Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The test conditions are acceptable for the assessment

Data source

Reference
Reference Type:
publication
Title:
Metabolism of ethers in the rabbit. 2. Nuclear-substituted anisoles
Author:
Bray HG, Craddock VM and Thorpe WV
Year:
1955
Bibliographic source:
Biochemical Journal, 60, 225-232

Materials and methods

Objective of study:
excretion
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Observation of excretion
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
no data

Test animals

Species:
rabbit
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.5-3.5 kg
- No. of rabbits per group: 6
- Fasting period before study: data not available
- Source, strain, sex, age at study initiation, housing, acclimation period: data not available
- food and water consumption: animals were maintained on a diet of rabbit pellets and water

ENVIRONMENTAL CONDITIONS (temperature, humidity, air changes, photoperiod): data not available

IN LIFE DATES: data not available

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
not applicable
Duration and frequency of treatment / exposure:
24 hour(s) (once)
Doses / concentrations
Remarks:
Doses / Concentrations:
0.7 g per rabbit
Control animals:
no
Positive control:
no
Details on study design:
not applicable
Details on dosing and sampling:
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: once 24 hr after treatment
Excretion of metabolites was determined on the days preceding and following the experiment.
General procedure for the examination of urines:
The 24-hr urine from 6 rabbits each dosed with 0.7 g PDMB/ rabbit, was adjusted to pH 6.5 and extracted with ether to give ether extract A. The aqueous residue, after addition of an equal volume of 10N H2SO4, was refluxed for 2 hr. A second ether extract, B, was obtained by exhaustive extraction of this hydrolysate with ether. The aqueous residue from this extraction was adjusted to pH 7 and again extracted with ether to yield a third ether extract, C.
The first extraction was omitted for the urines containing only small amounts of free phenol. The third extraction was carried out on the nitroanisole urines to collect any aminophenols present. Control experiments had shown that the hydrolysis of the urines with 5N-H2SO4 caused less than 0.3% of any of the anisoles to be converted into phenols. About 4% of p-cyanoanisole was, however, converted into anisic acid by this treatment. The ether extracts were examinated by paper chromatography and the major metabolites isolated by making appropriate derivatives. The examinated excretions recorded are based upon the amounts of recrystallized derivatives obtained and would almost certainly indicate less than the amount actually excreted.

Preparation of tissue:
liver were removed after the kill of the rabbit, put in Riger's solution. Tissue slices were cut and incubated in Krebs-Ringer bicarbonate solution. The substrate was dissolved in the solution of the 5 salts before the addition of the NaHCO3 solution.
The substrate dissolved in the medium (10 ml) was placed in a 50 ml flask and the air displaced by a stream of O2+CO2 (95:5) passed on to the surface of the liquid. The flask was then closed with a rubber bung and the mixture equilibrated by shaking at 38°C for 30 min. The tissue slices were then added and gas was passed again; the flask was closed and shaken in the thermostat for the required time. Appropriate controls were set up for each experiment. The reaction was stopped by the addition of 10% (w/v) trichloroacetic acid (6 ml) and the tissue slices and precipitated protein removed by filtration and dried to constant weight at 110°. The filtrate was refluxed with 10N-H2SO4 (equal vol.) for 1 hr and the Hydrolysate extracted continuously with ether for 6 hr. The extracted material was examined qualitatively or quantitatively for the presence of reaction products.

Examination of hydrolysed filtrates from deproteinized digests: Ether extracts of the hydrolysed filtrates were used for examination for phenols by paper chromatography. An ether extract of the hydrolysate of the filtrate from the medium incubated with tissue slices in the absence of substrate was used as a control. For quantitative analysis, water (10 ml.) was added to the ether extract of the hydrolysed filtrate, ether was removed by gentle evaporation and the residual solution was diluted to 15 ml. with water. Phenols were determined as described for urine.

CHARACTERISATION STUDIES: no data
Statistics:
no data

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on excretion:
1- The quantitative result of metabolites excreted after administration of substituted anisoles:
METABOLITES EXCRETED IN URINE AFTER ADMINISTRATION OF 0.7 g PDMB/RABBIT (results expressed as the average percentage of the dose excreted): ranges are given in parentheses
Ethereal sulphate: 27 (16-38)
Ether glucuronide: 63 (61-64)
Free phenol: 4 (2-5)
Average percentage of dose accounted: 94

Although PDMB is mainly excreted as conjugates of p-methoxyphenol and quinol.

The results, in conjunction with the qualitative findings, suggest that demethylation was the predominant reaction.
The higher metoxyl contents of light petroleum extracts of hydrolysed nitroanisole urines compared with those of the unhydrolysed urines suggest that small amounts of methoxy compound were excreted conjugated.
The rates of demethylation of the anisole derivatives were determined by measuring the phenols excreted at intervals during experiments lasting 14 hr. Graphs of the amounts of the dose excreted as ethereal sulphate and ether glucuronide plotted against time were of the form of exponenetial
curves, indicating that the reactions follow firstorder reaction kinetics with respect to the anisole derivative.
For PDMB the velocity constant is around 0.19 h-1

2-The urine examination of the different extracts A, B, C:
Paper chromatography revealed the presence of p-methoxyphenol and quinol in the B extracts of p-methoxyanisole urine. p-Methoxyphenol was
the chief metabolite and was isolated (estimated excretion 34% of the dose) as p-methoxyphenyl p-nitrobenzyl ether, m.p. 84°, unchanged by
admixture with an authentic sample. From paper chromatograms the amount of quinol seemed to be considerably greater than that present in
normal urine. p-Methoxyphenol itself gives rise to appreciable amounts of quinol in the rabbit.

3-Identification of products in rabbit liver slices were incubated 2h with PDMB and ether extracts of the hydrolysed incubation mixtures and
examinated for the corresponding phenols by means of paper chromatography
PDMB formed p-methoxyphenols.

4- Quantitative experiments:
Rabbit liver was more effective than kidney or intestine as a source of the demethylating system. Approximatively 10% (0.36-0.44 mg) of
p-methoxyanisole was demethylated by 100 mg (dry wt) rabbit-liver slices in 10 ml of 0.003M substrate in 2h at 38°C. Less than 3% was
demethylated by slices of kidney or intestine. No phenol was formed in control digests and 100 mg (dry wt) tissue contained less than 0.01 mg
phenol (calculated as p-methoxypbenol). The addition of glycocyamide (0.003M) or DL-methionine (0.003M) did not cause a significant increase in
the extent of phenol formation. As an indication of the relative initial rates of demethylation, the amount of phenol formed after 20 min. was
determined.
From eight experiments with p-methoxyanisole the average extent of demethylation corresponded to the removal of 17 µg/100 mg (dry wt) liver
(range 14-21).
Slices of rabbit liver were incubated with 0.003M p-methoxyanisole in Krebs-Ringer bicarbonate for 2 hr at
38°. Additions were made as indicated. Concentrations of KCN and ATP were 0.01M and 0.001M respectively. All flasks were gassed with 02+C02
mixture except when N2 was used. Incubation for 2 hr at 38°C.

5- Effect of KCN, adenosine triphosphate (ATP), 2,4-dinitrophenol and absence of O2. Experiments were performed in which the gas in the flasks
was N2 instead of O2+CO2 mixture. The effect of the addition of 0.01M KCN or 0.001M ATP to digests exposed to the O2+CO2 mixturewas also
examinated. Some results obtained with p-methoxyanisole as substrate are:
p-methoxyanisole demethylated/100 mg (dry wt) slices:
Exp.1: Without addition: 0.41 mg; with N2: 0 mg
Exp.2: Without addition: 0.44 mg; with N2: 0.05 mg; with KCN: 0.17 mg; with ATP: 0.55mg
Exp.3: Without addition: 0.37 mg; with ATP: 0.07 mg

Both replacement of O2 by N2 and the addition of KCN reduced the extent of demethylation, while ATP enhanced it. The formation of p-methoxy-
anisole in digests to which 2,4-dinitrophenol (0.01M) had been added could not be detected by paper chromatography.
Toxicokinetic parametersopen allclose all
Toxicokinetic parameters:
half-life 1st:
Toxicokinetic parameters:
half-life 2nd:
Toxicokinetic parameters:
half-life 3rd:

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
p-methoxyphenol and quinol.

Applicant's summary and conclusion