Diethyl phenylphosphonite

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: acceptable publication which meets basic scientific principles

Data source

Reference

Reference Type:

publication

Title:

Metabolism of phosphorus-containing compounds by pig liver microsomale FAD-contaning monooxygenase

Author:

Smyser, B. and Hodgson, E.

Year:

1985

Bibliographic source:

Biochemical Pharmacology 34 (8):1145-1150

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

In vitro enzyme activity measurement.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl phenylphosphonite

EC Number:

216-676-7

EC Name:

Diethyl phenylphosphonite

Cas Number:

1638-86-4

Molecular formula:

C10H15O2P

IUPAC Name:

diethyl phenylphosphonite

Test material form:

not specified

Details on test material:

- Name of test material: diethylphenylphosphine

Test animals

Species:

other: not applicable

Strain:

other: not applicable

Sex:

not specified

Details on test animals or test system and environmental conditions:

Pig liver microsomal FAD-containing monooxygenase was purified to homogeneity by the method of Sabourin et al.

Administration / exposure

Route of administration:

other: in vitro

Vehicle:

not specified

Details on exposure:

in vitro

Doses / concentrations

Remarks:

Doses / Concentrations:
1 g in 50 mL CHCl3

No. of animals per sex per dose / concentration:

not applicable

Positive control reference chemical:

not applicable

Details on study design:

The test substances have been examined as possible substrates for pig liver microsomal FAD-containing monooxygenase. The enzyme (purified to homogeneity) had a specific activity of 950 nmoles/min/mg protein.
FAD-containing monooxygenase activity was measured by substrate-dependent O2 uptake or NADPH oxidation at 37°C according to the procedure of Poulsen and Ziegler (L. L. Poulsen and D. M. Ziegler, 1. biol. Chem. 254, 6449, 1979) with the following modification: The reaction media contained 0.1 M potassium phosphate, pH 7.6, 3 mM n-octylamine, and either 0.1 mM NADPH or an NADPH-generating system. Reactions were started by adding the substrate. Kinetic constants were obtained using the KINFIT program of Knack and Rohm (I. Knack and K-H. Rohm, Hoppe Seyler's Z. physiol. Chem. 362, 1119,1981) which uses curve-fitting techniques to determine the kinetic constants which best fit the data to the Michaelis-Menten equation. In stoichiometry studies NADPH or O2 consumption rates prior to addition of substrate were extrapolated to the time of reaction completion. These values were then subtracted from total NADPH or O2 consumed.

Metabolite production:
Incubation mixtures contained 100 µM substrate in 5 mL reaction medium. Reactions were carried out in open 25 mL Erlenmeyer flasks shaken in a 37 °C water bath. Control incubations contained enzyme previously inactivated by heat treatment at 50 °C for 2.5 min. To determine reaction completion times, a parallel incubation was carried out in which NADPH oxidation was monitored. Reactions were complete within 15 min for diethylphenylphosphine and within 30 min for diethylphenylphosphine sulfide.

Isolation of metabolite:
After completion of the reaction, incubation mixtures were applied to C18-Sep-pak cartridges. The Sep-pak was washed with 5 ml water previously passed through a 0.45 µm filter and then eluted with 5 ml of a high performance liquid chromatography (HPLC) grade methanol. The methanol fraction contained the oxide.
HPLC was used to identify and quantify the metabolites.

Results and discussion

Main ADME results

Type:

metabolism

Results:

no determination of enyme activity possible due to fast hydrolysis of the substance.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

lt appears that the phosphonite is an excellent substrate for the enzyme but that its rapid nonenzymatic hydrolysis and/or oxidation prohibit calculation of stoichiometric and kinetic values. Other similar substances are good substrates for Pig liver microsomal FAD-containing monooxygenase.