4-trans-propenylveratrole

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in Iuclid Section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that the source and target substances have similar physico-chemical and toxicological properties because of their structural similarity (cis- and/or trans-isomers).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance is the trans isomer (E), as a mono-constituent substance. The source substance is a reaction mass, composed of two diastereoisomers (the target substance [trans] and its cis-isomer).

3. ANALOGUE APPROACH JUSTIFICATION
The source and the target substances have a common major constituent (trans-isomer) and the impurity of the target substance is the second major constituent of the source substance.
The available studies are well documented, meet generally accepted scientific principles, and are therefore acceptable for assessment. The test material was not clearly identified but it is assumed to represent the source substance in terms of constituents and impurities.
Therefore, based on the considerations above, it can be concluded that the results of the toxicokinetic studies conducted with the source substance are considered suitable to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VIII, 8.8.

4. DATA MATRIX
Cf. Iuclid Section 13. 

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

In this study, the oxidative metabolism of test material was studied using liver microsomes of rat, bovine, and human origin. Incubations of these microsomes with the test material provided phase I metabolites that were separated by high-performance liquid chromatography (HPLC) and identified by nuclear magnetic resonance (NMR) and UV-visible spectroscopy and/or liquid chromatography-mass spectrometry.

GLP compliance:

no

Radiolabelling:

no

Species:

other: rat, bovine and human microsomes

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Male Wistar rats were purchased from Charles River (Sulzbach, Germany). Animals were kept under a 12-h light/dark cycle and received water and commercial laboratory chow ad libitum. For inducing experiments, rats (b.wt. 180–250 g) were given a single dose of 500 mg/kg b.wt. Aroclor1254 i.p. from a stock solution of 200 mg/ml Aroclor1254 dissolved in corn oil. Five days after treatment the animals were sacrificed.
- Aroclor1254-treated rat (ARLM), noninduced rat (RLM), and bovine liver microsomes (BLM) were prepared from the liver of freshly sacrificed/slaughtered animals.
- Bovine livers were from 1.5- to 2-year-old female German Black Pied cattle (dairy cattle) and were obtained from a local slaughterhouse.
- Human liver microsomes (HLM) were purchased from BD Bioscience (Heidelberg, Germany) from a pool derived from 150 gender-mixed donors.

Vehicle:

DMSO

Remarks:

final concentration 5 %

Duration and frequency of treatment / exposure:

Incubations of microsomes with test substance with a NADPH-regenerating system for 24 h at 37 °C

Remarks:

Doses / Concentrations:
0.1, 0.5 and 2.5 mM

Details on dosing and sampling:

Incubations: Incubations of microsomes (2 mg of microsomal protein/mL) were performed with 0.1, 0.5, or 2.5 mM substrate dissolved in DMSO (final concentration 5%) with a NADPH-regenerating system. Incubations were carried out in various volumes (from 1 to 100 mL for preparative uses) and were gently shaken in an incubator at 37 °C. After preincubation of microsomes with the substrate for 5 min at 37 °C, the NADPH-generating system (1 mM NADPH, 0.5 units glucose-6-phosphate dehydrogenase, 5 mM glucose 6-phosphate, 3 mM magnesium chloride, and 50 mM phosphate buffer, pH 7.4) was added and the mixture was incubated for up to 24 h. Control incubations were carried out with heat-inactivated microsomes and with intact microsomes but without the NADPH- regenerating system, respectively. Incubations and time-course measurements were done in triplicate as independent experiments. The high concentration of DMSO (5%) was applied to improve solubility of the substrate, in particular of isolated metabolites. Although DMSO is known to act as a P450 inhibitor, concentrations of metabolites in control incubations of test material using 0.5% DMSO were found not to be significantly different (data not shown).

Sample Preparation. After incubation, samples were diluted with equal volumes of a ice-cold solution (-20 °C) of acetone containing 0.1 or 0.5 mM dihydromethyleugenol as an internal standard. Samples were cooled for 20 min on ice. The mixtures were shaken intensely, and the precipitated protein was removed by centrifugation at 13,000g for 5 min. For time-course measurements, the supernatants were used directly for HPLC analysis. The supernatants of single incubations were extracted five times with an equal volume of ethyl acetate. The solvent of the combined organic phases was removed, and the residue was taken up in a defined volume of methanol before further analysis.

Statistics:

Not applicable

Preliminary studies:

Not applicable

Details on absorption:

No data

Details on distribution in tissues:

No data

Details on excretion:

No data

Metabolites identified:

yes

Details on metabolites:

- Human, bovine, and rat (Aroclor1254-induced and non-induced) liver microsomes incubated with methyl isoeugenol in the presence of a NADPH generating system provided phase I metabolites that were separated by high-performance liquid chromatography (HPLC) and identified by NMR and UV-visible spectroscopy and/or liquid chromatography-mass spectrometry. Identity was confirmed by comparison with 1H NMR spectra of synthesized reference compounds. Formation of metabolites was quantified by HPLC/UV using dihydromethyleugenol synthesized as the internal standard.
- From incubations of ARLM with methyl isoeugenol, seven metabolites could be detected, with 3’-hydroxymethylisoeugenol, isoeugenol and isochavibetol, and 6-hydroxymethylisoeugenol being the main metabolites. Secondary metabolites derived from methyl isoeugenol were identified as the α, β - unsaturated aldehyde 3’-oxomethylisoeugenol and 1’,2’-dihydroxy-dihydromethylisoeugenol. It was also found that formation of allylic 6-hydroxymethyleugenol was observed starting at approximately 30 min after the beginning of incubations with ARLM.
- HLM did not form ring-hydroxylated metabolites but were most active in the formation of 3’-oxomethylisoeugenol and 1’,2’-dihydroxy-dihydromethylisoeugenol.
- ARLM incubations displayed the highest turnover rate and broadest metabolic pattern, presumably resulting from an increased expression of cytochrome P450 enzymes.
- Incubations without the NADPH-generating system did not produce metabolites in detectable amounts (data not shown). Relatively high concentrations (100 and 500 µM) of methyl isoeugenol were used with incubations to ensure detection of lesser-formed metabolites.

None

Conclusions:

Oxidative metabolism of methyl isoeugenol yielded several metabolites i.e., 3’-hydroxymethylisoeugenol, isoeugenol and isochavibetol, and 6-hydroxymethylisoeugenol being the main metabolites. Secondary metabolites derived from methyl isoeugenol were identified as the α, β - unsaturated aldehyde 3’-oxomethylisoeugenol and 1’,2’-dihydroxy-dihydromethylisoeugenol.

Executive summary:

In the metabolism study, the oxidative metabolism of test material was studied using liver microsomes of rat (Non-induced rat liver microsomes (RLM); Aroclor1254 induced rat liver microsomes (ARLM)), bovine (BLM), and human (HLM; pooled from 150 donors) origin. Incubations of these microsomes with substrate (0.1, 0.5, or 2.5 mM) in the presence of a NADPH generating system provided phase I metabolites that were separated by high-performance liquid chromatography (HPLC) and identified by nuclear magnetic resonance (NMR) and UV-visible spectroscopy and/or liquid chromatography-mass spectrometry. Formation of metabolites was quantified by HPLC/UV using dihydromethyleugenol synthesized as the internal standard.

 

From incubations of ARLM with methyl isoeugenol, seven metabolites could be detected, with 3’-hydroxymethylisoeugenol, isoeugenol and isochavibetol, and 6-hydroxymethylisoeugenol being the main metabolites. Secondary metabolites derived from methyl isoeugenol were identified as the α, β - unsaturated aldehyde 3’-oxomethylisoeugenol and 1’,2’-dihydroxy-dihydromethylisoeugenol. It was also found that formation of allylic 6-hydroxymethyleugenol was observed starting at approximately 30 min after the beginning of incubations with ARLM. HLM did not form ring-hydroxylated metabolites but were most active in the formation of the secondary metabolites 3’-oxomethylisoeugenol and 1’,2’-dihydroxy-dihydromethylisoeugenol. ARLM incubations displayed the highest turnover rate and broadest metabolic pattern, presumably resulting from an increased expression of cytochrome P450 enzymes; and lower turnover rate in HLM, BLM, and RLM.

Cross-reference 2

Reason / purpose for cross-reference:

read-across: supporting information

Materials and methods

Test material

Test material information

1

Reference substance name:

4-trans-propenylveratrole

EC Number:

228-958-7

EC Name:

4-trans-propenylveratrole

Cas Number:

6379-72-2

Molecular formula:

C11H14O2

IUPAC Name:

(E)-1,2-Dimethoxy-4-prop-1-en-1-ylbenzene

Results and discussion

Preliminary studies:

Not applicable

Toxicokinetic / pharmacokinetic studies

Details on absorption:

No data

Details on distribution in tissues:

No data

Details on excretion:

No data

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

- Human, bovine, and rat (Aroclor1254-induced and non-induced) liver microsomes incubated with methyl isoeugenol in the presence of a NADPH generating system provided phase I metabolites that were separated by high-performance liquid chromatography (HPLC) and identified by NMR and UV-visible spectroscopy and/or liquid chromatography-mass spectrometry. Identity was confirmed by comparison with 1H NMR spectra of synthesized reference compounds. Formation of metabolites was quantified by HPLC/UV using dihydromethyleugenol synthesized as the internal standard.
- From incubations of ARLM with methyl isoeugenol, seven metabolites could be detected, with 3’-hydroxymethylisoeugenol, isoeugenol and isochavibetol, and 6-hydroxymethylisoeugenol being the main metabolites. Secondary metabolites derived from methyl isoeugenol were identified as the α, β - unsaturated aldehyde 3’-oxomethylisoeugenol and 1’,2’-dihydroxy-dihydromethylisoeugenol. It was also found that formation of allylic 6-hydroxymethyleugenol was observed starting at approximately 30 min after the beginning of incubations with ARLM.
- HLM did not form ring-hydroxylated metabolites but were most active in the formation of 3’-oxomethylisoeugenol and 1’,2’-dihydroxy-dihydromethylisoeugenol.
- ARLM incubations displayed the highest turnover rate and broadest metabolic pattern, presumably resulting from an increased expression of cytochrome P450 enzymes.
- Incubations without the NADPH-generating system did not produce metabolites in detectable amounts (data not shown). Relatively high concentrations (100 and 500 µM) of methyl isoeugenol were used with incubations to ensure detection of lesser-formed metabolites.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Oxidative metabolism of the source substance (1,2-Dimethoxy-4-prop-1-en-1-ylbenzene) yielded several metabolites i.e., 3’-hydroxymethylisoeugenol, isoeugenol and isochavibetol, and 6-hydroxymethylisoeugenol being the main metabolites. Secondary metabolites derived from methyl isoeugenol were identified as the α, β - unsaturated aldehyde 3’-oxomethylisoeugenol and 1’,2’-dihydroxy-dihydromethylisoeugenol.

Executive summary:

In the metabolism study, the oxidative metabolism of the source substance (1,2-Dimethoxy-4-prop-1-en-1-ylbenzene) was studied using liver microsomes of rat (Non-induced rat liver microsomes (RLM); Aroclor1254 induced rat liver microsomes (ARLM)), bovine (BLM), and human (HLM; pooled from 150 donors) origin. Incubations of these microsomes with substrate (0.1, 0.5, or 2.5 mM) in the presence of a NADPH generating system provided phase I metabolites that were separated by high-performance liquid chromatography (HPLC) and identified by nuclear magnetic resonance (NMR) and UV-visible spectroscopy and/or liquid chromatography-mass spectrometry. Formation of metabolites was quantified by HPLC/UV using dihydromethyleugenol synthesized as the internal standard.

 

From incubations of ARLM with the source substance (1,2-Dimethoxy-4-prop-1-en-1-ylbenzene), seven metabolites could be detected, with 3’-hydroxymethylisoeugenol, isoeugenol and isochavibetol, and 6-hydroxymethylisoeugenol being the main metabolites. Secondary metabolites derived from methyl isoeugenol were identified as the α, β - unsaturated aldehyde 3’-oxomethylisoeugenol and 1’,2’-dihydroxy-dihydromethylisoeugenol. It was also found that formation of allylic 6-hydroxymethyleugenol was observed starting at approximately 30 min after the beginning of incubations with ARLM. HLM did not form ring-hydroxylated metabolites but were most active in the formation of the secondary metabolites 3’-oxomethylisoeugenol and 1’,2’-dihydroxy-dihydromethylisoeugenol. ARLM incubations displayed the highest turnover rate and broadest metabolic pattern, presumably resulting from an increased expression of cytochrome P450 enzymes; and lower turnover rate in HLM, BLM, and RLM.