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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

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
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2016

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
(R)-p-mentha-1,8-diene
EC Number:
227-813-5
EC Name:
(R)-p-mentha-1,8-diene
Cas Number:
5989-27-5
Molecular formula:
C10H16
IUPAC Name:
4-isopropenyl-1-methylcyclohexene
Test material form:
liquid
Radiolabelling:
no

Test animals

Species:
human
Sex:
male/female

Administration / exposure

Route of administration:
oral: capsule
Duration and frequency of treatment / exposure:
Single dose
Doses / concentrations
Dose / conc.:
9.3 other: mg
Remarks:
±0.4
No. of animals per sex per dose / concentration:
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

Results and discussion

Main ADME resultsopen allclose all
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.

Toxicokinetic / pharmacokinetic studies

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.

Metabolite characterisation studies

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.

Any other information on results incl. tables

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 

Applicant's summary and conclusion

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.