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Basic toxicokinetics

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basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No guideline available. The OECD guideline for toxicokinetic only applies to animals (this is an in vitro study). Study design was well conducted with sufficient details provided.

Data source

Reference Type:
Metabolism and cytotoxicity of eugenol in isolated rat hepatocytes
Thompson D.C., Constantin-Teodosiu D., and Moldeus P.
Bibliographic source:
Chemico-Biological Interactions. 1991; Volume 77: 137-147

Materials and methods

Objective of study:
Test guideline
no guideline available
Principles of method if other than guideline:
The test article (in DMSO) was incubated with hepatocytes obtained from male Sprague-Dawley rats.
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): Eugenol
Further details not reported.
[3H]eugenol (labeled on the methoxy group)

Test animals

other: in vitro
Details on test animals or test system and environmental conditions:
Hepatocytes obtained from male Sprague-Dawley rats.

Administration / exposure

Route of administration:
other: in vitro
Details on exposure:
Male Sprague Dawley rats (200 g), given food and water ad libitum, were used for these experiments. Hepatocytes were isolated by collagenase perfusion of the liver. Cells prepared by this method for use in these experiments were 85-90% viable. Cells were incubated at a concentration of 1 x 106 cells/mL in rotating round bottom flasks at 37°C in Krebs-Henseleit buffer (pH 7.4), supplemented with 12.5 mM Hepes. Eugenol was dissolved in DMSO before addition to the incubations in a volume not exceeding 1% (v/v). In some experiments, cells were pretreated with 0.5 mM diethylmaleate for 30 min to deplete intracellular glutathione. This treatment depleted cells of over 90% of their intracellular glutathione. The cells were washed and resuspended in buffer before use in subsequent experiments. Cell viability was assessed by trypan blue exclusion.
Duration and frequency of treatment / exposure:
In vitro incubation up to 5 hours
Doses / concentrations
Doses / Concentrations:
0.5, 1, and 1.5 mM eugenol
No. of animals per sex per dose / concentration:
Not applicable
Control animals:
other: not applicable
Positive control reference chemical:
Not applicable.
Details on study design:
Hepatocytes (1 x10^6 cells/mL) were incubated at 37°C in rotating round bottom flasks in modified Krebs-Henseleit buffer (pH 7.4) with various concentrations of eugenol for up to 5 hours. Test concentrations included 0, 0.5, 1.0, and 1.5 mM (with 1.5 mM eugenol+1mM N-acetylcysteine also tested). Furthermore, the effect of eugenol on the cytotoxicity of rat hepatocytes, the effect of eugenol on intracellular glutathione, and the amount of covalent binding of reactive metabolites of [3H]eugenol also was evaluated.
Details on dosing and sampling:
See details on exposure above
Not reported

Results and discussion

Main ADME results
Eugenol is actively metabolized by hepatocytes to form conjugates with glucuronic acid, sulfate and glutathione. The major metabolite formed in hepatocytes was the glucuronide.

Metabolite characterisation studies

Metabolites identified:
Details on metabolites:
Analysis of eugenol metabolites in the incubation media revealed that 3 major metabolites were being formed. These were conjugates with sulfate, glucuronic acid and glutathione. At a concentration of 1 mM eugenol, eugenol-glucuronide was the major metabolite formed, while the sulfate and glutathione conjugates were formed in much lower amounts.

Any other information on results incl. tables

The incubation of freshly isolated rat hepatocytes with eugenol elicited a cytotoxic response which was concentration and time-dependent. A concentration of 1 mM eugenol caused approximately 85% cell death over a 5-hour incubation period compared with 35% cell death in controls. At each concentration tested, the onset of cell death occurred at about 2 hours. The occurrence of cell death with 1 mM eugenol was prevented in the presence of 1 mM N-acetylcysteine.

The depletion of glutathione occurred rapidly and was less than 30% of control values after 2-hours incubation. Control cells maintained 90% of their original glutathione levels for up to 4 hours incubation but then began to lose significant amounts of glutathione between 4 and 5 hours. The addition of N-acetylcysteine was able to prevent loss of glutathione in incubations with 1mM eugenol similar to its protective effect on cytotoxicity.

Covalent binding of reactive metabolites of [3H]eugenol also was measured in these incubations. The amount of radioactivity covalently bound to cellular protein increased in a linear manner up to 3 h and then leveled off. The presence of N-acetylcysteine inhibited this covalent binding up to 3 hours. Separate experiments showed that at this time point the N-acetylcysteine was depleted which allowed the covalent binding to increase to control levels after the 3-hour time point. If N-acetylcysteine was replenished every 60 min the covalent binding was inhibited the entire 5-hour incubation period.

Applicant's summary and conclusion

Interpretation of results (migrated information): other: Toxicity of eugenol to isolated rat hepatocytes, as tested in vitro, is metabolism dependent and is due to the formation of a reactive intermediate, probably a quinone methide.
The major metabolite formed from the incubation of eugenol with rat hepatocytes was the glucuronic acid conjugate. Sulfate and glutathione conjugates also were formed. Covalent binding to cellular protein was observed using [3H]eugenol.
The authors have demonstrated that eugenol is actively metabolized by hepatocytes, that reactive intermediates are formed which covalently bind to cellular macromolecules, that eugenol is cytotoxic to hepatocytes in vitro, and the toxicity can be altered by agents which affect the reduced thiol status of these cells. These results suggest that the toxicity of eugenol to hepatocytes is metabolism dependent and is due to the formation of a reactive intermediate, probably a quinone methide.