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Reference
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:
other: No guideline available but good supporting information for method and results; no GLP compliance information. Supporting data for read-across justification (7.5.1; 7.8.2)
Objective of study:
metabolism
Qualifier:
no guideline available
Principles of method if other than guideline:
Metabolism of Ingestion-Correlated Amounts (MICA) Approach was used. Urine was collected for structural identification of metabolites on the basis of mass spectral analysis in combination with syntheses and NMR experiments (as indicated in OECD Guideline 417).
GLP compliance:
not specified
Radiolabelling:
no
Species:
human
Sex:
male/female
Route of administration:
other: oral
Vehicle:
other: Solution in full-fat milk
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: A single dose of carvone (Approximately 1 mg/kg of bodyweight) was ingested as a solution in full-fat milk (500 mL).

DIET PREPARATION
Volunteers received a controlled diet starting with lunch 24 h prior to the experiment to avoid uncontrolled intake of terpenes. Any intake of food,
sporting activity, and urine volumes were documented by the volunteers. The amounts were adjusted to the eating habits of each participant but were observed strictly throughout the following days of the experiment. Total diet period was 72 h. To avoid intake of terpenes from toothpaste, a terpene-free toothpaste was prepared

VEHICLE
- Justification for use and choice of vehicle (if other than water): Full-fat milk (administered in diet)
- Concentration in vehicle: 0.5 mmol
Duration and frequency of treatment / exposure:
One dose
Remarks:
Doses / Concentrations:
Approximately 1 mg/kg bodyweight
No. of animals per sex per dose / concentration:
3 males and 3 females (human)
Control animals:
other: After 24 h of diet adjustment, within the next 24 h the diet was continued and the 24 h urine (control) was collected
Details on dosing and sampling:
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled (delete / add / specify): urine
- Time and frequency of sampling: 24h after dosing
- From how many animals: 6 human samles; not pooled
- Method type(s) for identification: HRGC-MS, HRGC-High-Resolution MS, NMR.
- Limits of detection and quantification: No quanitifcation performed


Metabolites identified:
yes
Details on metabolites:
Major metabolites:
α,4-dimethyl-5-oxo-3-cyclohexene-1-acetic acid (dihydrocarvonic acid; M1) [Mass Spectra of Metabolites M1, M2, M3 - Figure 2];
α-methylene-4-methyl-5-oxo-3-cyclohexene-1-acetic acid (carvonic acid; M2) [Mass Spectra of Metabolites M1, M2, M3 - Figure 4],
5-(1,2-dihydroxy-1-methylethyl)-2-methyl-2-cyclohexen-1-one (uroterpenolone; M3) [Mass Spectra of Metabolites M1, M2, M3 - Figure 6].

Minor metabolites: carveol (M4) and dihydrocarveol (M5) (Not shown)

No quanitifcation was performed.

Study report attachments:

Mass Spectra of Metabolites M1, M2 M3 (Engel, 2001)

Figure 8 Proposed metabolism scheme (Engel, 2001)

Conclusions:
Interpretation of results (migrated information): other: No differences in metabolism between S-(+)- and R-(-)-carvone were detected.
The major in vivo metabolites of S-(+)- andR-(-)-carvone in a metabolism of ingestion correlated amounts (MICA) experiment were newly identified as α,4-dimethyl-5-oxo-3-cyclohexene-1-acetic acid (dihydrocarvonic acid), α-methylene-4-methyl-5-oxo-3-cyclohexene-1-acetic acid (carvonic acid), and 5-(1,2-dihydroxy-1-methylethyl)-2-methyl-2-cyclohexen-1-one (uroterpenolone) on the basis of mass spectral analysis in combination with syntheses and NMR experiments. Minor metabolites were identified as carveol and dihydrocarveol.
Executive summary:

In a metabolism study (Metabolism of ingestion-correlated amounts (MICA); Engel, 2001), S-(+) and R-(-)-carvone was administered to 3 human males/females in a single oral dietary dose of approximately 1 mg kg/bw.

All metabolites were identical after the application of either S-(+)-carvone or R-(-)-carvone. Additionally, no qualitative differences were observed among the six samples. The major metabolites were: α,4-dimethyl-5-oxo-3-cyclohexene-1-acetic acid (dihydrocarvonic acid), α-methylene-4-methyl-5-oxo-3-cyclohexene-1-acetic acid (carvonic acid), and 5-(1,2-dihydroxy-1-methylethyl)-2-methyl-2-cyclohexen-1-one (uroterpenolone). The minor metabolites were carveol and dihydrocarveol. No quantification of metabolites was performed.

This metabolism study in the humans is classified acceptable. Structural identification of metabolites on the basis of mass spectral analysis in combination with syntheses and NMR experiments was performed (as indicated in OECD Guideline 417).

Description of key information

L-carvone is a mono constituent organic liquid with a concentration range of 97%-100% and impurities present at 0%-3%. No impurity is present above 1%. A full ADME toxicokinetic study in the rat is not available. An in vivo study on metabolism in humans of L- and D-carvone is available (Metabolism of ingestion-correlated amounts (MICA) study).  Based on the physicochemical properties and information in the dossier (L-carvone studies and D-carvone read across study), L-carvone is expected to be absorbed after oral, inhalatory and dermal exposure and widely distributed. There is data to suggest the same metabolic pathway and metabolites for L-carvone and D-carvone. The default absorption rates of 50% (oral and dermal) and 100% (inhalation) are accepted for chemical risk assessment purposes.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
50
Absorption rate - dermal (%):
50
Absorption rate - inhalation (%):
100

Additional information

Physicochemical properties

In accordance with the ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7C Section R.7.12 (Endpoint Specific Guidance), the physicochemical properties can provide an insight into the potential behaviour of L-carvone in the body. D-carvone has the same physicochemical properties (within acceptable laboratory variation for measured values) as L-carvone.

Absorption

The molecular weight (150.2 g/mol) and octanol/water partition coefficient (log Kow) of L-carvone (2.74 at 37°C) favour absorption via passive diffusion. Based on the chemical structure, L-carvone has no groups which will dissociate in a relevant pH range (2-10) so is likely to be present in the body in a non-ionised form. These characteristics will facilitate transport of L-carvone across lipid cell membranes and therefore oral absorption. Due to the low vapour pressure of L-carvone (15.3Pa at 25°C; boiling point 231°C) respiratory exposure is expected to be low. L-carvone is slightly soluble in water (47.2 mg/L at 14°C) and has a moderate log Kow value which indicates favourable absorption directly across the respiratory tract epithelium by passive diffusion. The physical state of L-Carvone, low vapour pressure (15.3 Pa at 25°C), molecular weight (150.2 g/mol), water solubility (47.2 mg/L at 14°C) and moderate log Kow (2.74 at 37°C) are indicative of favourable dermal absorption.

Distribution/Metabolism

A wide distribution of L-carvone is favourable as it has a relatively low molecular weight, moderate lipophilicity and is present in a non-ionised form in the body. Based on the log Kow value (2.74 at 37°C), L-carvone is unlikely to accumulate with intermittent exposure. The physicochemical properties of L-carvone suggest it can cross the placenta. Based on the chemical structure, there are several possibilities for metabolic transformation e.g. Phase I reduction of the ketone followed by Phase II conjugation of the resulting alcohol with glucuronic acid or Phase I side-chain oxidation, which may be conjugated during Phase II to yield polar metabolites. The MICA study indicates a proposed metabolic scheme in humans (see below).

Information from other studies in the dossier and public literature

Absorption (oral)

Clinical effects were noted in the acute oral toxicity study in CFY Sprague-Dawley rats (OECD 401/GLP) with L-carvone at 3.2, 5.0, and 8.0 g/kg bw (LD50 (male/female): 5400 mg/kg bw (4600 - 6300 mg/kg bw; 95% C.I).

In the subchronic repeated dose toxicity study in B6C3F1 mice, (equivalent or similar to OECD 408; read across from d-carvone), the relative liver weights for male and female mice that received 750 mg/kg were significantly greater than those for vehicle controls. There were no other test substance-related effects in body weight, weight gain, organ weight or histopathology. The NOAEL (male/female) was 375 mg/kg bw/day (equivalent or similar to OECD 408).

In a pre-natal developmental toxicity study in Wistar CRL rats (OECD 414/GLP) with L-carvone, there was evidence of maternal toxicity at the highest dose, 500 mg/kg bw/day (clincal signs, decrease in body weight) but not at the low (125 mg/kg bw/day) or mid doses (250 mg/kg bw/day). There were no changes in reproduction parameters in treated groups. The examination of foetuses revealed a statistically significant decrease in the mean body weights at the dose 500 mg/kg b.w./day.  The decreased weight of foetuses could be associated with a negative influence of the test item on the growth of maternal animals. Examination of foetal skull and skeleton revealed anomalies in all treated groups. The incomplete ossification of the supraoccipital bone, could be associated with lower foetal body weight, especially at the highest dose level. Equally, transitional findings in the foetal skull, such as bipartite ossification of the supraoccipital bone and unossified supraoccipital bone could be associated with maternal toxicity, due to incidence at the dose 500 mg/kg b.w./day only. The increased incidence of transitional findings in all treated groups in comparison with control group – mainly hole in supraoccipital bone, asymmetric ossification of sternebra and bipartite and dumbbell ossification of vertebrae could be considered as adverse effect of the test item to foetuses, because no maternal toxicity was observed at the 125 and 250 mg/kg b.w./day dose levels. The malformations of ribs (absent) were observed only in treated groups. The increased incidence of transitional findings (in foetuses of treated females compared to control females) could be considered as an adverse effect of the test item to early prenatal development of organism in uterus. The effect of the test item on the incidence of malformation of ribs in treated groups is disputable. In a pre-natal developmental toxicity study in rats, the NOAEL for toxicity in pregnant females was 250 mg/kg b.w./day. The NOAEL for prenatal development is < 125 mg/kg b.w./day. The dose level 125 mg/kg b.w./day can be treated as the LOAEL in this study.

Based on these studies, there is evidence of oral absorption both acutely and on repeated exposure. For chemical safety assessment purposes, based on the physicochemical properties and information in the dossier, a default oral absorption of 50% is accepted.

Absorption (dermal)

A skin sensitisation study in the mouse indicated that L-Carvone is classified for skin sensitisation, which suggests that some systemic exposure occurs; no systemic toxicity was noted in the acute dermal toxicity study. The ECHA guidance criteria (Chapter R.7C) state that 10% dermal absorption is used when the molecular weight of the substance is >500 and the log Pow is <-1 or >4, otherwise 100% dermal absorption is used. In general, dermal absorption will not be higher than oral absorption, so for chemical safety assessment purposes a dermal absorption rate of 50% is accepted.

Absorption (inhalation)

There are no inhalation studies available for the substance. For chemical safety assessment purposes, based on the physicochemical properties, the default inhalation absorption rate of 100% is accepted.

Distribution/Metabolism

From the D-carvone repeated dose oral toxicity study, there is no evidence of specific target organ toxicity (liver weight changes in the 13 week study were not accompanied by histological alterations); it is assumed that similar distribution for L-carvone occurs. Based on the results of the pre-natal developmental toxicity study, L-carvone crosses the placenta and is exposed to the developing foetus.

An in vivo study on the metabolism of L- in humans using the MICA approach was carried out (Engel, 2001). No quantification of metabolites in the urine samples was performed. The major metabolites identified by GC-MS and NMR were α,4-dimethyl-5-oxo-3-cyclohexene-1-acetic acid (dihydrocarvonic acid), α-methylene-4-methyl-5-oxo-3-cyclohexene-1-acetic acid (carvonic acid), and 5-(1,2-dihydroxy-1-methylethyl)-2-methyl-2-cyclohexen-1-one (uroterpenolone). The minor metabolites were carveol and dihydrocarveol. The proposed metabolic pathways for L-carvone in humans is oxidation of the double bond in the side chain and to a minor extent 1,2- and 1,4 + 1,2-reduction of L-carvone.

Engel W (2001) In vivo studies on the metabolism of the monoterpenes S-(+)- and R-(-)-carvone in humans using the metabolism of ingestion-correlated amounts (MICA) approach. J Agric Food Chem. 2001 Aug;49(8):4069-75.