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Toxicokinetic evaluation Spearmint oil based on existing data

REACH indicates that an “assessment of the toxicokinetic behaviour of the substance to the extent that can be derived from the relevant available information” should be performed at Annex VIII level.


1.    General

Spearmint oil is an NCS (Natural Complex Substance (NCS) . This essential oil is obtained from the aerial part of Mentha spicata and/or Mentha cardiaca (Lamiaceae) by distillation,  Spearmint oil is an UVCB consisting of the following identified constituents:

















Germacrene D




















Sabinene hydrate




























The botanical relationship for the grouping of these particular NCSs is based on:

a)    The part of the plant used as source for the NCS, namely in all cases the fresh, above ground parts of the flowering plant.

b)    The common methods of production (cutting, chopping and steam distillation and other purification processes; see IUCLID chapter 3)

c)    The same dominant constituents: l-carvone (55-85%) and l-Limonene (0.01 – 24.9%).

d)    Same minor constituents in the same ranges (β-myrcene, Terpinen-4-ol, 1,8-Cineole, β-bourbonene, trans-Dihydrocarvone, Germacrene D, Sabinene hydrate, 3-Octanol, α-pinene, γ-terpinene, β-pinene, Linalool)


The main dominant constituents are laevus-(-) -Carvone (55-85%) and laevus-(-)-Limonene (0.01 – 24.9%). Together with minor constituents β-myrcene and cineole they add up to more than 80% of the total composition of both species (83.3% and 86.9% for M. spicata and M. cardiaca, respectively) and can therefore be considered representative of the whole substance. The repeated dose toxicity and reproductive toxicity of dexter(+)-carvone and dexter (+)-limonene are used as source for read across to laevus (-)-carvone and laevus (-)-limonene respectively, the latter being enantiomers of the former and the same toxicological profile being expected. Dexter (+)-Limonene is structurally very similar to carvone and read-across can be used in some cases, for example as an indicator of likely absorption, distribution and excretion.


2.    ADME data

 An initial draft risk assessment report (DAR) on Spearmint Oil was published by a designated rapporteur Member State (RMS), Sweden, in 2008. As no ADME studies on Spearmint Oil have been conducted, several studies from the public literature were reported. These studies provide some more information on the main constituent carvone. Furthermore, in 2014 a safety assessment of carvone was published by the EFSA Scientific Committee, in the EFSA Journal 2014;12(7):3806. For the hazard identification and characterisation of d- and l-carvone, extensive literature searches were performed to retrieve experimental in-vitro and in-vivo studies on the metabolism and toxicokinetics for d- and l-carvone in test species (dog, rat, mice, rabbit) and humans. The results of these literature searches on the toxicokinetics and toxicity of d- and l-carvone are summarised below:


2.1. Toxicokinetics Carvone and Limonene

In vivo toxicokinetics data on d-or l- carvone in test species (rat, mice, rabbit, dog) have not been reported in the literature. Hence, basic toxicokinetic parameters for d- and l-carvone are not available for any routes of exposure (ECHA, 2013). In humans, d-carvone pharmacokinetics was investigated in one study performed in 15 male volunteers who, after a 10 h fast, took 5 capsules of an immediate release formulation containing 20 mg caraway oil. Carvone concentrations in plasma were determined by GC/MS, with a limit of quantification of 0.5 ng/ml for carvone. Pharmacokinetic parameters were determined i.e. area-under the plasma-concentration curve (AUC) of 28.9± 20.0, plasma peak concentration (Cmax) of 14.8±10.4 ng/ml with a time to reach Cmax (Tmax) of 1.3 hours and a half-life of 2.4 hours. Inter-individual differences determined as the coefficients of variation in AUC, Cmax and t1/2 were 69%,74% and 50% respectively (Mascher et al., 2001).


 It is expected that l-carvone is absorbed, distributed and excreted in a similar way to structurally similar d-limonene. After oral administration in rats, the latter is rapidly absorbed and distributed in blood serum, liver and kidneys, along with its metabolites (1-2 h), followed by excretion (48 h) in the urine (60%), faeces (5%) and expired air (2%). After topical administration in rats, partial absorption (48% of the radioactivity was recovered in the skin) and subsequent distribution in the gastro-intestinal tract (0.1-0.4% dose/g), liver and kidneys (0.08- 0.2% dose/g), and thyroid and fat (0.02-0.06% dose/g), is also rapid (3-6 h), followed by excretion (24-72 h) in the urine (8-12%), faeces (1-3%) and expired air (14-18%). Although the d-limonene absorption, distribution and excretion data are indicative for l-carvone, they provide no direct information on the percentage absorption of l-carvone after oral, dermal or inhalation exposure, the distribution of l-carvone or the rate and routes of elimination from the body.


2.2.  Carvone Metabolism


2.2.1.Human data

- The main metabolites of carvone in humans in vivo are carvonic acid, dihydrocarvonic acid and uroterpenolone, with carveol and dihydrocarveol as minor products (figure 1). However, the study did not assess the stereospecificity of the metabolism of carvone in humans (Engel, 2001)


 2.2.2.    Animal data

               - In rabbits carvone was metabolised to (+)-9-hydroxycarvone and dihydrocarveol, which are excreted in urine (Fischer & Bielig, 1940; Williams, 1959; Ishida et al., 1989).

- In rat, carveol is likely to be the main metabolite. The evidence from in vitro studies suggests that metabolic conversion of carvone to carveol in female rat liver is likely to be very slow compared to male rat. (Shimada et al.; 2002)

- Glucuronidation of stereospecific d- and l-carvone and their other metabolites (carvonic acid, dihydrocarvonic acid and uroterpenolone, dihydrocarveol) has not been studied. Though in mice, treatment with racemic carvone and, to a lesser degee, other alpha,ß-unsaturated ketones increased the activity of glutathione S-transferase in all tissues by two to four times over that in controls and in animals treated with other carvyl derivatives. Carvone intake was associated with a decrease in glutathione content in the liver, lung, and large-bowel mucosa (Zheng et al., 1992). Carvone rapidly conjugated with glutathione in the absence of glutathione S-transferase (Portoghese et al., 1989).


2.2.3.    In-Vitro

- The findings of the in vitro and in vivo studies indicate that carvone is metabolised differently in humans and rats.

- In in-vitro studies, P450 was found to act as a catalyst, however it was not observedin-vivoin rats. (Madyastha & Raj,1990; Moorthy et al., 1989).

- There are in vitro indications for stereoselective reduction of carvone to carveol and stereospecific conjugation (only l-carveol is glucuronidated and with a four-fold higher rate in the rat compared with humans) (Jaeger, 2000)


 2.2.4 In-Silico

Because of the limited availability of experimental data, the EFSA Scientific Committee also empoyed different in silico methods, including (Q)SAR models, expert systems, and read-across, to predict 1) the metabolic fate of carvone in mammals and 2) the metabolic half-life, intestinal uptake, bioaccumulation, and/or toxicity of the predicted metabolites. The aim was to investigate whether there are other intermediates or metabolites of carvone in rats and human that are not reported in the literature.

- The in silico assessments predicted that carvone is metabolised at different sites of the molecule in rats and humans, providing further supporting evidence to the in vivo and in vitro metabolism data. In addition, the in silico assessments also predicted that carvone and its metabolites are readily absorbed by the GI tract and have similar metabolic half-lives.


2.2.5.    Conclusions on toxicokinetics and metabolism of Carvone

d-Carvone toxicokinetics in humans features rapid elimination with a half-life of 2.4 hours, no data are available for l-carvone. No toxicokinetic data on carvone in animals are available. It is expected that l-carvone is absorbed, distributed and excreted in a similar way to structurally similar d-limonene. The evidence from in vivo, in vitro and in silico assessments has shown that carvone metabolism is likely to be different in humans and rats – with further possible differences between metabolism in male and female rats. It is also evident that when compared with carvone itself, the metabolites are not likely to be different in terms of GI uptake or half-life in the body. Toxicokinetic data on other monoterpenes in the rat such as menthol suggest that metabolism involves conjugation to a glucuronide for which enterohepatic recirculation occurs in the rat but not in humans. Considering the molecular weight of glucuronidated carvone metabolites, they may undergo enterohepatic recirculation in rats but not in humans, making the rat more sensitive than humans for these compounds.


2.3. Limonene Metabolism


2.3.1.    Human data

Wade et al. (1966) reported that dietary limonene gives rise to uroterpenol (M-II: p-menth-1-ene-8,9-diol) in the urine of humans.


2.3.2.    Animal data

In rats and rabbits, the major urinary metabolites of d-limonene were identified as perillic acid-8,g-diol (M-IV) perillyl-P-D-glucopyranosiduronicacid (M-IX) in hamsters, pmenth-l-ene-8,9-diol (M-II) in dogs, and 8-hydroxy-p-menth-l-en-9-yl-B-D-glucopyra-nosiduronic (M-VI) acid in guinea pigs and hu- mans (Figure 2). Kodama and coworkers iso- lated five new metabolites from dog and rat urine after oral administration of radiolabeled d-limonene: 2-hydroxy-p-menth-8-en-7-oic acid (M-VII), perillylglycine (M-VIII), peri1lyl-fl-D- glucopyranosiduronic acid (M-1x1, p-mentha- l,&diene-g-ol (M-X), and probably p-menth-l- ene-6,8,9-triol (M-XI). (Kodama et al.,1976).


2.3.3.    In-Vitro

In vitro incubation with rat liver microsomes resulted in metabolism of d-limonene to d-limonene-l,2-diol and d-limo- nene-8,g-diol; intermediate products were iden- tified as d-limonene-l,2-epoxide and d-limo- nene-8,9-epoxide (Watabe et al., 1980,1981).




 Attached: EFSA Scientific Committee, 2014. Scientific Opinion on the safety assessment of carvone, considering all sources of exposure. EFSA Journal 2014;12(7):3806 74 pp. doi:10.2903/j.efsa.2014.3806 Available online: © European Food Safety Authority, 2014

Attached Draft Assessment Report RMS Sweden, 2008, Initial risk assessment provided by the for the existing active substance SPEARMINT OIL of the fourth stage of the review programme referred to in Article 8(2) of Council Directive 91/414/EEC Volume 3, Annex B, part 2, B.6


Mascher H, Kikuta C and Schiel H, 2001. Pharmacokinetics of menthol and carvone after administration of an enteric coated formulation containing peppermint oil and caraway oil. Arzneimittelforschung, 51(6):465-9


MEYER, F. & MEYER, E., 1959. Absorption of ethereal oils and substances contained in them through the skin. Arzneimittelforschung (Drug Research), 9: 516-519.


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. Journal of Agricultural and Food Chemistry, 49, 4069-4075


Engel W, 2002. Detection of a "nonaromatic" NIH shift during in vivo metabolism of the monoterpene carvone in humans. Journal of Agricultural and Food Chemistry, 50, 1686-1694

(Fischer & Bielig, 1940; Williams, 1959; Ishida et al., 1989)


Shimada T, Shindo M and Miyazawa M, 2002. Species Differences in the Metabolism of (+)- and (-)-Limonenes and their Metabolites, Carveols and Carvones, by Cytochrome P450 Enzymes in Liver Microsomes of Mice, Rats, Guinea Pigs, Rabbits, Dogs, Monkeys, and Humans. European Journal of Drug Metabolism and Pharmacokinetics, 17(6): 507-515


Zheng GQ, Kenney PM and Lam LK, 1992. Anethofuran, carvone, and limonene: potential cancer chemopreventive agents from dill weed oil and caraway oil. Planta Medica, 58(4):338-41


National toxicology program (NTP), 1989. Toxicology and Carcinogenesis Studies of d-Carvone (CAS No. 2244-16-8) B6C3F1 Mice and Toxicology Studies in F344/N Rats (Gavage Studies). (NTP/TR No. 381, Draft; NIH No. 90-2836). Washington, D.C.: U.S. Department of Health and Human Services. Draft report submitted to WHO by Flavor and Extract Manufacturer's Association of the United States, Washington, D.C., USA.


Moorthy, B., Madyastha, P. & Madyastha, M., 1989. Hepatotoxicity of pulegone in rats: its effects on microsomal enzymes in vivo. Toxicol., 55, 327-337.


Jaeger W, Mayer M, Platzer P, Reznicek G, Dietrich H and Buchbauer G, 2000. Stereoselective Metabolism of the Monoterpene Carvone by Rat and Human Liver Microsomes, Journal of Pharmacy and Pharmacology, 52, 191-197


Wade, A.P., Wilkinson, G.S., Dean, F.M. & Price, A.W., 1966. The isolation, characterization and structure of uroterpenol, a monoterpene from human urine.Biochem. J., 101: 727-734.


Kodama R, Yano T, Furukawa K, Noda K and Ide H, 1976. Studies on the metabolism of d-limonene (p-mentha-1,8-diene). IV. Isolation and characterization of new metabolites and species differences in metabolism. Xenobiotica. 6(6):377-89


Watabe, T., Hiratsuka, A., Isobe, M. & Ozawa, N. (1980). Metabolism of d-limonene by hepatic microsomes to non-mutagenic epoxides toward Salmonella typhimurium.Biochemical Pharmacology, 29: 1068-1071.

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