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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:
weight of evidence
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 Type:
review article or handbook
Title:
Roles of human CYP2A6 and rat CYP2B1 in the oxidation of (+)-fenchol by liver microsomes
Author:
Miyazawa M
Year:
2006
Bibliographic source:
Xenobiotica, September 2007; 37(9), pp 943–953.

Materials and methods

Objective of study:
metabolism
Principles of method if other than guideline:
- Principle of test: Analysis of the metabolism of the test item in human liver microsomes
- Short description of test conditions: see description below
- Parameters analysed / observed: Identification of metabolites of the test item and kinetic analysis
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Radiolabelling:
no

Test animals

Species:
other: Human
Sex:
not specified
Details on test animals and environmental conditions:
Human liver microsomes were obtained from Gentest Corporation (Woburn, MA, USA) (HG23, HG03, HH18, HG88, HG74, HG06, HK37, HH13, HG64, HH47) and were stored at -80ºC.
Recombinant CYPs1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 expressed in microsomes of Trichoplusia ni cells infected with a baculovirus containing human P450 cDNA were obtained from Gentest Co. (Woburn, MA, USA).

Administration / exposure

Route of administration:
other: In vitro incubation with human liver microsomes
Details on exposure:
Biotransformation of (+)-fenchol by P450 enzymes were determined as follows:
Standard reaction mixtures contained human liver microsomes (0.1mgml-1) or recombinant P450 (10 pmol ml-1) with 100 µM (+)-fenchol in a final volume of 0.5 ml of 100mM potassium phosphate buffer (pH 7.4) containing an NADPH-generating system consisting of 0.5mM NADP+, 5mM glucose 6-phosphate, and 0.5 unit of glucose 6-phosphate dehydrogenase ml-1.
Incubations were carried out at 37ºC for 30 min and terminated by adding 1.5 ml of dichloromethane. Formation of oxidation increased linearly with incubation time up to 30 min. The mixtures were mixed vigorously and the extracts (organic layer) collected by centrifugation at 3000 rpm for 5 min.
The organic phase was transferred to an insert for analysis by gas chromatography-mass spectrometry
Details on dosing and sampling:
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: human liver microsomes
- Method type(s) for identification: GC-MS

Statistics:
Kinetic parameters for (+)-fenchol metabolite formation by microsomes were estimated using a computer program (KaleidaGraph program from Synergy Software, Reading, PA, USA) designed for non-linear regression analysis of the hyperbolic Michaelis–Menten equation. Substrate concentrations used for the analysis of metabolism of (+)-fenchol were 5, 10, 50, 100, 200, 500 µM

Results and discussion

Main ADME results
Type:
metabolism
Results:
(+)-Fenchol was oxidized to fenchone by human liver microsomal P450 enzymes. CYP2A6 was identified as the major enzyme involved in the oxidation of (+)-fenchol by human liver microsomes.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Metabolite: Fenchone.
Kinetic analysis of (+)-fenchol oxidation catalysed by liver microsomes derived from human sample HG03 and recombinant CYP2A6 showed that the apparent Km values were 0.04 and 0.09mM, and the Vmax values were 0.29 and 6.96 nmol min-1 nmol-1 P450, respectively (See Table 1 below).
CYP2A6 is suggested to be the principal CYP enzyme in catalysing the oxidation in (+)-fenchol by human liver microsome.

Any other information on results incl. tables

Table 1. Kinetic analysis of the oxidation by human liver microsomes and recombinant P450 enzymes

expressed in Trichoplusia ni cells

Oxidation of (+)-Fenchol

 

Fenchone

Enzyme source

Km (1)

Vmax (2)

Vmax/km (3)

HG03

0.04

0.29

7.25

CYP2A6

0.09

6.96

77.3

*Substrate concentrations used were 10, 50, 100, 200, 500, 750, 1000 µM.

(1) mM

(2) nmol/min/nmol P450

(3) nM-1 min-1.

Applicant's summary and conclusion

Conclusions:
The test item was oxidized to fenchone by human liver microsomal P450 enzymes, with CYP2A6 identified as the major enzyme involved.
Executive summary:

The metabolism of the test item was studied by the analysis of incubations of in vitro-prepared human liver microsomes. Incubations were carried out at 37ºC for 30 min and contained human liver microsomes (0.1mgml-1) or recombinant P450 (10 pmol ml-1) with 100 mM test item in a final volume of 0.5 ml of 100mM potassium phosphate buffer (pH 7.4) containing an NADPH-generating system consisting

of 0.5mM NADP+, 5mM glucose 6-phosphate, and 0.5 unit of glucose 6-phosphate dehydrogenase ml-1. The biotransformation of the test item was investigated by gas chromatography-mass spectrometry (GC-MS). Under these conditions, the test item was found to be oxidized to fenchone, with CYP2A6 identified as the major enzyme involved.