4-allyl-2-methoxyphenyl acetate

Basic toxicokinetics

Administrative data

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

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

2004

Reference 2

Reference Type:

secondary source

Title:

Unnamed

Year:

2013

Materials and methods

Objective of study:

metabolism

Principles of method if other than guideline:

The rate of hydrolysis of eugenyl 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

Test material

Test material information

Constituent 1

Reference substance name:

4-allyl-2-methoxyphenyl acetate

EC Number:

202-235-6

EC Name:

4-allyl-2-methoxyphenyl acetate

Cas Number:

93-28-7

Molecular formula:

C12H14O3

IUPAC Name:

4-allyl-2-methoxyphenyl acetate

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): Eugenyl acetate
- Analytical purity: assumed to be as specified in Section 1.2

Radiolabelling:

no

Administration / exposure

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

Results and discussion

Main ADME results

Type:

metabolism

Results:

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

Metabolite characterisation studies

Metabolites identified:

not measured

Details on metabolites:

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

Any other information on results incl. tables

- Incubation of eugenyl acetate (500 μM) with microsomal protein (0.0125 mg/mL) revealed that hydrolysis was complete within approximately 20 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	114	216	0.5
Rat Skin Microsomes	638	223	2.9
Rat Hepatic S-9	60	173	0.4
Rat Hepatic Microsomes	3029	97	39.6
Human Male Microsomes	3656	89	41.3
Human Female Microsomes	2748	52	52.9

  

CL_int= intrinsic metabolic clearance (V_max/ K_m)

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

A study was performed to quantify the rate of hydrolysis of eugenyl acetate in rat skin cytosol, rat skin microsomes, rat hepatic S-9, rat hepatic microsomes and human microsomes (male and female). Eugenyl 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 approximately 20 minutes of incubation. Kinetic analysis of the hydrolytic reaction in hepatic microsomes (39 -970 μM, 3 minutes incubation) yielded Vmax (nmol/minute/mg of protein): 3829 (rat), 3656 (human male) and 2748 (human female); Km (μM): 97 (rat), 89 (human male) and 52 (human female); and CLint (mL/minute): 39.6 (rat), 41.3 (human male) and 52.9 (human female). Preliminary results demonstrated that rat plasma and preparations of rat skin also readily hydrolyse eugenyl acetate into eugenol. Kinetic analysis of the hydrolytic reaction in rat skin microsomes and cytosol (39 -970 μM, 15 minutes incubation) yielded Vmax (nmol/minute/mg of protein): 114 (skin cytosol ) and 505 (skin microsomes); Km (μM): 216 (skin cytosol) and 223 (skin microsomes) and CLint (mL/minute): 0.5 (skin cytosol) and 2.9 (skin microsomes). Kinetic analysis of the hydrolytic reaction in rat hepatic S-9 fraction (39 -970 μM, 3 minutes incubation) yielded Vmax = 60 nmol/minute/mg of protein, Km: 173 μM and CLint = 0.4 mL/minute.

Under the test conditions, eugenyl acetate was rapidly hydrolysed by liver, plasma and skin esterases to the corresponding alcohol, eugenol. The most extensive activity of the esterases was observed in the hepatic microsomal fraction. There was stoichiometric conversion to eugenol 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.