spirometer, which functioned as a variable reservoir.

No. of animals per group:

One human subject (no data on sex)

Control animals:

no

Details on study design:

Observation period: 24 hours

Signs and symptoms of toxicity:

no effects

Dermal irritation:

no effects

Total recovery:

A human subject immersed in a bath containing pine needle oil, exhaled terpenes, including 0.67 µL camphene, within 5 hr.
A constant of 81 µL/cm2/hr was calculated for the percutaneous absorption of camphene from the bath.
The total quantity of Kneipp pine oil extract absorbed through the skin of an adult person from a 30-min full bath containing 150 mL pine bath oil in 450 L water bath was estimated to be 0.04 mg, which must be considered to be within a range capable of producing therapeutic effects.

The test subject exhaled terpenes, including 0.67 µL camphene, within 5 hr. A constant of 81 µL/cm2/hr was calculated for the percutaneous absorption of camphene from the bath.

Conclusions:

The test subject exhaled terpenes, including 0.67 µL camphene, within 5 hr. A constant of 81 µL/cm2/hr was calculated for the percutaneous absorption of camphene from the bath.

Executive summary:

The permeability of human skin to terpenes was studied by Römmelt et al., who demonstrated percutaneous absorption from 30-min full baths (Kneipp) containing pine oil additives by measuring camphene and other terpene components in the expired air of one human subject. The results of dermal absorption were calculated with respect to the values obtained from the elimination of camphene through the lungs after intravenous administration. The test subject exhaled terpenes, including 0.67 µL camphene, within 5 hr. A constant of 81 µL/cm²/hr was calculated for the percutaneous absorption of camphene from the bath.

Reference 11

Endpoint:

dermal absorption in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance camphene which shares the same functional groups with the main components of the multi-constituent substance reaction mass of fenchene and laevo camphene and dextro camphene and laevo alpha pinene also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Based on read across from the analogue camphene, the reaction mass is expected to be readily absorbed following a topical application.

Conclusions:

Based on read across from the analogue camphene, the reaction mass is expected to be readily absorbed following a topical application.

Executive summary:

Based on read across from the analogue camphene, the reaction mass is expected to be readily absorbed following a topical application.

Reference 12

Endpoint:

dermal absorption in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance camphene which shares the same functional groups with the main components of the multi-constituent substance reaction mass of fenchene and laevo camphene and dextro camphene and laevo alpha pinene also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Signs and symptoms of toxicity:

no effects

Dermal irritation:

no effects

Total recovery:

Based on read across from the analogue camphene, a constant of 81 µL/cm2/hr was predicted for the percutaneous absorption of the reaction mass.

Conclusions:

Based on read across from the analogue camphene, a constant of 81 µL/cm2/hr was predicted for the percutaneous absorption of the reaction mass.

Executive summary:

The permeability of human skin to terpenes was studied by Römmelt et al., who demonstrated percutaneous absorption from 30-min full baths (Kneipp) containing pine oil additives by measuring camphene and other terpene components in the expired air of one human subject. The results of dermal absorption were calculated with respect to the values obtained from the elimination of camphene through the lungs after intravenous administration. The test subject exhaled terpenes, including 0.67 µL camphene, within 5 hr. A constant of 81 µL/cm²/hr was calculated for the percutaneous absorption of camphene from the bath. Based on these results, the read-across approach was applied and the reaction mass was predicted to have a constant of 81 µL/cm2/hr for percutaneous absorption.

Description of key information

Basic Toxicokinetics: Metabolism. Based on the experimental results obtained with the analogue substances d-camphene and l-alpha pinene, the main expected metabolites to be found in the urine might be glucuronic acid, cis-verbenol and trans-verbenol.

Basic Toxicokinetics: excretion. Based on the experimental results obtained with the analogue substance camphene, the reaction mass is expected to be mostly excreted through the respiratory air after intravenous administration. Also, based on the experimental results obtained with the analogue substance l-alpha pinene, the human metabolism of the reaction mass is expected to proceed fast and the metabolites leave the body 10 h after exposure.

Dermal absorption: Based on the experimental results obtained with the analogue substance camphene, the reaction mass is expected to be readily absorbed following a topical application.

Additional information

Read-across from experimental results with analogue substances d-camphene, camphene and l-alpha pinene:

Basic Toxicokinetics: Metabolism

Supporting study: The metabolism of camphene was studied on rabbits. The test substance was administered by gavage, with olive oil as vehicle. Camphene was the only studied terpene in rabbits that produced glucuronic acid as a metabolite in the urine. Based on these results, the read-across approach was applied and the reaction mass is expected to produced glucuronic acid as a metabolite in the urine.

Supporting study: The biotransformation of (-)-alpha-pinene was studied in albino rabbits orally administered 400-700 mg/kg bw of test item 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 (-)-alpha-pinene was (-)-trans-verbenol. Based on these results, the read-across approach was applied and the main urinary metabolite for the reaction mass is predicted to be (-)-trans-verbenol.

Basic Toxicokinetics: excretion.

Supporting study: To evaluate the elimination of injected camphene through respiration, a single dose of 0.6  μg/kg bw was intravenous administered to a human subject. After 30 minutes 90% of injected camphene was detected in the respiratory air. Based on these results, the read-across approach was applied and the reaction mass is expected to be mostly excreted through the respiratory air after intravenous administration.

Supporting study: In a metabolism study, four healthy human volunteers were orally exposed to a single dose of 9 mg of (1S,5S)-(−)-α- pinene via spiked gelatin capsules. In addition to the known and established alpha pinene metabolites cis-verbenol (cVER) and trans-verbenol, the relevance of myrtenol (MYR) and myrtenic acid (MYRA) as products of the human in vivo metabolism of alpha pinene was confirmed. Human metabolism of alpha pinene proceeds fast and the body is almost entirely cleared from the metabolites 10 h after exposure. Based on these results, the read-across approach was applied and the human metabolism of the reaction mass is expected to proceed fast and the metabolites leave the body 10 h after exposure.

Dermal absorption

Supporting study: The permeability of human skin to terpenes was studied by Römmelt et al., who demonstrated percutaneous absorption from 30-min full baths (Kneipp) containing pine oil additives by measuring camphene and other terpene components in the expired air of one human subject. The results of dermal absorption were calculated with respect to the values obtained from the elimination of camphene through the lungs after intravenous administration. The test subject exhaled terpenes, including 0.67 μL camphene, within 5 hr. A constant of 81 μL/cm2/hr was calculated for the percutaneous absorption of camphene from the bath. Based on these results, the read-across approach was applied and the reaction mass was predicted to have a constant of 81 μL/cm2/hr for percutaneous absorption..

Supporting study: Based on read across from the analogue camphene, the reaction mass is expected to be readily absorbed following a topical application.