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Endpoint:
basic toxicokinetics in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Objective of study:
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
In vivo metabolism study, following oral (gavage) administration in rats.
GLP compliance:
no
Radiolabelling:
no
Species:
rat
Strain:
not specified
Sex:
male
Route of administration:
other: oral - gastric intubation
Vehicle:
other: 1 % methyl cellulose
Duration and frequency of treatment / exposure:
Once daily for 20 days
Remarks:
Doses / Concentrations:
800 mg/kg bw/day
No. of animals per sex per dose:
No data
Control animals:
yes, concurrent vehicle
Type:
metabolism
Results:
hydroxylated derivatives of Cineol such as 2-hydroxy cineole and 3-hydroxy cineole are excreted as conjugates
Details on absorption:
Not applicable
Details on distribution in tissues:
Not applicable
Details on excretion:
Not applicable
Metabolites identified:
yes
Details on metabolites:
- The acid methyl esters (250 mg) were subjected to TLC which showed the presence of one major compound i.e., methyl ester of 1,8-dihydroxy -10-carboxy p-methane and three minor compounds. The TLC analysis of the neutral fraction (200 mg) showed the presence of one major i.e., 2-hydroxy cineole and two minor metabolites. The neutral fraction (500 mg) obtained from hydrolysed urine on TLC analysis revealed the presence of two major (similar to 2-hydroxy cineole and 3-hydroxy cineole) and two minor metabolites.
- Based on the results, it is rather difficult to predict the sequence of reactions taking place during the biotransformation of cineole. However, one can envisage the formation of 1,8-dihydroxy-10-carboxy-p-methane through the intermediary of p-methane 1,8-diol and further metabolism is possibly initiated by the oxygenation of the C-10 methyl group resulting in the formation of p-methane-1,8,10-triol which undergoes stepwise oxidation to the corresponding aldehydes and then to an acid.
Bioaccessibility testing results:
Not applicable

None

Conclusions:
Interpretation of results (migrated information): other: hydroxylated derivatives of Cineol such as 2-hydroxy cineole and 3-hydroxy cineole are excreted as conjugates
Cineole was metabolised to hydroxylated derivatives such as 2-hydroxy cineole and 3-hydroxy cineole in rats and are excreted as conjugates.
Executive summary:

In an in vivo metabolism study, 1,8-Cineole was administered by oral route (via gastric intubation) to male albino rats at the dose of 800 mg/kg bw/day once daily for 20 days as a suspension in 1 % methyl cellulose solution. Control rats were given only with vehicle (4 mL/kg bw/day). Urine samples collected daily for 20 days, were adjusted to pH 3-4 and extracted with ether. The aqueous portion containing conjugated metabolites was then subjected to acid hydrolysis and extracted with ether (Chadha and Madyastha 1984). Both the ether extracts were separated into neutral and acidic fractions. The total acidic fraction was methylated using diazomethane (Chadha and Madyastha 1984). Thin-layer chromatographic (TLC) analyses (silica gel G) of the metabolites were carried out using hexane-ethyl acetate. Separation and purification of the metabolites were accomplished by using a silica gel column and hexane-ethyl acetate as the eluent.

The acid methyl esters (250 mg) were subjected to TLC which showed the presence of one major compound i.e., methyl ester of 1,8-dihydroxy -10-carboxy p-methane and three minor compounds. The TLC analysis of the neutral fraction (200 mg) showed the presence of one major i.e., 2-hydroxy cineole and two minor metabolites. The neutral fraction (500 mg) obtained from hydrolysed urine on TLC analysis revealed the presence of two major (similar to 2-hydroxy cineole and 3-hydroxy cineole) and two minor metabolites. Based on the results, it is rather difficult to predict the sequence of reactions taking place during the biotransformation of cineole. However, one can envisage the formation of1,8-dihydroxy-10-carboxy-p-methane through the intermediary of p-methane 1,8-diol and further metabolism is possibly initiated by the oxygenation of the C-10 methyl group resulting in the formation of p-methane-1,8,10-triol which undergoes stepwise oxidation to the corresponding aldehydes and then to an acid. The opening of the ether bridge in cineole could result in the formation of a p-menthanoid cation with a positive charge either at C-1 or C-8 which further gets readily neutralized by the attack of a hydroxide ion to yield p- methane-1, 8-diol. The 2- and 3-hydroxy derivatives from cineole have been reported in bacterial (MacRae et al. 1979) and fungal systems (Nishimura et al. 1982) respectively. So it appears that the microbial systems are more specific while carrying out the hydroxylation of cineole unlike the mammalian system which hydroxylates at C-2 as well as C-3 position. Both these hydroxylated derivatives are excreted as conjugates.

 

1,8-Cineole was metabolised to hydroxylated derivatives such as 2-hydroxy cineole and 3-hydroxy cineole in rats and are excreted as conjugates.
Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Objective of study:
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
Biotransformation of 1,8 -Cineole in human liver microsomes.
GLP compliance:
no
Radiolabelling:
no
Species:
human
Sex:
not specified
Duration and frequency of treatment / exposure:
Not applicable
Remarks:
Doses / Concentrations:
200 µM concentration of 1, 8-Cineole
No. of animals per sex per dose:
Not applicable
Type:
metabolism
Results:
1, 8-Cineole was metabolised to 2-exo-hydroxy-1,8-cineole by human liver microsomes
Details on absorption:
Not applicable
Details on distribution in tissues:
Not applicable
Details on excretion:
Not applicable
Metabolites identified:
yes
Details on metabolites:
- 1,8 cineole was oxidized to 2-exo-hydroxy-1,8-cineole as an only one metabolite by human liver microsomes.
- The metabolite formation was studied under various conditions by changing incubation time, P450 level in liver microsomes and substrate concentration. On incubation of 1,8 cineole with human liver microsomes in the presence of an NADPH, the metabolite was found to be formed with the increase of incubation time, P450 levels, and substrate concentration. 1,8-cineole 2-hydroxylation activity was shown to be high for a short incubation time, a small quantity of P450 levels in liver microsomes, and 1,8 cineole at low concentration.
Bioaccessibility testing results:
Not applicable

None

Conclusions:
Interpretation of results (migrated information): other: 1,8 cineole was oxidized to 2-exo-hydroxy-1,8-cineole as an only one metabolite by human liver microsomes.
Under the test conditions, 1,8 cineole was oxidized to 2-exo-hydroxy-1,8-cineole as an only one metabolite by human liver microsomes.
Executive summary:

The biotransformation of 1,8 -Cineole has been investigated by using human liver microsomes. Standard reaction mixture contained human liver microsomes (0.025 mg/mL) with 200 µM concentration of 1,8 cineole in a final volume of 0.50 mL of 100 mM potassium phosphate buffer (pH 7.4) containing NADPH. Incubations were carried out at 37 °C for 30 min and terminated by adding 1.0 mL of ethyl acetate. The mixtures were stirred vigorously and the extracts (organic layer) were collected by centrifugation at 3000 rpm for 10 min. The organic phase was transferred to an insert for analysis by GC-MS.

 

1,8 cineole was oxidized to 2-exo-hydroxy-1,8-cineole as an only one metabolite by human liver microsomes. The metabolite formation was studied under various conditions by changing incubation time, P450 level in liver microsomes and substrate concentration. On incubation of 1,8 cineole with human liver microsomes in the presence of an NADPH, the metabolite was found to be formed with the increase of incubation time, P450 levels, and substrate concentration. 1,8-cineole 2-hydroxylation activity was shown to be high for a short incubation time, a small quantity of P450 levels in liver microsomes, and 1,8 cineole at low concentration.

Under the test conditions, 1,8 cineole was oxidized to 2-exo-hydroxy-1,8-cineole as an only one metabolite by human liver microsomes.
Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
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:
reference to same study
Reason / purpose:
reference to other study
Principles of method if other than guideline:
Skin absorption and elimination kinetics using human skin.
GLP compliance:
no
Radiolabelling:
no
Species:
human
Sex:
female
Details on test animals 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 beta-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

19.4 ± 5.6

28.8 ± 14.3

23.8 ± 15.7

SC II

10.5 ± 2.1

20.3 ± 11.0

12.2 ± 7.0

SC III

9.9 ± 5.4

23.5 ± 2.2

10.5 ± 3.0

SC total

39.8 ± 8.8

72.6 ± 12.1

46.5 ± 25.5

ED

89.0 ± 12.4

318.2 ± 103.4

417.8 ± 28.8

Skin total

128.8 ± 4.5

390.8 ± 106.7

464.3 ± 46.7

Table 2: Elimination of beta-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

19.4 ± 5.6

14.9 ± 6.2

12.0 ± 2.2

11.1 ± 4.2

4.8 ± 4.8

SC II

10.5 ± 2.1

6.6 ± 6.0

5.8 ± 5.0

5.5 ± 6.5

0

SC III

9.9 ± 5.4

5.1 ± 4.9

0

0

0

SC total

39.8 ± 8.8

26.6 ± 16.3

17.8 ± 7.1

16.6 ± 10.3

4.8 ± 4.8

ED

89.0 ± 12.4

84.0 ± 9.8

83.8 ± 7.4

78.1 ± 5.1

64.5 ± 4.5

Skin total

128.8 ± 4.5

110.6 ± 21.0

101.6 ± 1.7

94.7 ± 9.7

69.3 ± 8.0

Conclusions:
Beta-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. Beta-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, beta-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

Information gained from Eucalyptus oil and its major constituents showed that Eucalyptus globulus oil is bioavailable via oral route. Systemic absorption via inhalation and dermal route is anticipated. Following liver metabolism, Eucalyptus globulus oil is expected to be mainly excreted in urine.

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential

Additional information

There is no specific requirement to generate toxicokinetic information in REACH. Therefore, the toxicokinetic profile of Eucalyptus globulus oil (i.e absorption, distribution, metabolism and elimination) was derived from the relevant available information on the major constituents (i.e. 1,8-cineol, (d,l)-limonene, alpha-pinene). The physical chemical characteristics of its major constituents, the results obtained from acute and repeated-dose toxicity studies, as well as information gained from genotoxicity assays were used to predict the toxicokinetic behaviour of Eucalyptus globulus oil.

Physico chemical properties:

Eucalyptus globulus oil is an UVCB substance, mostly composed of 1,8-cineol, (d,l)-limonene and alpha-pinene. These three major constituents have a relatively low molecular weight (136.24 g/mol for (d,l)-limonene and alpha-pinene; 154.24 g/mol for 1,8-cineol). (d,l)-limonene and alpha-pinene are slightly soluble in water (2.65 and 1.82 mg/L, respectively) whereas 1,8-cineol is miscible in water (3500 mg/L). (d,l)-limonene and alpha-pinene are highly lipophilic (log Pow = 4.35 and 4.42, respectively) and 1,8-cineol is rather more lipophilic than hydrophilic without accumulative potential (log Pow = 2.84). 1,8-cineol and (d,l)-limonene are not volatile (vapour pressure = 253 and 207 Pa at 25°C, respectively) whereas alpha-pinene is volatile (vapour pressure = 633 Pa at 25°C).

Absorption:

Oral/GI absorption:

The physical chemical characteristics described above suggest that the three major constituents of Eucalyptus globulus oil are of adequate molecular size to participate in endogenous absorption mechanisms within the mammalian gastrointestinal tract. Due to its low molecular weight, moderate lipophilicity and high water solubility, 1,8-cineol may be absorbed in the gastro-intestinal tract through aqueous pores or carriage across membranes with the bulk passage of water. This is supported by the overall data detailed below. Alpha-pinene is readily absorbed through the intestines (Budavari et al., 1996; cited in HSDB, 2009). D-limonene, and by analogy l-limonene, is rapidly and almost completely taken up from the gastrointestinal tract in humans as well as in animals (Igimi et al., 1974; Kodama et al., 1976, cited in CICAD, 1998). Infusion of labeled d-limonene into the common bile duct of volunteers revealed that the chemical was very poorly absorbed from the biliary system (Igimi et al., 1991). However, two acute oral gavage toxicity studies conducted with Eucalyptus globulus oil identified no evidence of systemic toxicity, i.e. neither mortality nor clinical/macroscopic effects (LD50 ≥ 3320 mg/kg bw). The Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test using the oral route (gavage) showed a NOAEL of 300 mg/kg bw/day) based on systemic effects observed in pregnant female of the highest dose group. This value was based on a significant lower bodyweight gain of the dams within the gestation together with a significant lower food consumption that appeared associated with pregnancy status. It was not possible to link this effect to the taste of the substance since females had shown a significant duration of normal bodyweight and food performance prior to Day 6 of gestation and after birth of the pups. These latter observations appeared to indicate recovery in females. In addition males were not affected throughout the treatment period and therefore, the highest dose tested was considered as the NOAEL in males. Regarding the offspring, a significant lower body weight of the pups was observed at the highest dose during lactation. This effect may be associated with test material entering the milk. Indeed, fat soluble test materials have a higher chance of becoming incorporated in the milk and Eucalyptus globulus oil is fat soluble. However, a NOAEL at 300 mg/kg bw/d was determined for systemic effect in the offspring based on the magnitude of the weight reduction which was quite high. In any case, the effects on offspring body weight were not selective and have been observed at a dose producing maternal toxicity and therefore the substance was not considered to be a selective reproductive toxicant. Finally, the effects observed in the liver were considered to be a physiological adaptation and therefore non-adverse. The kidney effects were consistent with well-documented species-specific responses of the male rat in response to the administration with hydrocarbons. The lack of significant adverse findings following oral dosing may be due to limited gastrointestinal absorption of the test material and/or its metabolites, or to a very low inherent toxicity. However, the observation of systemic effects, even if of very low toxicological concern, indicates the oral bioavailability of Eucalyptus globulus oil and/or its metabolites.

Dermal absorption:

Dermal absorption of 1,8-cineol is expected to be significant based its log Pow and water solubility. (d,l)-limonene and alpha-pinene being more lipophilic (log Kow = 4.5), their rate of uptake into the stratum corneum is expected to be high while their rates of penetration is likely to be limited by their rate of transfer between the stratum corneum and the epidermis. It is assumed that the dermal uptake is also limited by their slight water solubility. However, (d,l)-limonene and alpha-pinene being both identified as skin sensitiser, some uptake must occur although it may only be a small fraction of the applied dose. Enhanced penetration might also be expected since both substances are skin irritants. Results demonstrate rapid penetration of alpha-pinene not only to the first stratum corneum layers but also to viable epidermis and dermis. In an in vitro study, 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 shaved mice, the dermal absorption of [3H] d/l-limonene from bathing water was rapid, reaching the maximum level in 10 minutes (von Schäfer & Schäfer, 1982). In one study (one hand exposed to 98% d-limonene for 2 hours), the dermal uptake of d-limonene in humans was reported to be low compared with that by inhalation (Falk et al., 1991, cited in CICAD, 1998); however, quantitative data were not provided. Nevertheless, the single-dose dermal application of Eucalyptus globulus oil resulted in no manifestations of systemic toxicity (LD50 > 5000 mg/kg bw). The lack of adverse findings following dermal dosing may be due to limited dermal absorption of Eucalyptus globulus oil constituents and/or their metabolites, or to a very low inherent systemic toxicity of Eucalyptus globulus oil constituents and/or their metabolites.

Respiratory absorption:

The potential for inhalation toxicity of Eucalyptus globulus oil was not evaluated in vivo. 1,8-cineol is not volatile. Physico-chemical properties of alpha-pinene indicate that it may be available as a vapour can be taken up by micellular solubilisation and reach the lower respiratory tract. Average pulmonary uptake of (+)-alpha-pinene and (-)-alpha-pinene amounted to 59% of the exposure concentration following humans exposure 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). Absolute uptake increased linearly with concentration. D-Limonene has a high partition coefficient between blood and air and is easily taken up in the blood at the alveolus (Falk et al., 1990, cited in CICAD, 1998). The net uptake of d-limonene in volunteers exposed to the chemical at concentrations of 450, 225, and 10 mg/m3 for 2 hours during light physical exercise averaged 65% (Falk Filipsson et al., 1993, cited in CICAD, 1998).

Distribution:

1,8-cineol is expected to readily diffuse through aqueous channels and pores and to be distributed into cells. Considering that (d,l)-limonene and alpha-pinene are highly lipophilic (log Pow >4) and slightly water soluble, it is suggested that, upon systemic absorption, they may be transported through the circulatory system in association with a carrier molecule such as a lipoprotein or other macromolecule. Afterwards, based on their lipophilic character, both substances will readily cross cellular barriers or will be distributed into fatty tissues with a low potential to accumulate. Following human inhalation exposure, blood alpha-pinene concentration increased rapidly at first then tapered off (Falk et al., 1990, cited in HSDB, 2009). Mean blood concentration at the end of exposure was linearly related to inhaled concentration. D-Limonene is rapidly distributed to different tissues in the body and is readily metabolized. Clearance from the blood was 1.1 litre/kg bw per hour in males exposed for 2 hours to d-limonene at 450 mg/m3 (Falk Filipsson et al., 1993, cited in CICAD, 1998). A high oil/blood partition coefficient and a long half-life during the slow elimination phase suggest high affinity to adipose tissues (Falk et al., 1990; Falk Filipsson et al., 1993, cited in CICAD, 1998). In rats, the tissue distribution of radioactivity was initially high in the liver, kidneys, and blood after the oral administration of [14C] d-limonene (Igimi et al., 1974, cited in CICAD, 1998); however, negligible amounts of radioactivity were found after 48 hours. Differences between species regarding the renal disposition and protein binding of d-limonene have been observed. For rats, there is also a sex-related variation (Lehman-McKeeman et al., 1989; Webb et al., 1989, cited in CICAD, 1998). The concentration of d -limonene equivalents was about 3 times higher in male rats than in females, and about 40% was reversibly bound to the male rat specific protein, alpha-2-microglobulin (Lehman-McKeeman et al., 1989; Lehman-McKeeman & Caudill, 1992, cited in CICAD, 1998)

Metabolism:

The results of the repeated oral toxicity study in the rat showed liver changes that are consistent with the increased metabolism associated with detoxification of a xenobiotic. Moreover, the liver induction confirmed that a non-negligible part of Eucalyptus globulus oil can be absorbed in gastrointestinal tract. 1,8-cineol undergoes oxidation in vivo with the formation of hydroxycineole which is excreted as glucuronide (Williams, 1959 cited in SCF, 2002). In rats, 2-hydroxycineole, 3-hydroxycineole and 1,8-dihydroxycineol-9-oic acid were identified as main urinary metabolites (Madyastha and Chadha, 1986). After oral administration to brushtail possums (Trichosurus vulpecula), p-cresol, 9-hydroxycineole and cineol-9-oic acid were found in urine (Southwell et al., 1980, cited in SCF, 2002). Rabbits given eucalyptol by gavage excreted 2-exo- and 2-endo-hydroxycineole as well as 3-exo- and 3-endo-hydroxycineole in the urine (Miyazawa et al., 1989) The biotransformation of (+)-, (-)-, and (+/-)-alpha-pinenes was investigated in rabbits. The major metabolite was (-)-trans-verbenol (Ishidada, 1981, cited in HSDB, 2009). The biotransformation of d-limonene has been studied in many species, showing metabolic differences between species with respect to the metabolites present in both plasma and urine. About 25–30% of an oral dose of d-limonene in humans was found in urine as d -limonene-8,9-diol and its glucuronide; about 7–11% was eliminated as perillic acid (4-(1-methylethenyl)-1-cyclohexene-1-carboxylic acid) and its metabolites (Smith et al., 1969; Kodama et al., 1976, cited in CICAD, 1998). D-Limonene-8,9-diol is probably formed via d-limonene-8,9-epoxide (Kodama et al., 1976; Watabe et al., 1981, cited in CICAD, 1998). In another study, perillic acid was reported to be the principal metabolite in plasma in both rats and humans (Crowell et al., 1992, cited in CICAD, 1998). Other reported pathways of limonene metabolism involve ring hydroxylation and oxidation of the methyl group (Kodama et al., 1976, cited in CICAD, 1998).

Excretion:

The three major constituents of Eucalyptus globulus oil having a molecular weight lower than 300, they are expected to be mainly excreted in urine and no more than 5-10% may be excreted in bile as described in humans (see § absorption) . Urinary excretion is supported by microscopic kidney changes identified as hyaline droplet nephropathy in the kidneys, accompanied by tubular casts and/or tubular degeneration/regeneration in treated males, together with increased kidney weights in males. Any substance that is not absorbed from the gastro-intestinal tract, following oral ingestion, will be excreted in the faeces. Alpha-pinene is also excreted as vapours in exhaled air. 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 2009). Similarly, the renal elimination of verbenols after experimental exposure to (+) 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 2009). Following the inhalation exposure of volunteers to d-limonene at 450 mg/m3 for 2 hours, three phases of elimination were observed in the blood, with half-lives of about 3, 33, and 750 minutes, respectively (Falk Filipsson et al., 1993, cited in CICAD, 1998). About 1% of the amount taken up was eliminated unchanged in exhaled air, whereas about 0.003% was eliminated unchanged in the urine. When male volunteers were administered (per os) 1.6 g [14C] d-limonene, 50–80% of the radioactivity was eliminated in the urine within 2 days (Kodama et al., 1976, cited in CICAD, 1998). Limonene has been detected, but not quantified, in breast milk of non-occupationally exposed mothers (Pellizzari et al., 1982, cited in CICAD, 1998).

REFERENCES:

[CICAD, 1998] Falk Filipsson, A., 1998. Concise International Chemical Assessment Document 5 - Limonene, 32 p.Chapitre 7. Comparative kinetics and metabolism in laboratory animals and humans.

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

[SCF, 2002] Opinion of the Scientific Committee on Food on eucalyptol (expressed on 17 April 2002). European Commission. SCF/CS/FLAV/FLAVOUR/20 ADD2 Final.