Registration Dossier

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:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Metabolism of 2-phenylethylamine and phenylacetaldehyde by precision-cut guinea pig fresh liver slices
Author:
Panoutsopoulos et al.
Year:
2004
Bibliographic source:
European Journal of Drug Metabolism and Pharmacokinetics, Vol. 29, pp. 111-118

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
In the study the metabolism of 2-phenylethylamine to phenylacetaldehyde was examined in liver slices and the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activity was compared in the oxidation of the test substance with precision-cut fresh liver slices in the presence/absence of specific inhibitors of each enzyme.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Radiolabelling:
no

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 450-950 g
- Diet: FD1 pellets supplemented with ascorbic acid and hay three times weekly, ad libitum
- Water: ad libitum

No further data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18±1
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
other: ex vivo incubation in vials
Vehicle:
not specified
Details on exposure:
Cylindrical cores, measuring 1 cm in diameter and approximately 1 cm in depth, were removed from the isolated livers using a sharpened stainless steel tube attached to a circular handle. The prepared liver slices were stored in oxygenated Krebs-Henseleit buffer pH 7.4 containing 0.024 M bicarbonate at 4 °C until required. 4 fresh liver slices in a total volume of 3 mL Krebs-Henseleit buffer pH 7.4 containing 0.024 M bicarbonate per 20 mL vials were incubated with 0.001 M phenylacetaldehyde at 37 °C in a shaking water bath. The medium was oxygenated with 95% O2 and 5% CO2 initially and every subsequent hour. 0.2 mL aliquots were removed after 0, 5, 10, 15, 30, 45, 60 and 90 min and analysed by HPLC. In addition, incubations without liver slices were performed as control incubations. To determine the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activity in the oxidation of the test substance, liver slices were co-incubated in the presence of specific inhibitors of each enzyme for 15, 30 and 45 min. The inhibitors used were 0.001 M isovanillin for aldehyde oxidase, 0.0001 M allopurinol for xanthine oxidase and 0.0001 M disulfiram for aldehyde dehydrogenase activity. After analysis, the slices were blotted dry and weighed to determine the total weight of liver used for each incubation. For comparison reasons, the results on the effect of inhibitors in liver slices have been normalized per 100 mg of liver.
Duration and frequency of treatment / exposure:
4 liver slices per vial were incubated with test substance for 0, 5, 10, 15, 30, 45, 60 and 90 min
Doses / concentrations
Remarks:
Doses / Concentrations:
0.001 M
No. of animals per sex per dose:
not data
Control animals:
no

Results and discussion

Any other information on results incl. tables

Incubation of the test substance with fresh guinea pig liver slices:

When the test substance (0.001 M) was incubated with freshly prepared guinea pig liver slices, three metabolites appeared on the chromatograms. The major metabolite was phenylacetic acid with small amounts of 2-phenylethanol and a third more polar metabolite, which eluted at 3.2 min and was thought to be phenyl-2-hydroxyacetic acid. However, the identity of the third compound could not be clearly verified. 4-hydroxyphenylacetic acid or N-(phenylacetyl)glycine was also considered as possible metabolites.

 

Incubation of the test substance with fresh guinea pig liver slices in the presence of inhibitors:

Isovanillin (0.001 M) only slightly reduced (about 14%, t= 30 min) the production of phenylacetic acid although breakdown of the test substance was significantly inhibited. A small increase in the formation of the other metabolites was observed.

In the presence of allopurinol (0.0001 M) there was a slight enhancement of phenylacetic acid production. There was also a positive enhancement in both the production of 4-hydroxyphenylacetic and 2-phenylethanol.

The presence of disulfiram (0.0001 M) in test substance incubations caused a 70-80 % inhibition in the production of phenylacetic acid over the incubation time period. It was shown that upon inhibition with disulfiram there was a significant increase in 2-phenylethanol production.

 

Conclusion:

The effects of inhibitors tested in the liver slice incubations showed that disulfiram (specific inhibitor for aldehyde dehydrogenase) almost totally inhibited oxidation of the test substance, whereas isovanillin (specific inhibitor for aldehyde oxidase) inhibited to lesser extent and allopurinol (specific inhibitor for xanthine oxidase) had little or no effect. The results indicate that the test substance is metabolized mainly by aldehyde dehydrogenase and aldehyde oxidase with little or no contribution from xanthine oxidase.

Applicant's summary and conclusion