Carvacrol

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Objective of study:

metabolism

Qualifier:

no guideline available

Principles of method if other than guideline:

The aim of the study was to investigate the role of cytochrome P450s (CYPs) in the metabolism of thymol and carvacrol. After incubation with human liver microsomes (HLMs) in the presence of NADPH, a new metabolite and two metabolites were detected for thymol and carvacrol, respectively. A combination of chemical inhibition studies and assays with recombinant CYP isoforms was used to investigate what aret the predominant drug-metabolizing enzyme involved in the metabolism of thymol and carvacrol.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Carvacrol; Aladdin Corp. (Shanghai, China)
- Purity: 99%

- Source of test material: Thymol; Aladdin Corp. (Shanghai, China0
- Purity: 99%

Radiolabelling:

no

Type:

metabolism

Results:

CYP2A6 was demonstrated to be the major drug-metabolizing enzyme involved in the metabolism of thymol and carvacrol.

Metabolites identified:

no

Details on metabolites:

After incubation with HLMs in the presence of NADPH, a new metabolite and two metabolites were detected for thymol and carvacrol, respectively. These metabolies were not identified.

Detection of thymol and carvacrol metabolites in HLM

After incubation of thymol with HLMs and NADPH-generating system, a new peak was eluted at 14.5 min (M) for thymol (Fig. 2A). This new peak was not detected in the negative controls without NADPH, without substrate, and without microsomes. For the metabolism of carvacrol, two new peaks were eluted at 14.7 min (M-1) and 15.3 min (M-2) (Fig. 2B). These two new peaks were not detected in the negative controls without NADPH, without substrate, and without microsomes.

Kinetic study

Under the experimental conditions used, the metabolism of thymol in HLMs obeyed typical Michaelis-Menten kinetics, as evidenced by Eadie-Hofstee plots (Fig. 3A). The kinetic parameters (apparent Vmax and Km) were calculated to be 0.58±0.02 nmol/min/mg pro and 19.8±2.2µM. The metabolism of carvacrol in HLMs obeyed the typical Michaelis-Menten kinetics, as evidenced by Eadie-Hofstee plots (Fig. 3B and Fig. 3C).The kinetic parameters (Km and Vmax) were 9.8µM and 0.78 nmol/min/mg pro for M-1, and 9.3 µM and 0.37 nmol/min/mg pro for M-2.

Chemical inhibition study

The effect of various chemical inhibitors on the metabolism of thymol was investigated in pooled HLMs (Fig. 4A). ABT, the broad CYP inhibitor, strongly inhibited the formation of thymol metabolite, suggesting that CYPs were the major drug-metabolizing enzymes involved in the metabolism of thymol. Among the selective inhibitors of nine CYP iso- forms, 8-methoxypsolaren (the selective inhibitor of CYP2A6) almost completely inhibited the formation of thymol metabolite. As shown in Fig. 4B, ABT, the broad CYP inhibitor, strongly inhibited the formation of carvacrol metabolites (M-1, M-2), suggesting that CYPs were the major drug metabolizing enzymes involved in the metabolism of carvacrol. 8-methoxypsolaren (the selective inhibitor of CYP2A6) almost completely inhibited the formation of M-1 and M-2.

Assay with human recombinant CYP isoforms

Nine recombinant CYP isoforms were employed to identify the CYP isoforms involved in the metabolism of thymol and carvacrol. The results (Fig. 5A) showed that CYP1A2, CYP2A6 and CYP2B6 could catalyze the formation of thymol metabolite. The levels of involvement of other CYP isoforms in the metabolism of thymol were negligible. For the metabolism of carvacrol, the results (Fig. 5B) showed that CYP1A2, and CYP2A6 could catalyze the formation of M-1, and CYP1A2, CYP2A6 and CYP2B6 could catalyze the formation of M-2. The levels of involvement of other CYP isoforms in the metabolism of carvacrol were negligible.

Conclusions:

After incubation with human liver microsomes (HLMs) in the presence of NADPH, a new metabolite and two metabolites were detected for thymol and carvacrol, respectively. These metabolites were not identified. A combination of chemical inhibition studies and assays with recombinant CYP isoforms demonstrated that CYP2A6 was the predominant drug-metabolizing enzyme involved in the metabolism of thymol and carvacrol.

Executive summary:

In an vitro enzyme assay (Dong et al., 2012), the role of cytochrome P450s (CYPs) in the metabolism of thymol and carvacrol was investigated.

After incubation with human liver microsomes (HLMs) in the presence of NADPH, a new metabolite and two metabolites were detected for thymol and carvacrol, respectively. These metabolites were not identified. A combination of chemical inhibition studies and assays with recombinant CYP isoforms demonstrated that CYP2A6 was the predominant drug-metabolizing enzyme involved in the metabolism of thymol and carvacrol.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

This paper studied the metabolism of Carvacrol in male albino Wistar rats using capillary gas chromatography–mass spectrometry. The rats weighed between 250 and 350 g at the start of the experiment. Food and water were given ad libitum. Carvacrol, 1 mmol/kg dissolved in 1 to 2 ml of propylene glycol, was given by gavage. Control animals received solvent only. Urine samples were collected and stored at −10°C at 24 hr intervals unitl 72 hrs.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source.of test material: Carvacrol - Fluka (Buchs, Switzerland)
- Purity: >98%

- Source.of test material: Thymol - Norsk Medisinaldepot (Bergen, Norway).
- Purity: >99%

2,5-Dihydroxy-p-cymene was obtained from Pfaltz and Bauer, Stamford, CT, U.S.A. was used as the reference compound in the study.

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Mellegaards Breeding Centre Ltd., Ejby, Denmark
- Weight at study initiation: 250-350 g
- Diet: They were switched from a standard pellet diet to a purified diet (Scheline 1968) two days before dosing and during the experiments in order to reduce the number of normal chromatographic peaks in the urine extracts. The diet was provided ad libitum.
- Water: ad libitum

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Carvacrol, 1 mmol/kg was dissolved in 1 to 2 ml of propylene glycol, was given by gavage. Control animals received solvent only.

Duration and frequency of treatment / exposure:

Once

Dose / conc.:

1 other: mmol/kg

No. of animals per sex per dose / concentration:

Caravacrol - 5 males
Thymol - 6 males

Control animals:

yes, concurrent vehicle

Details on dosing and sampling:

Urine samples were collected and stored at −10°C at 24 hr intervals, unti; 72 hrs.

Type:

metabolism

Results:

7 metabolites of Carvacrol were identified in the urine. The major eluate was propan-1-ol derivative whereas Carvacrol was less abundant. The 48 hr samples contained Carvacrol and 2 of its metabolites.

Type:

excretion

Results:

No metabolites were detected in the 72 h sample so the authors concluded that Carvacrol and its metabolites undergo rapid excretion.

Type:

metabolism

Results:

Urine samples contained 6 Thymol metabolites. Thymol was the most prominent urinary component at 24 h, followed by the propan-1-ol derivative. Only small amounts of Thymol were detected at 48 h.

Type:

excretion

Results:

No metabolites were found in the 72 hr samples so the authors concluded that Thymol and its metabolites undergo rapid excretion.

Details on excretion:

No metabolites were detected in the 48 or 72 h sample so the authors concluded that Carvacrol and Thymol and their metabolites undergo rapid excretion.

Metabolites identified:

yes

Details on metabolites:

Table 1 lists the prominent mass spectral fragments of carvacrol and thymol together with those from p-cymene, the reference compound used in this study. It is evident that the two phenols, which differ from p-cymene only by the presence of a hydroxyl group in the ring, show similar patterns of fragmentation. Thus, the molecular ion and base peak are shifted upwards by the expected 16 mass units in the phenols. Furthermore, this picture is also seen with the TMS-derivatives of carvacrol and thymol which have the two highest fragments 72 mass units (TMS) higher than the parent phenols and 88 units (OTMS) higher than p-cymene. This similarity in fragmentation patterns among the three compounds, and similarly among their metabolites, is the factor which allows the metabolites detected in the present study to be identified on the basis of the known structures of the p-cymene metabolites described earlier (Walde et al. 1983).

Conclusions:

In a metabolic assay in rats, Carvacrol and Thymol and their metabolies were rapidly excreted within 72 hours in the urine.

Executive summary:

In a metabolic assay (Austgulen et al., 1987), male Wistar rats (5-6/group) were treated with Carvacrol (>98%) or Thymol (>99%) in propylene glycol by oral gavage. Control animals received solvent only. Urine samples were collected at 24 hr intervals, for 72 hrs. Metabolites were studied using capillary gas chromatography–mass spectrometry.

In addition to the parent phenols themselves, seven carvacrol metabolites (C1-C7) and six thymol metabolites (T1-T6) were identified.  The semi quantitative data revealed that the major carvacrol metabolite was C3 (2-(3-Hydroxy-4-methyIphenyl)propan1-ol) while the major thymol metabolite was the unchanged parent substance, closely followed by the propan-1-ol derivative T2 (2-(2-Hydroxy-4-methyIphenyl)propan1-ol). The urinary metabolites of both substances show considerable similarity: C1 and T1 were dihydroxy derivatives of p-cymene, thymol metabolites T2-T6 correspond to the carvacrol metabolites C3-C7 and only the tertiary alcohol corresponding to C2 was absent from the urinary samples from thymol-treated rats. The results show that carvacrol and thymol and their metabolites undergo rapid excretion as no metabolites were found in the 72 hr samples.

Overall, large quantities of carvacrol and, especially, thymol were excreted unchanged (or as their glucuronide and sulphate conjugates) and extensive oxidation of the methyl and isopropyl groups also occurred. This resulted in the formation of derivatives of benzyl alcohol and 2-phenylpropanol and their corresponding carboxylic acids. In contrast, ring hydroxylation of the two phenols was a minor reaction.

Description of key information

A toxicokinetic assessment was conducted in accordance with REACH Annex VIII 8.8.1. The substance Carvacrol is an organic mono-constituent with a purity of >99% to <100% with a typical concentration of 99.5%.

A full ADME toxicokinetic study in the rat is not available.  The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has indicated a metabolic pathway for Carvacrol (JECFA, 2001a, b). Two publications are available describing the proposed metabolic pathway and excretion of Carvacrol in (Austgulen et al., 1987; Dong et al., 2012). The toxicokinetic analysis is based on these publications, physicochemical and in vivo toxicological data. In vivo studies in rats covering the oral routes are available (acute oral toxicity study, read-across combined repeated dose toxicity and reproduction/developmental toxicity screening study from Thymol (CAS No. 89-83-8)). Carvacrol is corrosive to skin based on an in vivo skin irritation study in rabbits. No inhalational toxicity study data is available. Further details on endpoints are available in the IUCLID 6 registration dossier.

Based on the physicochemical properties and information in the dossier, Carvacrol is expected to be absorbed via the oral, dermal and inhalation routes. It will be distributed throughout the body and large quantities of Carvacrol are excreted unchanged (or as their glucuronide and sulphate conjugates); extensive oxidation of the methyl and isopropyl groups also occurs. These are water soluble innocuous metabolites and are rapidly excreted in the urine.

The absorption rates of 50% (oral), 50% (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

1.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 Carvacrol in the body.

Absorption - oral

The molecular weight of Carvacrol is 150.2 g/mol and is in the range for favourable oral absorption (<500 g/mol). The log Pow of Carvacrol (3.3) indicates it is moderately lipophilic and water solubility (0.330 g/L at 20°C) for Carvacrol indicates it is moderately soluble in water. These characteristics will facilitate transport of Carvacrol via passive diffusion.

Absorption – dermal

The log Kow and water solubility of Carvacrol are in the optimal range for dermal absorption and dermal uptake will be enhanced as it is corrosive to the skin.

Absorption – inhalation

Carvacrol has a low vapour pressure (6.67 Pa at 25˚C), however due to the end use as a fragrance in air-care and cosmetic products, exposure via the inhalation route will be considered. The log Pow of Carvacrol indicates it is sufficiently lipophilic to cross the alveolar and capillary membranes and the water solubility indicates it is not too hydrophilic,  therefore absorption via passive diffusion is expected.

Distribution/Metabolism/Excretion

Based on the molecular weight, water solubility and log Kow, Carvacrol is likely to be widely distributed, may accumulate on repeated exposure and will be excreted as the parent substance or metabolites in the urine.

2. Other data in the literature

Carvacrol is a member of the ‘Phenol and Phenol Derivatives’ group as evaluated by the JECFA (JECFA 2001a, b). All of the substances in this group are expected to be metabolized to innocuous products. Detoxification primarily involves conjugation of the hydroxyl group with sulfate and glucuronic acid. Other metabolic pathyways, observed mainly at high doses, included hydroxylation of the phenol ring and oxidation of side chains.

In a metabolic assay (Austgulen et al., 1987), male Wistar rats (5-6/group) were treated with Carvacrol (>98%) in propylene glycol by oral gavage. Control animals received solvent only. Urine samples were collected at 24 hr intervals, for 72 hrs. Metabolites were studied using capillary gas chromatography–mass spectrometry. In addition to the parent phenol, seven Carvacrol metabolites (C1-C7) were identified.  The semi quantitative data revealed that the major Carvacrol metabolite was C3 (2-(3-Hydroxy-4-methyIphenyl)propan1-ol). The results show that Carvacrol and metabolites undergo rapid excretion as no metabolites were found in the 72 hr samples. Overall, large quantities of Carvacrol were excreted unchanged (or as their glucuronide and sulphate conjugates) and extensive oxidation of the methyl and isopropyl groups also occurred. This resulted in the formation of derivatives of benzyl alcohol and 2-phenylpropanol and their corresponding carboxylic acids. In contrast, ring hydroxylation was a minor reaction. The proposed metabolic pathway of Carvacrol is indicated in Figure 1 (Austgulen et al., 1987).

In an vitro enzyme assay (Dong et al., 2012), the role of cytochrome P450s (CYPs) in the metabolism of Carvacrol was investigated. After incubation with human liver microsomes in the presence of NADPH, two metabolites were detected for Carvacrol. These metabolites were not identified. A combination of chemical inhibition studies and assays with recombinant CYP isoforms demonstrated that CYP2A6 was the predominant drug-metabolizing enzyme involved in the metabolism of Carvacrol.

3. Information from other studies in the dossier

Absorption - oral

In the acute oral gavage toxicity study in rats, deaths occurred between 1 hr and 3 days. Depression was evident within 10 mins and coma within 1 hour. The LD50 (male/female) was 810mg/kg bw with 95% confidence limits of 710-920 mg/kg bw.

A read-across combined repeated dose toxicity and reproduction/developmental toxicity screening study from Thymol is available. In this study (OECD 422/GLP), Thymol (99.6%) was administered to four groups of Crl:CD (SD) rats (10 animals/sex/group) by gavage in 3% gum arabic solution at dose levels of 0, 8, 40 and 200 mg/kg bw/day, 7 days per week, for 43 days (males) and 14 days before mating to day 3 of lactation (females).

One male in the 200 mg/kg bw/day group died 43 days after the start of administration. Although there was no change in the general condition of this animal, autopsy revealed thickening of the anterior stomach wall, dilation of the atrium, congestion of the liver and lung. Pathological examination revealed mild proliferation of the forestomachial epithelium, mild congestion of the liver, moderate congestive edema in the lung and infiltration of mild inflammatory cells.  One female in the 200 mg/kg bw/day group died due to mis-delivery on the 18th gestation (33 days after starting administration).

There was no significant difference in males in the 200 mg/kg bw/day group, but the body weight and the weight gain increased slightly lower than that of the control group after 14 days from the start of administration and showed a tendency to suppress weight gain. In the 200 mg/kg bw/day group, transient locomotor activity reduction and walking ataxia after administration in a small number of females were observed continuously or intermittently from 1 to 13 days after the start of administration. In addition, transient salivation immediately after administration continued from 13th day after the start of administration in females, intermittently from 0th day of gestation (15th day after starting of administration) in females, and until the end of administration, males In almost all cases, half of the females.

Statistically significant changes in the % of lymphocytes and monocytes at higher doses in males were within the historical data of the lab and are not treatment-related. A statistically significant decrease in triglycerides at 8 mg/kg bw/day was not seen at other doses and was not considered treatment-related. There was no significant difference between the control group and the test substance-administered group in absolute weight and relative weight in either organ in both sexes.

Upon necropsy, thickening of the anterior stomach wall was found in 9 males and 1 female in the 200 mg/kg bw/day group. The surface of the thickened forestomach mucosa became whitened and exhibited roughness. In addition, miniaturization of the thymus was observed in 1 case in females in the 40 and 200 mg/kg bw/day group and in 2 females in the 200 mg/kg bw/day group of whitening of the adrenal glands.

Changes caused by the test substance were observed in the forestomach, thymus and adrenal glands upon histopathological analysis. In the forestomach, changes mainly consisting of mucosal epithelial hyperplasia were observed in both males and females in the 40 and 200 mg/kg bw/day groups, and the forestomach mucosa was thickened by stratified squamous epithelium hyperplasia and hyperkeratosis. A few cases were found in submucosal tissues with invasion of inflammatory cells and edema in males and few in females. Regression in the thymus was observed in each case in 40 and 200 mg/kg bw/day females. These two cases were examples showing the miniaturization of the thymus at necropsy. In the adrenal gland, an increase in lipid droplets in the cortical bundle band was observed in one female in the 200 mg/kg bw/day group. This example was one out of two cases showing macroscopically whitening of the adrenal glands. No change was observed in the other animal of the same group and female adrenal glands of the 8 and 40 mg/kg bw/day groups.

Read-across from Thymol - subacute NOEL (male/female, rat): 8 mg/kg bw/day (OECD 422/GLP)

The substance exerted no effects on reproductive parameters such as the estrous cycle, mating index, fertility index, gestation length, number of corpora lutea or implantations, the implantation index, gestation index, delivery index, parturition or maternal behaviour. Birth weight and body weight gain tended to be low in the 200 mg/kg bw/day group neonates. There were no significant differences in numbers of offspring or live offspring at birth, the sex ratio, live birth index or viability index. No abnormal findings ascribable to the compound were found on external examination, or in terms of clinical signs or necropsy finding for the neonates.

Read-across from Thymol - subacute NOAEL (parental): 200 mg/kg bw/day (OECD 422/GLP)

Read-across from Thymol - subacute NOAEL (offspring): 40 mg/kg bw/day (OECD 422/GLP)

Based on the physicochemical data, literature and available in vivo toxicological data, there is systemic absorption after oral administration. For chemical safety assessment purposes, based on the physicochemical properties and information in the dossier, an oral absorption rate of 50% is accepted.

Absorption – dermal

Carvacrol is corrosive to skin based on an in vivo skin irritation study in rabbits. 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

No inhalational toxicity study data is available. Carvacrol is corrosive to the skin and no animal testing was permitted. For chemical safety assessment purposes, an inhalation absorption rate of 100% is accepted using a conservative approach.

Distribution/Metabolism/Excretion

Based on the physicochemical data, literature and available in vivo toxicological data, Carvacrol will be distributed throughout the body. Large quantities of Carvacrol are excreted unchanged (or as their glucuronide and sulphate conjugates); extensive oxidation of the methyl and isopropyl groups also occurs. These are water soluble innocuous metabolites and are rapidly excreted in the urine.

JECFA, 2001a. Evaluation of certain food additives and contaminants. WHO Technical Report Series: 901. Fifty-fifth report of JECFA. World Health Organization, Geneva.

JECFA, 2001b. Safety evaluation of certain food additives and contaminants. WHO food additives series: 46. Prepared by the Fifty-fifth meeting of the JECFA. World Health Organization, Geneva, 2001.

Austgulen, Solheim, Scheline (1987). Metabolism in rats of p-cymene derivatives: carvacrol and thymol. Pharmacol Toxicol. 1987 Aug;61(2):98-102. (No guideline; Klimisch Code: RL4, Not assignable).

Dong, Fang, Zhu, Ge, Cao, Li, Hu, Yang, Liu (2012). Identification of CYP isoforms involved in the metabolism of thymol and carvacrol in human liver microsomes (HLMs). Pharmazie.Dec;67(12):1002-6. (No guideline; Klimisch Code: RL4, Not assignable).