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Reference
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:
reference to same study
Reason / purpose:
reference to other study
Objective of study:
excretion
metabolism
Principles of method if other than guideline:
Metabolism and excretion of d-limonene was studied in volunteers.
GLP compliance:
no
Radiolabelling:
no
Species:
human
Sex:
male/female
Route of administration:
oral: capsule
Duration and frequency of treatment / exposure:
Single dose
Dose / conc.:
9.3 other: mg
Remarks:
±0.4
No. of animals per sex per dose:
Four healthy human volunteers
Details on dosing and sampling:
TEST ITEM ADMINISTRATION
- Four healthy human volunteers (3 men and 1 woman, mean age 33 ± 11 years, mean body weight 80 ± 8 kg) were orally exposed to 9.3 ± 0.4 mg (68 ± 3 μmol, M = 136.23 mg/mmol) via spiked gelatin capsule.
- The volunteers ingested the capsule in the morning on an empty stomach, directly after the collection of one pre-exposure urine and blood samples. After the exposure, they fasted for 1 h. During the remaining time of the experiment, they were allowed to eat and drink normally. However, they were encouraged to avoid food rich in limonene content (citrus fruits, lemon-flavoured foods and drinks).

METABOLITE CHARACTERISATION STUDIES
In frequent intervals within 24h, urine samples were collected by every volunteer. Blood samples were drawn hourly from two volunteers until 5h post-exposure. 6 samples were collected from each of the two volunteers.
- Storage: all samples were stored frozen at −20 °C until analysis.
- Method type(s) for identification:
R-limonene analysis in blood (HS-GC-MS): The samples were analysed using headspace gas chromatography-mass spectrometry (HS-GC-MS). Three ion traces were detected by selected ion monitoring (SIM): m/z 136 was used as quantifier ion, and m/z 93 and 68 were used as qualifier ions. The limit of detection and quantification was 4 µg/L and 12 µg/L, respectively. Assessment of spiked quality control samples revealed a precision range of 3-7% and an accuracy of 91-94%.

R-limonene metabolite analysis in blood and urine by Gas chromatographic–mass spectrometry (GC–PCI–MS/MS): Urine samples were analysed according to the procedure of Schmidt et al. (2013). Following this procedure, the urine samples were enzymatically hydrolysed for 12 h with beta-glucuronidase/arylsulphatase, acidified with HCl, and extracted by solid-supported liquid-liquid extraction using dichloromethane. After sylilation with BSTFA and TSIM, the extracts were analysed by GC–PCI–MS/MS. For identification of unknown limonene metabolites, pre-exposure urine samples and urine samples with maximum metabolite excretion rates were analysed with GC-PCI-MS full scan. Additional peaks which became apparent in the post-exposure samples were analysed regarding their PCI-MS mass spectra.
Statistics:
None
Type:
metabolism
Results:
cis- and trans-carveol, perillic acid, (1S, 2S, 4R)-limonene-1,2-diol (LMN-1,2-OL) and limonene-8,9-diol (LMN-8,9-OL) were detected at much higher concentrations in urine than in blood
Type:
excretion
Results:
Human limonene metabolism proceeds fast and the body is almost entirely cleared of metabolites within 24 h post-oral exposre.
Details on excretion:
- The whole process of uptake and elimination is almost finished within 10h after exposure due to the short elimination half-lives of metabolites.
- Human limonene metabolism proceeds fast and the body is almost entirely cleared of metabolites within 24 h post-oral exposre.
Metabolites identified:
yes
Details on metabolites:
- None of the volunteers reported adverse effects due to the exposure. But all mentioned a distinct smell of the exhaled breath, which emerged about 1h post-exposure and vanished about 2-3 h after exposure.
- unmetabolised R-limonene was not detected in blood and urine.
- Metabolites concentrations were much higher in urine than in blood.
- An additional metabolite could be identified in urine which would most likely be an isomer of dihydroperillic acid (DHPA).
The absence of limonene associated with the clear presence of metabolites in blood is evidence of a rapid hepatic or intestinal first-pass metabolism.
Human in vivo metabolism of limonene is characterised by oxidation reactions on the allylic methyl side chain yielding in carboxylic acids, whereas the endocyclic oxidation plays a minor role. In contrast to the bicyclic terpenes, limonene offers an exocyclic double bond, whose oxidation is by far the most prominent metabolism pathway.

Table 7.1.1/1: blood kinetics of R-limonene metabolites after oral exposure

   

 

Cmax

(µg/L)

Cmax

(nM)

Tmax

(h)

T1/2

(h)

AUC5h

(nM x h)

Cis-carveol

0.6

4

1

1

2

Trans-carveol

0.3

2

1

1

3

Perillyl alcohol

<LOD

-

-

-

-

Perillic acid

5.0

45

2

4

27

LMN-1,2-OH

10.8

64

2

4

133

LMN-8,9-OH

27.0

158

2

2

265

 

Table 7.1.1/2: renal elimination kinetics of R-limonene metabolites after oral exposure

 

RE,max

(µg/h)

Tmax

(h)

T1/2

(h)

Kel

(h-1)

AUC24h

(µmol)

Fraction of oral dose (%)

Conjugation rate (%)

Cis-carveol

6.8 ± 0.9

0.9 ± 0.5

0.9 ± 0.1

0.801

0.1 ± 0.1

0.2 ± <0.1

88-94

Trans-carveol

15 ± 6.4

0.8 ± 0.5

0.7 ± 0.1

1.005

0.2 ± 0.1

0.2 ± <0.1

93-99

Perillyl alcohol

0.8 ± 0.1

2.7 ± 2.5

1.2 ± 0.1

0.572

0.1 ± <0.1

<0.1 ± <0.1

79-83

Perillic acid

80 ± 24

1.5 ± 0.7

1.9 ± 0.2

0.374

1.4 ± 0.2

2.0 ± <0.1

91-96

LMN-1,2-OH

110 ± 43

1.7 ± 0.7

2.5 ± 0.1

0.273

2.9 ± 0.6

4.3 ± 0.9

81-96

LMN-8,9-OH

1400 ± 460

1.5 ± 0.7

1.6 ± 0.1

0.439

22 ± 2.6

32 ± 4.0

99-100

RE,max: maximal renal excretion rate ;Tmax: time to RE,max;T1/2:elimination half-life;Kel:elimination rate constant; AUC24h: area under the mean renal elimination versus time curve (24h)

Table 7.1.1/3: Relative proportions of metabolites in urine

Relative % of metabolites in urine

 Carveol (Cis+trans)

 0.2

Perillyc acid + peryllil alcohol + DHPA

 18

 LMN-1,2 -OH

 10

 LMN-8,9 -OH

 72

Table 7.1.1/4: Relative proportions of metabolites related to the dose applied

 

Relative % of metabolites

 Carveol (Cis+ trans)

0.4 -0.5 

 Perillyl alcohol

<LOD 

 Perillic acid

1.7 -2.5 

 DHPA

 LMN-1,2 -OH

 3.4 -5.5

 LMN-8,9 -OH

29.2 -31.9 

Conclusions:
Human in vivo metabolism of d-limonene is characterised by oxidation reactions on the allylic methyl side chain yielding in carboxylic acids, whereas the endocyclic oxidation plays a minor role. In contrast to the bicyclic terpenes, d-limonene offers an exocyclic double bond, whose oxidation is by far the most prominent metabolism pathway. Human d-limonene metabolism proceeds fast and the body is almost entirely cleared of metabolites within 24 h post-oral exposure.
Executive summary:

In a metabolism study, four healthy human volunteers were orally exposed to a single dose of 9.3 mg of d-limonene 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 from two volunteers were also collected within 5h post-administration. d-Limonene was analysed with HS-GC-MS in blood. d-Limonene in urine and its metabolites in blood and urine were analysed with GC-PCI-MS/MS.

None of the volunteers reported adverse effects due to the exposure. But all mentioned a distinct smell of the exhaled breath, which emerged about 1h post-exposure and vanished about 2-3 h after exposure. Unmetabolised d-limonene was not detected in blood and urine. cis- and trans-carveol, perillic acid, (1S, 2S, 4R)-limonene-1,2-diol (LMN-1,2-OL) and limonene-8,9-diol (LMN-8,9-OL) were detected in blood and urine but not perillyl alcohol. Metabolites concentrations were much higher in urine than in blood. The whole process of uptake and elimination is almost finished within 10h after exposure due to the short elimination half-lives of metabolites. The absence of limonene associated with the clear presence of metabolites in blood is evidence of a rapid hepatic or intestinal first-pass metabolism. Thus, human in vivo metabolism of d-limonene is characterised by oxidation reactions on the allylic methyl side chain yielding in carboxylic acids, whereas the endocyclic oxidation plays a minor role. In contrast to the bicyclic terpenes, d-limonene offers an exocyclic double bond, whose oxidation is by far the most prominent metabolism pathway.

Therefore, human d-limonene metabolism proceeds fast and the body is almost entirely cleared of metabolites within 24 h post-oral exposre.

Description of key information

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
100
Absorption rate - inhalation (%):
100

Additional information

ABSORPTION:

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). 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). Orally administered d-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). Infusion of labelled 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). 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); however, quantitative data were not provided.

DISTRIBUTION:

d-Limonene is rapidly distributed to different tissues in the body and is readily metabolized. Limonene has been detected, but not quantified, in breast milk of non-occupationally exposed mothers (Pellizzari et al., 1982).

EXCRETION:

Clearance from the blood was 1.1 litre/kg body weight per hour in males exposed for 2 hours to d-limonene at 450 mg/m3 (Falk Filipsson et al., 1993). 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). 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); 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). 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, "2:-globulin (Lehman-McKeeman et al., 1989; Lehman-McKeeman & Caudill, 1992). 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). In a recent metabolism study in human volunteers orally exposed to a single dose of 9.3 mg R-limonene, the whole process of uptake and elimination was almost finished within 10h after exposure due to the short elimination half-lives of metabolites. Human limonene metabolism proceeds fast and the body is almost entirely cleared of metabolites within 24 h post-oral exposure (Schmidt et al., 2016).

METABOLISM:

The biotransformation of d-limonene has been studied in many species, with several possible pathways of metabolism. Metabolic differences between species have been observed 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). d-Limonene-8,9-diol is probably formed via d-limonene- 8,9-epoxide (Kodama et al., 1976; Watabe et al., 1981). In another study, perillic acid was reported to be the principal metabolite in plasma in both rats and humans (Crowell et al., 1992). Other reported pathways of limonene metabolism involve ring hydroxylation and oxidation of the methyl group (Kodama et al., 1976). 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). About 1% of the amount taken up was eliminated unchanged in exhaled air, whereas about 0.003% was eliminated unchanged in the urine. In a recent metabolism study in human volunteers orally exposed to a single dose of 9.3 mg R-limonene, unmetabolised R-limonene was not detected in blood and urine. cis- and trans-carveol, perillic acid, (1S, 2S, 4R)-limonene-1,2-diol (LMN-1,2-OL) and limonene-8,9-diol (LMN-8,9-OL) were detected in blood and urine but not perillyl alcohol. Metabolites concentrations were much higher in urine than in blood. The absence of limonene associated with the clear presence of metabolites in blood was evidence of a rapid hepatic or intestinal first-pass metabolism. Thus, human in vivo metabolism of limonene is characterised by oxidation reactions on the allylic methyl side chain yielding in carboxylic acids, whereas the endocyclic oxidation plays a minor role. In contrast to the bicyclic terpenes, limonene offers an exocyclic double bond, whose oxidation is by far the most prominent metabolism pathway (Schmidt et al., 2016).

See complete references in the monograph:

Falk Filipsson, A., 1998. Concise International Chemical Assessment Document 5 - Limonene, 32 p.

Chapitre 7. Comparative kinetics and metabolism in laboratory animals and humans.