(E)-2-methoxy-4-(prop-1-enyl)phenol

Endpoint summary

Description of key information

Isoeugenol is rapidly metabolised and eliminated. Oral toxicokinetic studies show no signs of metabolic saturation. Studies on the metabolism of isoeugenol show that sulphate conjugation and glucuronidation followed by excretion into the urine, constitute the major route of elimination. Skin penetration studies in vitro and in vivo show isoeugenol rapidly penetrates the skin.

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

In accordance with the section 8.1.1 of Annex VIII of Regulation (EC) No 1907/2006 (REACH), the toxicokinetic profile of the substance (i.e. absorption, distribution, metabolism and elimination) was derived from the relevant available information collated in the dossier.

The HERA assessment (HERA, 2005), the CLH report (CLH, 2015), and the NTP technical report (NTP, 2010) on Isoeugenol (mixture of cis- and trans-Isoeugenol) were used to support this toxicokinetic assessment on Trans-isoeugenol.

In addition, the physical chemical characteristics of Trans-Isoeugenol and Isoeugenol (mixture of cis- and trans-Isoeugenol) and the results obtained from toxicological studies were used to predict its toxicokinetic behaviour.

Physical-chemical properties:

The substance is a mono-constituent, having a relatively low molecular weight of 164.21 g/mol. The substance is a moderately water soluble liquid (709 mg/L) and is moderately lipophilic based on the octanol/water partition coefficient (Log Kow 2.1 at 25°C). The substance has low volatility according to its vapour pressure (1 Pa at 25°C).

The hydrolysis study indicates that the target substance is stable at acidic and neutral pHs.

Absorption:

Oral/GI absorption

In rats:

The uptake and excretion of isoeugenol is both thorough and rapid. Following a single oral dose of 14C-isoeugenol to male F344 rats (156 mg/kg; 50 μCi/kg), more than 85% was absorbed in 72 hours (NTP, 2010).

In an oral toxicokinetic study carried out under the US National Toxicology Program, after administration of a single 150 mg/kg dose of test material in corn oil to female Fischer 344/N rats, there was no evidence for saturation of isoeugenol metabolism in female rats by the gavage route of administration. The maximum plasma concentration (Cmax) was 2.64 (μg/mL). The area under the curve (AUC) was 5.93 (μg-hr/mL at 8 hours). Plasma concentration-versus time profiles for isoeugenol revealed that isoeugenol is absorbed rapidly from the gastrointestinal tract (the absorption half-life was approximately 15-20 minutes post-dose). Although saturation of metabolism was not determined by comparing the pharmacokinetics at different dose levels, the short half-life provided evidence that lack of saturation occurred at 150 mg/kg. Administration of 37 mg/kg isoeugenol to another ten female rats gave a Cmax of 1.07 (μg/mL) and the AUC was 1.57 (μg-hr/mL at 8 hours). Bioavailability was approximately 20% (HERA, 2005).

In another study, also carried out under the US National Toxicology Program, male and female Fischer 344 rats were given three different doses of Trans-isoeugenol as a single gavage bolus in corn oil. At doses of 140, 70 and 17 mg/kg, the time variation of the concentration in plasma of Trans-isoeugenol was characterized by an early absorption phase occurring within 5-20 minutes post dosing.followed by at least one secondary peak which prevented estimation of toxicokinetic parameters. Maximum plasma levels (Cmax) values increased with dose. The areas under the curves (AUCs) were significantly higher for females than males but increased supraproportionately with dose for both sexes. At doses of 140, 70 and 35 mg/kg, the clearance [Cl(app)] values were significantly greater for males as compared to females and also increased proportionately with dose for both sexes. At the lowest dose of 17 mg/kg, bioavailability was significantly greater in female rats (17%) as compared to male rats (11%). Overall, low bioavailability was evident at all doses and only a small amount of the administered dose reached systemic circulation. Trans-isoeugenol was rapidly cleared from circulation suggestive of extensive metabolism and/or excretion (HERA, 2005 & NTP, 2010).

In mice:

In another study, also carried out under the US National Toxicology Program, administration of a single 150 mg/kg dose of test material in corn oil to female B6C3F1 mice, produced no evidence for saturation of isoeugenol metabolism in female mice by the gavage route of administration. Maximum plasma level (Cmax) was 5.43 μg/mL and the area under the curve (AUC) was 9.48 μg-hr/mL at 8 hours. Plasma concentration-versus time profiles for isoeugenol revealed that isoeugenol is absorbed rapidly (the absorption half-life was approximately 15-20 minutes post-dose) from the gastrointestinal tract. Administration of 37 mg/kg isoeugenol to another group of female mice gave a Cmax of 2.18 μg/mL and an AUC of 1.86 μg-hr/mL at 8 hours. Bioavailability was approximately 20% (HERA, 2005).

In another study, also carried out under the US National Toxicology Program, forty-two male and female B6C3F1 mice were given three different doses of Trans-isoeugenol as a single gavage bolus in corn oil. At doses of 140, 70 and 35 mg/kg, the time variation of the concentration in plasma of Trans-isoeugenol was characterized by an early absorption phase occurring within 5-20 minutes post dosing followed by at least one secondary peak which prevented estimation of toxicokinetic parameters. AUCs were significantly higher for females than males but increased proportionately with dose for both sexes. The clearance [Cl(app)] values were significantly greater for males as compared to females. Overall, low bioavailability revealed that only a small amount of the administered dose reached systemic circulation, and the compound was rapidly cleared from circulation suggestive of extensive metabolism and/or excretion. Bioavailability at a dose of 35 mg/kg was not significantly different between the sexes [33.9%/36.2% (M/F)] (HERA, 2005 & NTP, 2010).

Despite that these data indicate low bioavailability via the oral route the registered substance was conservatively assumed to be 100% bioavailable by the oral route for the purpose of human health risk assessment.

Dermal absorption

Human

In an in vitro study on freshly excised human skin, a dose of 92.2 mg/cm2 radio-labelled isoeugenol (184 mg/cm3) was applied to the surface of the skin and radioactivity was measured in the skin, on the surface of the skin and in receptor fluid. The recovery of radioactivity in the receptor fluid after 72 hours was 30% and an additional 8.4% was detected in the skin. Total uptake was 38.4% and total recovery was 60% (HERA, 2005).

Application of 10 mM 14 of C-isoeugenol to human cadaver skin using various vehicles (ethanol:water, propylene glycol, liquid paraffin, lotions, white petrolatum, or macrogol ointment) resulted in penetration values ranging from 0.29% to 4% (water- based vehicles) and 0.05% to 11% (lotions and ointments) (NTP, 2010).

In addition, a dermal absorption of 63.6% was estimated by IH SkinPerm, which is not a worst-case since the irritant propoerties of Isoeugenol is not integrated in the model.

In light of these data, the registered substance was conservatively assumed to be 100 % bioavailable in humans for the purpose of human health risk assessment.

Rats

In vivo, a single application of radio-labelled isoeugenol to the skin of rats showed absorption after 24 hours of between 36.6% and 48.7% of the applied dose. A total of 25% of applied dose was recovered as radioactive urinary metabolites (HERA, 2005).

Despite that these data indicate moderate bioavailability via the dermal route, it was not possible to assess the reliability of the study. Therefore, the registered substance was assumed to be 100% bioavailable in rats by the dermal route for the purposes of human health risk assessment.

Respiratory absorption

The potential for inhalation toxicity was not evaluated in toxicokinetic studies.

The vapour pressure of the substance (Vp = 1 Pa at 25°C) indicated a low volatility and inhalability and therefore any anticipated exposure by inhalation is negligible. Thus, at ambient temperature, no respiratory absorption is expected under normal use and handling of the substance.

However, when used as a vapour in aerosol, the substance is expected to be directly absorbed across the respiratory tract epithelium by passive diffusion. This assumption is supported by in vivo toxicity studies by inhalation route showing systemic effects as summarized below:

- In a preliminary 3-day exposure study in rats (6 hours/day) run on a Trans-Isoeugenol (WIL, 2012, rel.2), at 750 mg/m3, the animals were observed to have decreased body weights over the 3-day exposure period and significant clinical observations included rales and laboured respiration. At 300 mg/m3, the mean body weight was decreased.

- In a respiratory lymph node assay (RIVM, 2007, rel.3), mice were exposed nose-only with supporting substance (Isoeugenol, mixture of cis- and trans-Isoeugenol, see Iuclid section 13 for read-across justification). Some mice died, other mice displayed several signs of distress.

In light of these data the registered substance was conservatively assumed to be 100% bioavailable by the inhalation route for the purposes of human health risk assessment.

Distribution:

In the toxicokinetic study carried out by the NTP with single intravenous administration of Isoeugenol, there was evidence for extensive distribution to extravascular tissues and/or high tissue uptake/binding following isoeugenol administration because the apparent volume of distribution (Vapp) values greatly exceeded the total body water volume of 0.668 L/kg. Following gavage administration, the absorption was rapid and consequently extensive distribution to extravascular tissues may be assumed. However, based on the toxicokinetic studies and the reported studies on metabolism, Isoeugenol is rapidly and extensively eliminated and thus this substance is not expected to bioaccumulate.

This is supported by the physicochemical properties of the substance that indicate a low potential for bioaccumulation (log Kow <4).

Metabolism:

Metabolism of Isoeugenol was investigated in the male Fischer 344 rat. Following a single oral dose of 14[C] isoeugenol (156 mg/kg bw, 50 microCi/kg bw), the parent compound isoeugenol was not detected in the blood at any of the time points analysed (0.25 to 72 hours). Following intravenous administration (15.6 mg/kg bw, 100 microCi/kg bw), isoeugenol disappeared rapidly from the blood. Based on the findings of this study, it can be concluded that isoeugenol is rapidly metabolised in the rat and is excreted predominantly in the urine as phase II conjugates of the parent compound (NTP, 2010).

In the toxicokinetic study carried out by the NTP with single administration of Trans-isoeugenol by oral gavage routes in male and female Fischer344/N rats (140, 70 and 17 mg/kg) and B6C3F1 mice (140, 70 and 35 mg/kg), Trans-isoeugenol was eliminated from systemic circulation rapidly and extensively. The collective toxicokinetic evidence indicates that the low bioavailability is the result of extensive first-pass metabolism. There was no evidence of saturation in either male or female rats or mice (HERA, 2005 & NTP, 2010).

Studies on the biotransformation of isoeugenol following intravenous and oral administration to rats have shown that isoeugenol is metabolized by direct sulphation and glucuronidation of the phenolic hydroxyl group. The distal carbon of the propenyl group is also hydroxylated and this metabolite is sulphated and may be subsequently methylated. After oral administration, the major urinary metabolites were found to be the 4-sulphate (33.8%) followed by the glucuronide (20%) and the 3’-sulphate (19.1%) (HERA, 2005).

The relative extent of sulfation and glucuronidation of the major urinary metabolites of isoeugenol was evaluated in the in vivo skin absorption study reported above. At a dose of 2.6 mg/cm2, sulphate conjugates accounted for 88.9% of the urinary metabolites while 11.1% were glucuronide conjugates. The sulfate-conjugated metabolites were 42.1% of conjugated isoeugenol, 4.6% of conjugated 3,4-dihydroxypropylbenzene, 3.5% of conjugated 3,4-diyhdroxyallylbenzene and about 50% unknowns. Unchanged isoeugenol accounted for under 1% (HERA, 2005).

The cytochrome P450 system catalyzes the metabolism of allylbenzene analogs by three competing pathways (NTP, 2010):

- ω-hydroxylation of the terminal methyl group to form the corresponding 3-hydroxy-1-phenylpropene (note that the 3-hydroxy metabolite of isoeugenol is naturally occurring coniferyl alcohol),

- oxidation of the propenyl double bond to form the corresponding 1,2-oxide,

- and O-dealkylation.

Additionally, like allylbenzenes, propenylbenzenes that have a free phenolic group may form glucuronide or sulfonate conjugates and be excreted without undergoing phase I metabolism.

Although isoeugenol is detoxified by phase II conjugation of its free phenolic group, direct single-electron oxidation is a fifth pathway that results in formation of the quinone-methide metabolite (NTP, 2010).

Excretion:

Metabolism of Isoeugenol was investigated in the male Fischer 344 rat. Following a single oral dose of 14[C] isoeugenol (156 mg/kg bw, 50 microCi/kg bw), greater than 85% of the administered dose was excreted in the urine predominantly as sulfate or glucuronide metabolites by 72 hours. Approximately 10% was recovered in the faeces, and less than 0.1% was recovered as CO₂or expired organics. The parent compound isoeugenol was not detected in the blood at any of the time points analysed (0.25 to 72 hours). Following intravenous administration (15.6 mg/kg bw, 100 microCi/kg bw), isoeugenol disappeared rapidly from the blood. The half-life was 12 minutes, the volume of distribution was 13.96 l/kg, mean residence time (MRT) was 11.6 minutes and the systemic clearance was 1.9 l/min/kg. Excretion characteristics were similar to those seen following oral administration. The total amount of radioactivity remaining in selected tissues (heart, kidneys, liver, muscle, subcutaneous adipose tissue and testicular adipose tissue) by 72 hours was less than 0.25% of the dose following both oral and intravenous administration. Based on the findings of this study, it can be concluded that isoeugenol is rapidly metabolised in the rat and is excreted predominantly in the urine as phase II conjugates of the parent compound (NTP, 2010).

In a toxicokinetic study carried out by the NTP, gavage administration of 156 mg/kg [14C]-Isoeugenol (μCi/kg) in corn oil to male Fischer 344-rats, led to approximately 10% of the administered dose being recovered in the faeces (possibly following absorption and then biliary excretion), less than 0.1% was recovered as CO₂or expired organics and less than 0.2% was detected in selected tissues. About 85% was detected in urine; a level that was reached after 24 hours. No parent Isoeugenol was detected in the blood at any to the time-points analysed. Incubation of urine samples with B-glucuronidase caused dramatic peak shifts in the HPLC profile. Isoeugenol is rapidly metabolized and is excreted predominantly in the urine as phase II conjugates of the parent compound (HERA, 2005 & NTP, 2010).

Studies on the metabolism of isoeugenol show that sulphate conjugation and glucuronidation followed by excretion into the urine, constitute the major route of elimination. By all routes studied, elimination is rapid and extensive. In oral toxicokinetic studies, there is no evidence of saturation of metabolism which was rapid and led to a low bioavailability of parent isoeugenol with only a small proportion (down to 10% in male rats) reaching the systemic circulation. Slightly higher bioavailability was seen in mice (HERA, 2005 & NTP, 2010).

References:

CLH, 2015. Proposal for Harmonised Classification and Labelling Based on Regulation (EC) No 1272/2008 (CLP Regulation), Annex VI, Part 2. Substance Name: 2-methoxy-4-(prop-1-enyl)phenol; 2-methoxy-4-((E)prop-1-enyl)phenol; 2-methoxy-4-((Z)prop-1-enyl)phenol

HERA, 2005: ISOEUGENOL 4-Hydroxy-3-methoxy-1-propen-1-yl benzene (CAS 97-54-1). Prepared by the Human and Environmental Risk Assessment on ingredients of Household Cleaning Products (HERA).

NTP, 2010: NTP technical report on the toxicology and carcinogenesis studies of Isoeugenol (CAS NO. 97-54-1) in F344/N rats and B6C3F1 mice (gavage studies). Prepared for the National Toxicology Program (NTP), in September 2010, NTP TR 551, National Institutes of Health (NIH) Publication No. 10-5892.