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Toxicological information

Basic toxicokinetics

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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

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
2004
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 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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
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:
Isoeugenyl acetate was extensively hydrolysed by tissue esterases (liver, plasma and skin enzymes) to the corresponding alcohol, isoeugenol.

Metabolite characterisation studies

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.

Any other information on results incl. tables

- 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

  

CLint= intrinsic metabolic clearance (Vmax/ Km)

Applicant's summary and conclusion

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.