4-trans-propenylveratrole

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1976

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 metabolism study, metabolites of Isoeugenol methyl ether in the rat was identified and quantitatively determined by GLC and GLC-Mass spectrometry.

GLP compliance:

no

Species:

rat

Strain:

Wistar

Sex:

male

Route of administration:

other: oral and intraperitoneal

Vehicle:

not specified

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Control animals:

no

Positive control reference chemical:

Not applicable

Details on dosing and sampling:

- Animals were treated as described previously (Solheim & Scheline, 1973) except that 200 mg/kg bw doses were used in the quantitative experiments.
METABOLITE CHARACTERISATION STUDIES
- Extraction of metabolites: The urine and bile samples were treated with Glusulase and extracted with ether according to the method described previously (Solheim & Scheline, 1973).
- Quantitative determination of metabolites: p-Methoxybenzoic acid (2 mg) was used as internal standard and was added to the thawed urine samples. The urines were treated as described previously (Solheim & Scheline, 1973). Experiments were also carried out to assess the degree of recovery of the major metabolites in comparison with that of the internal standard when employing the extraction method described (Solheim & Scheline, 1973).
- Incubation with caecal extracts: Incubation of the test compounds under anaerobic conditions with rat caecal micro-organisms was carried out as described previously (Scheline, 1966, 1968).
- Method type(s) for identification: GLC and GLC-mass spectrometry

Statistics:

None

Preliminary studies:

None

Type:

metabolism

Results:

When administered to rats by oral and intraperitoneal routes, the substance extensively metabolized in vivo and eliminated as transformation products

Details on absorption:

None

Details on distribution in tissues:

None

Details on excretion:

None

Metabolites identified:

yes

Details on metabolites:

- The metabolites of 3,4-dimethoxypropenylbenzene (isoeugenol methyl ether) in the rat were identified and quantitatively determined by g.l.c. and g.l.c.-mass spectrometry.
- The major metabolic pathway of 3,4-dimethoxypropenylbenzene was via the cinnamoyl derivatives, leading to the formation of 4-hydroxy-3-methoxycinnamic acid (ferulic acid), 3,4-dimethoxycinnamic acid and 3,4-dimethoxybenzoic acid, the two latter being excreted largely as their glycine conjugates. Other reactions were O-demethylation to 4-hydroxy-3-methoxypropenylbenzene (isoeugenol) and 3-hydroxy-4-methoxypropenylbenzene in equal amounts, and oxidation to 3,4-dimethoxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetone. Epoxidation of the side chain appeared to be a minor metabolic reaction with the propenyl derivative. The biliary metabolites of 3,4-dimethoxypropenylbenzene were identified and most of the urinary metabolites were also found in the bile. 1-(3,4-Dihydroxyphenyl)propane was detected in the extracts of incubates of 3,4- dimethoxypropenylbenzene with rat caecal microorganisms.
- Major urinary metabolites were excreted within 24 h, i.e., 53 and 79 % of metabolites from untreated and hydrolysed urine samples, respectively (oral administration); 55 and 90 % of metabolites from untreated and hydrolysed urine samples, respectively (i.p. administration). Some of these metabolites were detected in trace amounts only, after 24 h.

Bioaccessibility (or Bioavailability) testing results:

None

None

Conclusions:

When administered to rats by oral and intraperitoneal routes, the substance extensively metabolized in vivo and eliminated as transformation products.

Executive summary:

In this metabolism study, rats were administered with 3,4-dimethoxypropenylbenzene (isoeugenol methyl ether) at 100, 200 and 400 mg/kg bw by oral or intraperitoneal route. Metabolites in urine and bile samples were identified and quantitatively determined by GLC and GLC-Mass spectrometry.

 

The major metabolic pathway of 3,4-dimethoxypropenylbenzene was via the cinnamoyl derivatives, leading to the formation of 4-hydroxy-3-methoxycinnamic acid (ferulic acid), 3,4-dimethoxycinnamic acid and 3,4-dimethoxybenzoic acid, the two latter being excreted largely as their glycine conjugates. Other reactions were O-demethylation to 4-hydroxy-3-methoxypropenylbenzene (isoeugenol) and 3-hydroxy-4-methoxypropenylbenzene in equal amounts, and oxidation to 3,4-dimethoxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetone. Epoxidation of the side chain appeared to be a minor metabolic reaction with the propenyl derivative. Major urinary metabolites were excreted within 24 h, i.e., 53 and 79 % of metabolites from untreated and hydrolysed urine samples, respectively (oral administration); 55 and 90 % of metabolites from untreated and hydrolysed urine samples, respectively (i.p. administration).The biliary metabolites of 3,4-dimethoxypropenylbenzene were identified and most of the urinary metabolites were also found in the bile. 1-(3,4-Dihydroxyphenyl)propane was detected in the extracts of incubates of 3,4 - dimethoxypropenylbenzene with rat caecal microorganisms.

When administered to rats by oral and intraperitoneal routes, the substance extensively metabolized in vivo and eliminated as transformation products.

Reference 3

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. 

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

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 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.

Reference 4

Endpoint:

basic toxicokinetics in 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. 

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Preliminary studies:

None

Type:

metabolism

Results:

When administered to rats by oral and intraperitoneal routes, the substance extensively metabolized in vivo and eliminated as transformation products

Details on absorption:

None

Details on distribution in tissues:

None

Details on excretion:

None

Metabolites identified:

yes

Details on metabolites:

- The metabolites of 3,4-dimethoxypropenylbenzene (isoeugenol methyl ether) in the rat were identified and quantitatively determined by g.l.c. and g.l.c.-mass spectrometry.
- The major metabolic pathway of 3,4-dimethoxypropenylbenzene was via the cinnamoyl derivatives, leading to the formation of 4-hydroxy-3-methoxycinnamic acid (ferulic acid), 3,4-dimethoxycinnamic acid and 3,4-dimethoxybenzoic acid, the two latter being excreted largely as their glycine conjugates. Other reactions were O-demethylation to 4-hydroxy-3-methoxypropenylbenzene (isoeugenol) and 3-hydroxy-4-methoxypropenylbenzene in equal amounts, and oxidation to 3,4-dimethoxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetone. Epoxidation of the side chain appeared to be a minor metabolic reaction with the propenyl derivative. The biliary metabolites of 3,4-dimethoxypropenylbenzene were identified and most of the urinary metabolites were also found in the bile. 1-(3,4-Dihydroxyphenyl)propane was detected in the extracts of incubates of 3,4- dimethoxypropenylbenzene with rat caecal microorganisms.
- Major urinary metabolites were excreted within 24 h, i.e., 53 and 79 % of metabolites from untreated and hydrolysed urine samples, respectively (oral administration); 55 and 90 % of metabolites from untreated and hydrolysed urine samples, respectively (i.p. administration). Some of these metabolites were detected in trace amounts only, after 24 h.

Bioaccessibility (or Bioavailability) testing results:

None

None

Conclusions:

When administered to rats by oral and intraperitoneal routes, based on the available data on the source substance, the target substance is expected to be extensively metabolized in vivo and eliminated as transformation products.

Executive summary:

In this metabolism study, rats were administered with the source substance (1,2-Dimethoxy-4-prop-1-en-1-ylbenzene) at 100, 200 and 400 mg/kg bw by oral or intraperitoneal route. Metabolites in urine and bile samples were identified and quantitatively determined by GLC and GLC-Mass spectrometry.

 

The major metabolic pathway of 3,4-dimethoxypropenylbenzene was via the cinnamoyl derivatives, leading to the formation of 4-hydroxy-3-methoxycinnamic acid (ferulic acid), 3,4-dimethoxycinnamic acid and 3,4-dimethoxybenzoic acid, the two latter being excreted largely as their glycine conjugates. Other reactions were O-demethylation to 4-hydroxy-3-methoxypropenylbenzene (isoeugenol) and 3-hydroxy-4-methoxypropenylbenzene in equal amounts, and oxidation to 3,4-dimethoxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetone. Epoxidation of the side chain appeared to be a minor metabolic reaction with the propenyl derivative. Major urinary metabolites were excreted within 24 h, i.e., 53 and 79 % of metabolites from untreated and hydrolysed urine samples, respectively (oral administration); 55 and 90 % of metabolites from untreated and hydrolysed urine samples, respectively (i.p. administration).The biliary metabolites of 3,4-dimethoxypropenylbenzene were identified and most of the urinary metabolites were also found in the bile. 1-(3,4-Dihydroxyphenyl)propane was detected in the extracts of incubates of 3,4 - dimethoxypropenylbenzene with rat caecal microorganisms.

When administered to rats by oral and intraperitoneal routes, based on the available data on the source substance, the target substance is expected to be extensively metabolized in vivo and eliminated as transformation products.

Description of key information

The available evidence suggests that the substance is bioavailable via the oral and dermal route. Systemic absorption of this substance via inhalation route is expected but to a limited extent. The substance is expected to be mainly excreted in urine.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

In accordance with the section 8.1.1 of Annex VIII of Regulation (EC) No 1907/2006 (REACH), the toxicokinetic profile of the substance (i.e. absorption, distribution, metabolism and elimination) was derived from the relevant available information collated in the dossier. The physical chemical characteristics of the substance, the results obtained from acute, repeated-dose, and reproductive toxicity studies, as well as information gained from genotoxicity assays were used to predict its toxicokinetic behaviour.

The JECFA evaluation (JECFA, 2004) was also used to support this toxicokinetic assessment.

Physical-chemical properties:

The substance is a mono-constituent, having a relatively low molecular weight of 178.23 g/mol. The substance is a moderately water soluble liquid (297 mg/L) and is moderately lipophilic based on the octanol/water partition coefficient (Log Kow = 2.9). The substance has low volatility according to its vapour pressure (0.5 Pa at 25°C).

Absorption:

Oral/GI absorption

The physical chemical characteristics described above suggest that the substance is absorbed in the gastro-intestinal tract by passive diffusion. This hypothesis is supported by oral toxicity data, as summarized below:

- In an acute oral gavage toxicity study, mortality was observed at high dose levels (≥ 2000 mg/kg bw; LD50 = 2500 mg/kg bw).

- The dietary 28-day repeated dose toxicity gave a NOAEL of equivalent to 275 mg/kg bw/day in male rats and 264 mg/kg bw/day in female rats (the highest dose-level tested in this study). Non-adverse treatment-related changes observed in liver, kidneys and haematological parameters suggest the systemic absorption of the test ite following oral administration.

- In the Reproduction/Developmental Toxicity Screening test, the NOAEL for males and females was 4500 ppm (the mid-dose level, equivalent to daily intakes between 295 and 702 mg/kg bw/day depending on the age and weight of the animals) on the basis that the animals of the 15000 ppm dose group suffered adverse effects on food consumption and weight gain, which were not fully alleviated in the recovery period. However, these effects are considered to be palatability-associated secondary effects so they are not directly relevant to support oral absorption of the test item.

Taken together, the observation of systemic effects indicates the oral bioavailability of the substance and/or its metabolites. In light of these data, and the lack of specific information on oral absorption, the substance was assumed to be 100% bioavailable by oral route for the purposes of human health risk assessment.

Dermal absorption

Regarding dermal absorption, systemic absorption by the dermal route is expected to be moderate to high based on the Log Kow and the water solubility values. This is supported by the mortality observed in the acute dermal toxicity study (LD50 > 5000 mg/kg bw).

An in vitro skin absorption study indicated that the percutaneous absorption level of the source substance (1,2-Dimethoxy-4-prop-1-en-1-ylbenzene) was not significant (Jimbo, 1983). Following 72 hours exposure, 0.327 ± 0.021 % of the applied dose (mean ± standard error) had permeated into the receptor phase. However this result is not considered as accurate due to the poor reliability of this study (Klimisch score = 3) which is strengthened by the different dermal absorption percentage reported by Shen et al. (2014) for other substances mentioned in Jimbo’s publication:

Isoeugenol 0.489 ± 0.029 % vs 38.4%;

Methyl eugenol 0.511 ± 0.038 % vs 49.7%;

Dihydroeugenol 1.364 ± 0.109 % vs 22.6%.

In light of these data, the substance was conservatively assumed to be 100% bioavailable by dermal route for the purposes of human health risk assessment.

Respiratory absorption

The potential for inhalation toxicity was not evaluated in vivo.

The vapour pressure of the substance (Vp = 0.5 Pa at 25°C) indicated a low volatility and inhalability and any exposure by inhalation is anticipated to be minimal. Thus, at ambient temperature, no respiratory absorption is expected under normal use and handling of the substance.

However, when used as a vapour in aerosol, the substance is expected to be directly absorbed across the respiratory tract epithelium by passive diffusion.

In light of these data, and the lack of specific information on respiratory absorption, the substance was conservatively assumed to be 100% bioavailable by inhalation for the purposes of human health risk assessment.

Distribution:

Any material that is absorbed will be distributed via the blood to the liver, and other organs and tissues. The moderate water solubility of the substance would limit distribution in the body via the water channels. The log Kow would suggest that the substance would pass through the biological cell membrane. Due to the expected metabolization, the substance as such would not accumulate in the body fat.

Metabolism:

The predominant metabolic pathways of the substance include O-demethylation leading to an isoeugenol derivative, and omega-oxidation of the terminal methyl group leading to a benzoic acid derivative. In O-demethylation, either (m)- or (p)-methoxy-substituted isoeugenol is converted to the corresponding isoeugenol derivative containing a free phenolic OH group. The phenol is then excreted in the urine as the sulfate or glucuronic acid conjugate. Carbon dioxide produced by O-demethylation is eliminated in the expired air. At low doses, this is the predominant detoxication pathway in animals. As doses increase, omega-oxidation, and to a lesser extent, epoxidation of the propenyl side-chain compete favourably with O-demethylation. Other minor metabolites of these three substituted isoeugenol derivatives can be formed. (JECFA, 2004)

In a study involving its administration to rats by oral and intraperitoneal routes, the source substance (1,2-Dimethoxy-4-prop-1-en-1-ylbenzene) was extensively metabolized in vivo and eliminated as transformation products (Solheim and Scheline, 1976). No unchanged compound was recovered from the urine compared to relatively large amounts of transformation products that could be isolated. The major metabolic pathway of 3,4-dimethoxypropenylbenzene was via the cinnamoyl derivatives, leading to the formation of 4-hydroxy-3-methoxycinnamic acid (ferulic acid), 3,4-dimethoxycinnamic acid and 3,4-dimethoxybenzoic acid, the two latter being excreted largely as their glycine conjugates. Other reactions were O-demethylation to 4-hydroxy-3-methoxypropenylbenzene (isoeugenol) and 3-hydroxy-4-methoxypropenylbenzene in equal amounts, and oxidation to 3,4-dimethoxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetone. Epoxidation of the side chain appeared to be a minor metabolic reaction with the propenyl derivative.

(The scheme for the proposed metabolism pathway described by Solheim & Scheline is attached to this endpoint summary into Iuclid section 7.1)

In an in vitro study (Cartus et al., 2011), the oxidative metabolism of the source substance (1,2-Dimethoxy-4-prop-1-en-1-ylbenzene) was studied using liver microsomes of non-induced rat, Aroclor1254 induced rat, bovine, and human origin. Formation of metabolites was quantified by HPLC/UV. From incubations of Aroclor 1254 induced rat liver microsomes 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 Aroclor1254 induced rat. Human liver microsomes did not form ring-hydroxylated metabolites but were most active in the formation of the secondary metabolites 3’-oxomethylisoeugenol and 1’,2’-dihydroxy-dihydromethylisoeugenol. Incubations with Aroclor1254-induced rat liver microsomes displayed the highest turnover rate and broadest metabolic pattern, presumably resulting from an increased expression of cytochrome P450 enzymes; and lower turnover rate in liver microsomes of non-induced rat, bovine and human origin.

Taken together, these data support the conclusion that the substance is rapidly absorbed, metabolized, and eliminated.

Excretion:

The substance, with a molecular weight lower than 300 g/mol, is expected to be mainly excreted in urine and no more than 5-10% may be excreted in bile. Any substance that is not absorbed from the gastro-intestinal tract, following oral ingestion, will be excreted in the faeces.

In groups of male Wistar rats given a single dose of 200 mg/kg bw of the source substance (1,2-Dimethoxy-4-prop-1-en-1-ylbenzene) by oral or 400 mg/kg bw by intraperitoneal administration, 79% and 90% of the administered dose, respectively, was eliminated in the urine within 24 hours (Solheim and Scheline, 1976). Subsequent analysis of the urine failed to detect any parent compound.

Carbon dioxide produced by O-demethylation is eliminated in the expired air.

Accumulative potential:

Based on the available data demonstrating the rapid absorption of the substance and its efficient metabolism and excretion, and taking into consideration the low molecular weight, moderate log Kow and water solubility values, the substance is not expected to bioaccumulate.

References:

- Cartus AT, Merz KH and Schrenk D. (2011). Metabolism of Methylisoeugenol in Liver Microsomes of Human, Rat, and Bovine Origin. Drug Metab Dispos. 39(9):1727-33.

- JECFA (2004). Safety evaluation of certain food additives and contaminants. Prepared by the Sixty-first meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). WHO FOOD ADDITIVES SERIES: 52.

- Jimbo Y (1983). Penetration of Fragrance Compounds Through Human Epidermis. The Journal of Dermatology, 10:229-239

- Shen J, Kromidas L, Bhata S (2014). An in silico skin absorption model for fragrance materials. Food and Chemical Toxicology, 74: 164-176.

- Solheim E and Scheline RR (1976). Metabolism of Alkenebenzene Derivatives in the Rat. II. Eugenol and Isoeugenol Methyl Ethers. Xenobiotica. 6(3):137-50.