(E)-2-Methoxy-4-prop-1-en-1-ylphenyl acetate

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:

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

Objective of study:

metabolism

Principles of method if other than guideline:

The rate of hydrolysis of isoeugenyl acetate in rat skin cytosol, rat skin microsomes, rat hepatic S-9, rat hepatic microsomes, human hepatic microsomes was quantified by HPLC and kinetic constants Vmax, Km and CLint (defined as the Vmax/Km ratio) were determined.

GLP compliance:

not specified

Radiolabelling:

no

Details on study design:

- Isoeugenyl acetate (500 µM) was incubated with subcellular fractions of rat skin cytosol, rat skin microsomes, rat hepatic S-9, rat hepatic microsomes, human microsomes (male & female) s at 37 °C.
- Kinetic analysis by HPLC was performed under following conditions:
Column: Reversed-phase alpha bond C18 column
Mobile phase: Acidified water and acetonitrile
Monitoring wavelengths: 254 nm and 265 nm
- Following parameters were selected for kinetic analysis:
Rat:
Skin protein concentrations: 0.0375 (microsomes) and 0.1 mg/mL (cytosol)
Hepatic protein concentrations: 0.0125 (microsomes) and 0.1 mg/mL (S-9 fraction)
Dosing Range: 5-495 µM
Duration of incubation: skin (15 minutes) and hepatic (3 minutes)
Kinetic Data was normalized to nmol / minute / mg of protein
- Kinetic constants Vmax, Km and CLint (intrinsic metabolic clearance defined as the Vmax/Km ratio) were determined.

Statistics:

None

Type:

metabolism

Results:

Isoeugenyl acetate was extensively hydrolysed by tissue esterases (liver, plasma and skin enzymes) to the corresponding alcohol, isoeugenol.

Metabolites identified:

not measured

Details on metabolites:

Isoeugenyl acetate was extensively hydrolysed by tissue esterases (liver, plasma and skin enzymes) to the corresponding alcohol, isoeugenol.

- Incubation of isoeugenyl acetate (500 μM) with microsomal protein (0.0125 mg/mL) revealed that hydrolysis was complete within 15 minutes of incubation.

Table 7.1.1/1: Kinetic Constants for Alcohol Formation

 

Subcellular Fraction	Vmax (nmol/min/mg protein)	Km(µM)	CLint(ml/min)
Rat Skin Cytosol	97	137	0.7
Rat Skin Microsomes	505	190	2.7
Rat Hepatic S-9	52	110	0.5
Rat Hepatic Microsomes	3822	91	42.0
Human Male Microsomes	3795	72	52.5
Human Female Microsomes	3072	51	60.1

  

CL_int= intrinsic metabolic clearance (V_max/ K_m)

Conclusions:

Isoeugenyl acetate was rapidly hydrolysed by liver, plasma and skin esterases to the corresponding alcohol, isoeugenol.

Executive summary:

A study was performed to quantify the rate of hydrolysis of isoeugenyl acetate in rat skin cytosol, rat skin microsomes, rat hepatic S-9, rat hepatic microsomes and human microsomes (male and female). Isoeugenyl acetate (500 µM) was incubated with microsomal protein (0.0125 mg/mL for microsomes and 0.1 mg/mL for S-9 fraction for 3 minutes) and skin protein (0.0375 mg/mL for microsomes and 0.1 mg/mL for cytosol for 15 minutes) at 37 °C. Kinetic constants Vmax, Km and CLint (defined as the Vmax/Km ratio) were determined by HPLC.

Incubation of isoeugenyl acetate (500 μM) with microsomal protein (0.0125 mg/mL) revealed that hydrolysis was complete within 15 minutes of incubation. Kinetic analysis of the hydrolytic reaction in hepatic microsomes (5-495 μM, 3 minutes incubation) yielded Vmax (nmol/minute/mg of protein): 3822 (rat), 3795 (human male) and 3072 (human female); Km (μM): 91 (rat), 72 (human male) and 51(human female); and CLint (mL/minute): 42 (rat), 52.5 (human male) and 60.1 (human female). Preliminary results demonstrated that rat plasma and preparations of rat skin also readily hydrolyse isoeugenyl acetate into isoeugenol. Kinetic analysis of the hydrolytic reaction in rat skin microsomes and cytosol (5-495 μM, 15 minutes incubation) yielded Vmax (nmol/minute/mg of protein): 97 (skin cytosol ) and 505 (skin microsomes); Km (μM): 137 (skin cytosol) and 190 (skin microsomes) and CLint (mL/minute): 0.7 (skin cytosol) and 2.7 (skin microsomes). Kinetic analysis of the hydrolytic reaction in rat hepatic S-9 fraction (5-495 μM, 3 minutes incubation) yielded Vmax = 52 nmol/minute/mg of protein, Km: 110 μM and CLint = 0.5 mL/minute.

Under the test conditions, isoeugenyl acetate was rapidly hydrolysed by liver, plasma and skin esterases to the corresponding alcohol, isoeugenol. The most extensive activity of the esterases was observed in the hepatic microsomal fraction. There was stoichiometric conversion to isoeugenol of hepatic, blood and skin preparations incubated with the test material. The most extensive activity was observed in the hepatic microsomal fraction. Slow rate of hydrolysis of esters in skin correlates with their decreased sensitization potential. Under the conditions of this study, test material absorption into the systemic circulation would be minimal following dermal or oral exposure. Rapid hydrolytic conversion by enzymes in the liver, blood and skin, as well as in the intestinal fluid and intestinal cells, would limit systemic exposure to the parent molecules.

Reference 2

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in Iuclid Section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, toxicological and environmental fate properties because of their structural similarity (cis- and trans-isomers).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance is the trans isomer (E), as a mono-constituent substance. The source substance is a reaction mass, composed of two diastereoisomers (the source substance [trans] and its cis-isomer).

3. ANALOGUE APPROACH JUSTIFICATION
The source and the target substances have a common major constituent (trans-isomer). Hydrolysis of the source substance by liver, plasma and skin esterases was rapid and led to the corresponding alcohol, Isoeugenol. The test material was not clearly identified but it is assumed to represent the source substance in terms of constituents and impurities.
Therefore, based on the considerations above, it can be concluded that the results of the in vitro hydrolysis test conducted with the source substance are highly likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VIII, 8.8.

4. DATA MATRIX
Cf. Iuclid Section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Type:

metabolism

Results:

Isoeugenyl acetate was extensively hydrolysed by tissue esterases (liver, plasma and skin enzymes) to the corresponding alcohol, isoeugenol.

Metabolites identified:

not measured

Details on metabolites:

Isoeugenyl acetate was extensively hydrolysed by tissue esterases (liver, plasma and skin enzymes) to the corresponding alcohol, isoeugenol.

- Incubation of isoeugenyl acetate (500 μM) with microsomal protein (0.0125 mg/mL) revealed that hydrolysis was complete within 15 minutes of incubation.

Table 7.1.1/1: Kinetic Constants for Alcohol Formation

 

Subcellular Fraction	Vmax (nmol/min/mg protein)	Km(µM)	CLint(ml/min)
Rat Skin Cytosol	97	137	0.7
Rat Skin Microsomes	505	190	2.7
Rat Hepatic S-9	52	110	0.5
Rat Hepatic Microsomes	3822	91	42.0
Human Male Microsomes	3795	72	52.5
Human Female Microsomes	3072	51	60.1

  

CL_int= intrinsic metabolic clearance (V_max/ K_m)

Conclusions:

Isoeugenyl acetate was rapidly hydrolysed by liver, plasma and skin esterases to the corresponding alcohol, isoeugenol.

Executive summary:

A study was performed to quantify the rate of hydrolysis of isoeugenyl acetate in rat skin cytosol, rat skin microsomes, rat hepatic S-9, rat hepatic microsomes and human microsomes (male and female). Isoeugenyl acetate (500 µM) was incubated with microsomal protein (0.0125 mg/mL for microsomes and 0.1 mg/mL for S-9 fraction for 3 minutes) and skin protein (0.0375 mg/mL for microsomes and 0.1 mg/mL for cytosol for 15 minutes) at 37 °C. Kinetic constants Vmax, Km and CLint (defined as the Vmax/Km ratio) were determined by HPLC.

Incubation of isoeugenyl acetate (500 μM) with microsomal protein (0.0125 mg/mL) revealed that hydrolysis was complete within 15 minutes of incubation. Kinetic analysis of the hydrolytic reaction in hepatic microsomes (5-495 μM, 3 minutes incubation) yielded Vmax (nmol/minute/mg of protein): 3822 (rat), 3795 (human male) and 3072 (human female); Km (μM): 91 (rat), 72 (human male) and 51(human female); and CLint (mL/minute): 42 (rat), 52.5 (human male) and 60.1 (human female). Preliminary results demonstrated that rat plasma and preparations of rat skin also readily hydrolyse isoeugenyl acetate into isoeugenol. Kinetic analysis of the hydrolytic reaction in rat skin microsomes and cytosol (5-495 μM, 15 minutes incubation) yielded Vmax (nmol/minute/mg of protein): 97 (skin cytosol ) and 505 (skin microsomes); Km (μM): 137 (skin cytosol) and 190 (skin microsomes) and CLint (mL/minute): 0.7 (skin cytosol) and 2.7 (skin microsomes). Kinetic analysis of the hydrolytic reaction in rat hepatic S-9 fraction (5-495 μM, 3 minutes incubation) yielded Vmax = 52 nmol/minute/mg of protein, Km: 110 μM and CLint = 0.5 mL/minute.

Under the test conditions, isoeugenyl acetate was rapidly hydrolysed by liver, plasma and skin esterases to the corresponding alcohol, isoeugenol. The most extensive activity of the esterases was observed in the hepatic microsomal fraction. There was stoichiometric conversion to isoeugenol of hepatic, blood and skin preparations incubated with the test material. The most extensive activity was observed in the hepatic microsomal fraction. Slow rate of hydrolysis of esters in skin correlates with their decreased sensitization potential. Under the conditions of this study, test material absorption into the systemic circulation would be minimal following dermal or oral exposure. Rapid hydrolytic conversion by enzymes in the liver, blood and skin, as well as in the intestinal fluid and intestinal cells, would limit systemic exposure to the parent molecules.

Description of key information

The available evidence suggests that the substance is rapidly hydrolysed by liver and plasma esterases and to a lower extent by skin esterases to the corresponding alcohol Isoeugenol. Therefore it is not highly bioavailable via the oral, dermal and inhalation route. The substance is expected to be mainly excreted in urine.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

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 physical chemical characteristics of the registered substance and supporting substances (see Section 13 for read-across justification) and the results obtained from toxicological studies were used to predict its toxicokinetic behaviour.

 

As detailed in the metabolism section below, Isoeugenyl acetate (reaction-mass of cis- and trans-isomers) is rapidly hydrolysed by liver and plasma esterases and to a lower extent by skin esterases to the corresponding alcohol, Isoeugenol. Therefore, the toxicokinetics profile of Isoeugenol was used to assess the toxicokinetic behaviour of Isoeugenyl acetate.

 

Physical-chemical properties:

The substance is a mono-constituent, having a relatively low molecular weight of 206.24 g/mol. The substance is a slightly water soluble powder (44.6 mg/L) and is moderately lipophilic based on the octanol/water partition coefficient (Log Kow 2.9 at 35°C). The substance has low volatility according to its vapour pressure (0.07 Pa at 25°C).

 

Absorption:

As detailed in the metabolism section below, Isoeugenyl acetate (reaction mass of cis- and trans-isomers) is rapidly hydrolysed by liver, plasma and skin esterases to the corresponding alcohol, Isoeugenol. There was stoichiometric conversion to Isoeugenol of hepatic, blood and skin preparations incubated with the test material. Under the conditions of the study (Castro, 2004), test material absorption into the systemic circulation would be minimal following dermal or oral exposure. Rapid hydrolytic conversion by enzymes in the liver, blood and skin, as well as in the intestinal fluid and intestinal cells, would limit systemic exposure to the parent molecules (i.e. Isoeugenyl acetate).

 

Oral/GI absorption

- Trans-isoeugenyl acetate

In light of the lack of specific information, the parent molecule was assumed to be 100% bioavailable by oral route for the purpose of human health risk assessment.

 - Isoeugenol

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

- Trans-isoeugenyl acetate

Regarding dermal absorption, systemic absorption by the dermal route is expected to be low to moderate based on the Log Kow and the water solubility values. This is supported by the in vitro hydrolysis study.

 In light of these data, and the lack of specific information on any the registered substance or its supporting substances, a dermal absorption of 100% was conservatively assumed for the purposes of human health risk assessment.

- Isoeugenol

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

- Isoeugenyl acetate

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

The substance has a low volatility but the particle size indicated that 13% of the particles have a particle size below 100 µm, i.e. inhalable. However, granulometry test did not show any respirable particle (i.e. below 10 µm) meaning that particles do not reach the alveoli, the major site of absorption in the respiratory tract. Therefore if any absorption can occur, it should be limited.

When used as a vapour in aerosol, the substance is expected to be directly absorbed across the respiratory tract epithelium by passive diffusion.

 In light of these data, and the lack of specific information on respiratory absorption, the substance was conservatively assumed to be 100% bioavailable by inhalation for the purposes of human health risk assessment.

-Isoeugenol:

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

In vivo toxicity studies by inhalation route showed 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:

- Isoeugenyl acetate

As detailed in the metabolism section below, Isoeugenyl acetate is rapidly hydrolysed by liver, plasma and skin esterases to the corresponding alcohol, isoeugenol. There was stoichiometric conversion to isoeugenol of hepatic, blood and skin preparations incubated with the test material. Rapid hydrolytic conversion by enzymes in the liver, blood and skin, as well as in the intestinal fluid and intestinal cells, would limit systemic exposure to the parent molecules (i.e. Isoeugenyl acetate).

Based on the toxicokinetic studies, Isoeugenol is quickly and extensively eliminated and thus is not expected to bioaccumulate.

This is supported by the physicochemical properties of Isoeugenyl acetate with a low potential for bioaccumulation (log Kow <4).

- Isoeugenol

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:

- Isoeugenyl acetate:

A study was performed to quantify the rate of hydrolysis of Isoeugenyl acetate in rat skin cytosol, rat skin microsomes, rat hepatic S-9, rat hepatic microsomes and human microsomes (male and female) (Castro, 2004). Isoeugenyl acetate (500 µM) was incubated with microsomal protein (0.0125 mg/mL for microsomes and 0.1 mg/mL for S-9 fraction for 3 minutes) and skin protein (0.0375 mg/mL for microsomes and 0.1 mg/mL for cytosol for 15 minutes) at 37 °C. Kinetic constants Vmax, Km and CLint (defined as the Vmax/Km ratio) were determined by HPLC.

Incubation of Isoeugenyl acetate (500μM) with microsomal protein (0.0125 mg/mL) revealed that hydrolysis was complete within 15 minutes of incubation. Kinetic analysis of the hydrolytic reaction in hepatic microsomes (5-495μM, 3 minutes incubation) yielded Vmax (nmol/minute/mg of protein): 3822 (rat), 3795 (human male) and 3072 (human female); Km (μM): 91 (rat), 72 (human male) and 51(human female); and CLint (mL/minute): 42 (rat), 52.5 (human male) and 60.1 (human female). Preliminary results demonstrated that rat plasma and preparations of rat skin also readily hydrolyse Isoeugenyl acetate into isoeugenol. Kinetic analysis of the hydrolytic reaction in rat skin microsomes and cytosol (5-495μM, 15 minutes incubation) yielded Vmax (nmol/minute/mg of protein): 97 (skin cytosol ) and 505 (skin microsomes); Km (μM): 137 (skin cytosol) and 190 (skin microsomes) and CLint (mL/minute): 0.7 (skin cytosol) and 2.7 (skin microsomes). Kinetic analysis of the hydrolytic reaction in rat hepatic S-9 fraction (5-495μM, 3 minutes incubation) yielded Vmax = 52 nmol/minute/mg of protein, Km: 110μM and CLint = 0.5 mL/minute.

Under the test conditions, Isoeugenyl acetate is rapidly hydrolysed by liver, plasma and skin esterases to the corresponding alcohol, isoeugenol. There was stoichiometric conversion to isoeugenol of hepatic, blood and skin preparations incubated with the test material. Under the conditions of the study (Castro, 2004), test material absorption into the systemic circulation would be minimal following dermal or oral exposure. Rapid hydrolytic conversion by enzymes in the liver, blood and skin, as well as in the intestinal fluid and intestinal cells, would limit systemic exposure to the parent molecules.

Slow rate of hydrolysis of esters in skin correlates with their decreased sensitization potential. As detailed in the IDEA workshop on pre- and pro-haptens in Fragrance, in vivo skin sensitisation data suggests that Isoeugenyl Acetate does not hydrolyse rapidly enough in skin that it should be considered equivalent to Isoeugenol with regard to sensitisation induction. Indeed, with EC3 values between 0.5 and 3.8, Isoeugenol is considered to be a strong skin sensitizer (skin sensitizer Category 1A) (CLH report) whereas Isoeugenyl acetate did not have the potential to induce skin sensitization up to 25% (highest concentration tested, all SI < 1, no dose-relationship).

- Isoeugenol:

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:

As detailed in the metabolism section, Isoeugenyl acetate is rapidly hydrolysed to the corresponding alcohol, isoeugenol. There was stoichiometric conversion to isoeugenol of hepatic, blood and skin preparations incubated with the test material.

 

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:

Castro DJ, Sweet CJ, Kuester RK and Sipes G. Hydrolysis of Isoeugenyl-acetate and Eugenyl-acetate by Rat and Human Hepatic Microsomes. 2004, Abstract No. 1446, The Toxicologist- a supplement to Toxicological Sciences, 78 (S-1), 298.

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).

IDEA Workshop - Risk assessment of pre- & pro-haptens (May 28-29, 2013). The in vitro hydrolysis of isoeugenyl acetate and eugenyl acetate.

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.