4-allylveratrole

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Objective of study:

absorption

distribution

excretion

metabolism

toxicokinetics

Principles of method if other than guideline:

NTP study on toxicokinetics, absorption, distribution, metabolism and elimination.

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Elan Chemical Company (Newark, NJ) / Lot: 9224705

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The bulk chemical was stored at room temperature, protected from light in amber glass bottles with Teflon®-lined caps.
- Stability under test conditions: Accelerated stability test: Methyleugenol is stable as bulk chemical for 2 weeks when stored protected from light at temperatures up to 60 ºC.

Radiolabelling:

yes

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

F344/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 5 to 6 weeks old
- Housing: Individually (toxicokinetic tests) or three (males) or five (females) per cage (core test), in polycarbonate cages containing hardwood bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Between 11 and 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24 ºC,
- Humidity (%): 40-65%
- Air changes (per hr): minimum of 10/hour
- Photoperiod (hrs dark / hrs light): 12:12 hour light/dark cycle

Route of administration:

other: oral gavage and intravenous injection

Vehicle:

methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Toxicokinetics:
Oral gavage: The dose formulations were prepared by mixing methyleugenol with 0.5% aqueous methylcellulose to give the required concentrations.
Intravenous injection: Dose formulations were prepared by mixing methyleugenol with water:emulphor:ethanol (8:1:1).

Metabolism:
Oral gavage: The dose formulations were prepared by mixing [14C]-methyleugenol with corn oil.
Intravenous injection: Dose formulations were prepared by mixing [14C]-methyleugenol with ethanol:Emulphor:saline (10:10:80)

DOSE VOLUME: 2 mL/kg body weight by intravenous injection and 5 mL/kg by gavage.

Vehicle: Methylcellulose (USP/FCC grade)
Source: Fisher Scientific Company (St. Louis, MO, and Pittsburgh, PA)
Lots: 876672 and 946150

Homogeneity and stability were confirmed.

Duration and frequency of treatment / exposure:

Singe dose toxicokinetics and metabolism test: Single dose.
Core test: 5 days per week for 12 months and for the single-administration gavage studies in aged animals.

Dose / conc.:

37 mg/kg bw/day (nominal)

Remarks:

(intravenous injection or gavage)

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

(gavage)

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

(gavage)

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

(gavage)

Dose / conc.:

118 other: mg/kg (50 μCi/kg)

Remarks:

(gavage [14C]-methyleugenol)

Dose / conc.:

11.8 other: mg/kg (120 μCi/kg)

Remarks:

(intravenous [14C]-methyleugenol)

No. of animals per sex per dose / concentration:

Core study: 10 rats per sex and dose + 12-15 aged rats per sex
Toxicokinetics: 12 rats per sex and dose.
Metabolism: 3 male rats per dose (x3 = Metabolism, urinary metabolism profile, metabolites).

Control animals:

no

Details on dosing and sampling:

Core toxicokinetic test withinn the 2 year toxicity test:
10 male and 10 female rats administered 37, 75, 150, or 300 mg/kg were designated for toxicokinetic studies.
At 18 months, 12 to 15 previously undosed male and female rats and mice were given a single dose of 150 mg/kg (rats) for toxicokinetic studies in aged animals.

Singe dose toxicokinetic and metabolism test:
Toxicokinetics: Groups of 12 male and 12 female rats were administered a single intravenous injection of 37 mg/kg bw.
Toxicokinetics: Groups of 12 male and 12 female rats were administered a single dose of 37, 75, or 150 mg/kg bw by gavage.
Metabolism: A single dose of [14C]-methyleugenol (118 mg/kg, 50 μCi/kg) in corn oil (5 mL/kg) was administered orally to three male rats.
Metabolism: A single dose of [14C]-methyleugenol (11.8 mg/kg, 120 μCi/kg) in ethanol:Emulphor:saline (10:10:80, 2 mL/kg) was administered intravenously to three rats via an indwelling jugular vein cannula.

TOXICOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, serum or other tissues, cage washes, bile .
- Time and frequency of sampling:

Core toxicokinetics:
Blood was collected from rats at 6, 12, and 18 months at the following time points after dosing:
Rats
6 and 12 months:
37 mg/kg: 5, 15, 30, 60, 90, and/or 120 minutes
75 mg/kg: 5, 30, 90, 120, 240, 360, and/or 450 minutes
150 mg/kg: 5, 30, 90, 240, 360, 480, and/or 600 minutes
300 mg/kg: 5, 60, 120, 240, 360, 540, and/or 780 minutes
18 months:
Male:
37 mg/kg: 5, 15, 30, 60, 90, 120, and 240 minutes
75 mg/kg: 5, 15, 30, 60, 90, 120, 240, and 360 minutes
150 mg/kg: 5, 30, 90, 240, 360, 480, and 600 minutes
Female:
37 mg/kg: 5, 10, 15, 30, 45, 60, 90, 120, 180, and
240 minutes 75 mg/kg: 5, 15, 30, 60, 90, 120, 240, and 360 minutes
150 mg/kg: 5, 30, 60, 90, 120, 240, 360, 480, and 600 minutes

Single-Dose Toxicokinetics in Aged Animals:
Males: 5, 15, 30, 60, 120, 240, 360, 480, and 600 minutes
Females: 5, 10, 15, 30, 45, 60, 120, 240, 360, and 480 minutes

Singe dose toxicokinetics:
In the rat intravenous injection study, blood was collected from three males and three females per time point at 2, 5, 15, 30, 45, 90, 180, and 360 minutes after methyleugenol administration. In the rat gavage study, blood was collected from three males and three females per time point at 5, 15, 30, 60, 90, 120, 240, and 360 minutes after methyleugenol administration.

The red cell fraction was separated from the plasma by centrifugation, and the plasma was stored at -20 ºC until analysis for methyleugenol concentration.

Singe dose metabolism:
In the gavage study, urine (6, 12, 24, 48, and 72 hr) and feces (24, 48, and 72 hr) were collected and measured for radioactivity. Expired CO2 and organics were also analyzed for the presence of [14C]-equivalents over the 72-hour time course. At study termination various tissues were collected and then stored at -80 ºC until analysis. Blood, feces, and tissue samples were analyzed for total radioactivity using liquid scintillation counting of oxidized samples. Urine was also analyzed for the presence of parent and metabolites by HPLC.

In the intravenous study, blood samples were collected via the jugular cannula at selected time points (0, 1, 4, 8, 12, 15, 20, 30, 40, and 50 min, and 1, 6, 12, 24, 48, and 72 hr). The samples were either counted for radioactivity or extracted with ethyl acetate and immediately analyzed by HPLC. At study termination, blood was collected immediately from the posterior vena cava into a heparinized syringe and stored at -80 ºC until analysis. Blood and feces were analyzed for total radioactivity by scintillation counting of oxidized samples. Urine was also analyzed for the presence of parent and metabolites by HPLC.

Urinary metabolism:
Urine was collected at 6, 12, 24, 48, and 72 hours. Samples from individual animals were pooled for each time point and then analyzed by reverse phase HPLC.

Urinary matabolic profile:
Urine was collected at 6, 12, 24, 48, and 72 hours. Samples from individual animals from both 6 and 12 hours were pooled and then analyzed by HPLC using a Phenomenex Prodigy 5μ column. Fractions corresponding to the major metabolites were then collected, lyophilized, and subjected to LC-MS analysis. Putative structures were then assigned based on mass-spectral data for each metabolite, when possible.

Statistics:

Plasma concentration values were recorded for individual animals, and the mean ± standard error was calculated by sex, dose group, and time point using tables and graphic illustrations. Graphic illustrations include semilog plots of concentration versus time and area under the curve (AUC) versus dose. Values for AUC were calculated for each concentration-versus-time profile using the trapezoidal method. A software program (Sigma Plot, Version 5.0) was used to calculate the AUC values. Reported toxicokinetic parameters, i.e., Cmax, Tmax, and t½, are observed values only.

Details on absorption:

Absorption from oral doses was rapid, with peak plasma levels achieved within the first 5 minutes for all doses in males and females.

Details on distribution in tissues:

Methyleugenol and its metabolites were distributed preferentially to the liver 72 hours after gavage or intravenous administration of [14C]-methyleugenol to males. Blood ratios of methyleugenol-derived radioactivity were 2 to 3 in the liver, 0.9 to 1.4 in the kidney, and significantly less than 1 in all other tissues examined after 72 hours.

Details on excretion:

Approximately 85% of methyleugenol administered orally to males was eliminated in urine as parent or metabolites. Elimination of methyleugenol from the bloodstream was rapid and multiphasic, with initial half-lives on the order of 5 minutes and terminal half-lives on the order of 1 to 2 hours in males and females. No difference in the elimination of the parent compound between naive males and females was apparent with either young or aged animals. Male core study animals eliminated methyleugenol more rapidly at 6 and 12 months, with areas under the concentration versus time curve (AUCs) generally less than those for the naive animals. Females at all time points and males at 18 months had AUCs similar to those of naive animals. This suggests that metabolic induction may occur to a greater extent in males than in females. Plots of AUC versus dose were sublinear in males at 6 and 12 months, indicative of metabolic saturation at the higher doses at these time points, but approximately linear at 18 months. The increase in AUCs with age in the core study males and females is suggestive of an age-related decrease in methyleugenol metabolic capability.

Metabolites identified:

yes

Details on metabolites:

Methyleugenol was rapidly metabolized. Approximately 85% of methyleugenol orally administered to males was eliminated in urine as metabolites by 72 hours after dosing. Bioavailability of methyleugenol was low in both males and females, with less than 6% bioavailability at 37 mg/kg. This increased to approximately 13% at 75 mg/kg and 15% to 20% at 150 mg/kg. These findings suggest a strong, but saturable, first-pass metabolic effect, leading to a nonlinear relationship between dose and parent chemical dosimetry. No parent methyleugenol was found in urine from males dosed with methyleugenol orally or by intravenous injection. Hydroxylated, sulfated, and glucuronidated metabolites constituted the majority of metabolites detected in urine.

Toxicokinetic Parameters in Rats at the 6-, 12-, and 18-Month Interim Evaluations in the 2-Year Gavage Study of Methyleugenol:

	Dose (mg/kg)	Cmax (μg/mL)	Tmax (minutes)	AUC (μg/mL*min)
Male
Month 6	37	0.51	5	0.40
	75	0.43	5	0.82
	150	1.34	5	3.11
	300	4.03	5	7.57
Month 12	37	0.57	15	0.39
	75	0.51	5	1.03
	150	0.97	5	4.96
	300	22.5	5	14.3
Month 18	37	0.71	30	0.80
	75	8.31	5	2.55
	150	2.70	5	3.92
Female
Month 6	37	1.41	5	0.55
	75	2.46	5	1.34
	150	0.84	5	1.35
	300	3.11	5	3.24
Month 12	37	0.89	5	0.71
	75	2.05	5	1.74
	150	0.79	5	2.11
	300	1.78	5	4.26
Month 18	37	1.15	5	0.77
	75	1.29	5	0.81
	150	0.75	5	1.92

Cmax = maximum mean concentration;

Tmax = time of maximum mean concentration;

AUC = area under the curve calculated using the trapezoidal rule

Toxicokinetic Parameters in Aged Rats after a Single Gavage Dose of 150 mg/kg Methyleugenol:

	Cmax (μg/mL)	Tmax (minutes)	AUC (μg/mL*min)
Male	7.44	15	11.1
Female	13.0	5	12.5

Cmax = maximum mean concentration;

Tmax = time of maximum mean concentration;

AUC = area under the curve calculated using the trapezoidal rule

Summary of Toxicokinetic Data from a Single-Dose Intravenous and Oral Gavage Methyleugenol Study in F344/N Rats:

Route	Dose (mg/kg)	Cmax (μg/mL)	Tmax (minutes)	T1/2 (minutes)	AUC (μg/mL*min)	Absolute bioavailability (%)
Male
Intravenous	37	44.3	2	75	581.4	-
Gavage	37	0.656	5	60	33.5	5.8
Gavage	75	1.52	5	75	459.5	13.2
Gavage	150	3.84	5	115		19.5
Female
Intravenous	37	47.1	2	75	495.4	-
Gavage	37	1.14	5	95	27.0	5.5
Gavage	75	3.22	5	80	133.1	13.3
Gavage	150	8.30	5	105	307.9	15.3

Cmax = maximum mean concentration;

Tmax = time of maximum mean concentration;

t½ = elimination half-life;

AUC = area under the curve calculated using the trapezoidal rule

Bioavailability calculated as AUCoral/AUCIV × DoseIV/Doseoral × 100

- :Not applicable to intravenous dosing

Oral Studies of Methyleugenol in the Male Fischer 344 Rat:

Approximately 72% of the total administered radioactivity was excreted in the urine by 72 hours. Approximately 13% was recovered in feces, and less than 0.1% was recovered as [14C]-CO2 or expired [14C]-organics. [14C]-Equivalents determined in tissues accounted for less than 0.4% of the administered dose. Blood collected at 72 hours and oxidized contained approximately 0.1% of the administered dose. Less than 0.6% of the administered dose was accounted for at any time point in blood samples counted for radioactivity. Neither methyleugenol nor its metabolites were detected in the extracted blood samples analyzed by HPLC.

Intravenous Disposition and Metabolism of Methyleugenol in the Male Fischer 344 Rat:

Approximately 86% of the total administered radioactivity was excreted in the urine by 72 hours. Approximately 9% was recovered in feces. [14C]-Equivalents determined in tissues accounted for less than 0.4% of the administered dose. Blood collected at 72 hours contained approximately 0.1% of the administered dose. After 12 hours, radioactivity was still present in the blood for up to 48 hours, but at very low levels (200 to 400 dpm total). Parent methyleugenol was present in blood samples counted for radioactivity for the first six hours and not thereafter. During these 6 hours, the curves for total radioactivity and methyleugenol in blood were parallel. Possible metabolites were detected in the blood through 50 minutes but never exceeded 2.5% of the administered dose at any of the time points analyzed. Maximum metabolite recovery occurred at 8 minutes.

Urinary Metabolic Profiles for Methyleugenol:

With either route of administration, the majority of the metabolites were excreted in the urine within 24 hours of dosing.

Methyleugenol Metabolism:

Due to low amounts of radioactivity in the urine beginning at 24 hours, samples from 24 hours and later were not analyzed by HPLC. The metabolites suggest that methyleugenol can undergo demethylation, ring or side chain hydroxylation, and that these hydroxylated metabolites are subjected to sulfation or glucuronidation.

Conclusions:

Methyleugenol is rapidly absorbed following oral administration to rats and mice. The kinetic data are consistent with rapid clearance from the blood, metabolism in the liver, and excretion of the parent and various metabolites in the urine.

Executive summary:

Toxicokinetics studies of methyleugenol in male and female F244/N rats were conducted by National Toxicology Program (US Deptartment of Health and Human Serviced). Bioavailability studies, including intravenous administration, were also performed to determine blood levels of parent and major metabolites in the Fischer 344 rat. Tissue levels of radiolabeled compound following oral administration were determined. During the two years gavage study of methyleugenol, special study groups of 10 male and 10 female F344/N rats administered 37, 75, 150, or 300 mg/kg bw/day were designated for toxicokinetic studies. Toxicokinetcis parameters were evaluated at 6, 12 and 18 (except 300 mg/kg bw/day) months of exposure. Moreover, single-dose intravenous and oral gavage toxicokinetic studies of methyleugenol were performed in male and female F344/N. Groups of 12 male and 12 female rats were administered a single intravenous injection of 37 mg/kg bw; or a single dose of 37, 75, or 150 mg/kg bw. Blood was collected from three males and three females per time point. In a parallel, the absorption, distribution, metabolism, and excretion of methyleugenol following oral and intravenous exposure in male Fischer 344 rats were determined. A single dose of [14C]-methyleugenol (118 mg/kg, 50 μCi/kg) in corn oil (5 mL/kg) was administered orally to three male Fischer 344 rats. Urine and feces were collected and measured for radioactivity. Moreover, A single dose of [14C]-methyleugenol (11.8 mg/kg, 120 μCi/kg) in ethanol:Emulphor:saline (10:10:80, 2 mL/kg) was administered intravenously to three male Fischer 344 rats via an indwelling jugular vein cannula. Blood samples were collected via the jugular cannula at selected time points. The samples were either counted for radioactivity or extracted with ethyl acetate and immediately analyzed by HPLC. Blood and feces were analyzed for total radioactivity by scintillation counting of oxidized samples. Urine was also analyzed for the presence of parent and metabolites by HPLC. Major metabolites eliminated in the urine following oral administration were determined. Fractions corresponding to the major metabolites were then collected, lyophilized, and subjected to LC-MS analysis. Absorption from oral doses was rapid, with peak plasma levels achieved within the first 5 minutes for all doses in males and females. Methyleugenol and its metabolites were distributed preferentially to the liver 72 hours after gavage or intravenous administration of [14C]-methyleugenol to males. Blood ratios of methyleugenol-derived radioactivity were 2 to 3 in the liver, 0.9 to 1.4 in the kidney, and significantly less than 1 in all other tissues examined after 72 hours. Approximately 85% of methyleugenol administered orally to males was eliminated in urine as parent or metabolites. Elimination of methyleugenol from the bloodstream was rapid and multiphasic, with initial half-lives on the order of 5 minutes and terminal half-lives on the order of 1 to 2 hours in males and females. No difference in the elimination of the parent compound between naive males and females was apparent with either young or aged animals. Male core study animals eliminated methyleugenol more rapidly at 6 and 12 months, with areas under the concentration versus time curve (AUCs) generally less than those for the naive animals. Females at all time points and males at 18 months had AUCs similar to those of naive animals. This suggests that metabolic induction may occur to a greater extent in males than in females. Plots of AUC versus dose were sublinear in males at 6 and 12 months, indicative of metabolic saturation at the higher doses at these time points, but approximately linear at 18 months. The increase in AUCs with age in the core study males and females is suggestive of an age-related decrease in methyleugenol metabolic capability. Methyleugenol was rapidly metabolized. Approximately 85% of methyleugenol orally administered to males was eliminated in urine as metabolites by 72 hours after dosing. Bioavailability of methyleugenol was low in both males and females, with less than 6% bioavailability at 37 mg/kg. This increased to approximately 13% at 75 mg/kg and 15% to 20% at 150 mg/kg. These findings suggest a strong, but saturable, first-pass metabolic effect, leading to a nonlinear relationship between dose and parent chemical dosimetry. No parent methyleugenol was found in urine from males dosed with methyleugenol orally or by intravenous injection. Hydroxylated, sulfated, and glucuronidated metabolites constituted the majority of metabolites detected in urine. It can be concluded that Methyleugenol is rapidly absorbed following oral administration to rats and mice. The kinetic data are consistent with rapid clearance from the blood, metabolism in the liver, and excretion of the parent and various metabolites in the urine.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Objective of study:

absorption

distribution

excretion

metabolism

toxicokinetics

Principles of method if other than guideline:

NTP study on toxicokinetics, absorption, distribution, metabolism and elimination.

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Elan Chemical Company (Newark, NJ) / Lot: 9224705

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The bulk chemical was stored at room temperature, protected from light in amber glass bottles with Teflon®-lined caps.
- Stability under test conditions: Accelerated stability test: Methyleugenol is stable as bulk chemical for 2 weeks when stored protected from light at temperatures up to 60 ºC.

Radiolabelling:

yes

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Portage, MI)
- Age at study initiation: 6 to 7 weeks old
- Housing: Individually (except famales of the core test, 5 by cage), in polycarbonate cages containing hardwood bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: between 11 and 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24 ºC,
- Humidity (%): 40-65%
- Air changes (per hr): minimum of 10/hour
- Photoperiod (hrs dark / hrs light): 12:12 hour light/dark cycle

Route of administration:

other: oral gavage and intravenous injection

Vehicle:

methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Core test/Singe-dose toxicokinetics:
Oral gavage: The dose formulations were prepared by mixing methyleugenol with 0.5% aqueous methylcellulose to give the required concentrations.
Intravenous injection: Dose formulations were prepared by mixing methyleugenol with water:emulphor:ethanol (8:1:1).

Metabolism:
Oral gavage: The dose formulations were prepared by mixing [14C]-methyleugenol with corn oil.
Intravenous injection: Dose formulations were prepared by mixing [14C]-methyleugenol with ethanol:Emulphor:saline (10:10:80)

DOSE VOLUME:
Core test: 2 mL/kg body weight by intravenous injection and 5 mL/kg by gavage.
Singe-dose toxicokinetics: 4 mL/kg body weight by intravenous injection and 10 mL/kg body weight by gavage.

Vehicle: Methylcellulose (USP/FCC grade)
Source: Fisher Scientific Company (St. Louis, MO, and Pittsburgh, PA)
Lots: 876672 and 946150

Homogeneity and stability were confirmed.

Duration and frequency of treatment / exposure:

Singe dose toxicokinetics and metabolism test: Single dose.
Core test: 5 days per week for 12 months and for the single-administration gavage studies in aged animals.

Dose / conc.:

37 mg/kg bw/day (nominal)

Remarks:

(core test: intravenous injection or gavage)

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

(core test: gavage)

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

(core test: gavage)

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

(single dose toxicokinetics: intravenous injection or gavage)

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

(single dose toxicokinetics: gavage)

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

(single dose toxicokinetics: gavage)

Dose / conc.:

118 other: mg/kg (50 μCi/kg)

Remarks:

(metabolism: gavage [14C]-methyleugenol)

No. of animals per sex per dose / concentration:

Core study: 10 mice per sex and dose + 12-15 aged rats per sex.
Toxicokinetics: 24 mice per sex and dose.
Metabolism: 3 female mice per dose (x3 = Metabolism, urinary metabolism profile, metabolites).

Control animals:

no

Details on dosing and sampling:

Core toxicokinetic test withinn the 2 year toxicity test:
10 male and 10 female mice administered 37, 75 and 150 mg/kg were designated for toxicokinetic studies.
At 18 months, 12 to 15 previously undosed male and female mice and mice were given a single dose of 75 mg/kg for toxicokinetic studies in aged animals.

Singe dose toxicokinetic and metabolism test:
Toxicokinetics: Groups of 24 male and 24 female mice were administered a single intravenous injection of 25 mg/kg bw.
Toxicokinetics: Groups of 24 male and 24 female mice were administered a single dose of 25, 50, or 75 mg/kg bw by gavage.
Metabolism: A single dose of [14C]-methyleugenol (118 mg/kg, 50 μCi/kg) in corn oil (5 mL/kg) was administered orally to three female mice.

TOXICOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, serum or other tissues, cage washes, bile .
- Time and frequency of sampling:

Core toxicokinetics:
Blood was collected from mice at 12 months at the following time points after dosing:
Male:
37 mg/kg: 5, 10, 20, 30, 40, 60, and 75 minutes
75 mg/kg: 5, 15, 30, 45, 60, 90, and 150 minutes
150 mg/kg: 5, 15, 40, 75, 120, 210, and 300 minutes
Female:
37 mg/kg: 5, 10, 20, 30, 40, 50, and 60 minutes
75 mg/kg: 5, 15, 30, 45, 60, 90, and 150 minutes
150 mg/kg: 5, 15, 40, 120, 180, and 240 minutes

Single-Dose Toxicokinetics in Aged Animals:
Male and female: 5, 20, 50, 90, and 150 minutes

Singe dose toxicokinetics:
In the mouse intravenous injection study, blood was collected from two to four mice per time point at 2, 5, 15, 30, 45, 60, 180, and 300 minutes after methyleugenol administration. In the mouse gavage study, blood was collected from three males and three females per time point at 5, 15, 30, 45, 60, 90, 120, and 240 minutes after methyleugenol administration.

The red cell fraction was separated from the plasma by centrifugation, and the plasma was stored at -20 ºC until analysis for methyleugenol concentration.

Singe dose metabolism:
In the gavage study, urine (6, 12, 24, 48, and 72 hr) and feces (24, 48, and 72 hr) were collected and measured for radioactivity. Expired CO2 and organics were also analyzed for the presence of [14C]-equivalents over the 72-hour time course. At study termination various tissues were collected and then stored at -80 ºC until analysis. Blood, feces, and tissue samples were analyzed for total radioactivity using liquid scintillation counting of oxidized samples. Urine was also analyzed for the presence of parent and metabolites by HPLC.

Urinary metabolism:
Urine was collected at 6, 12, 24, 48, and 72 hours. Samples from individual animals were pooled for each time point and then analyzed by reverse phase HPLC.

Urinary matabolic profile:
Urine was collected at 6, 12, 24, 48, and 72 hours. Samples from individual animals from both 6 and 12 hours were pooled and then analyzed by HPLC using a Phenomenex Prodigy 5μ column. Fractions corresponding to the major metabolites were then collected, lyophilized, and subjected to LC-MS analysis. Putative structures were then assigned based on mass-spectral data for each metabolite, when possible.

Statistics:

Plasma concentration values were recorded for individual animals, and the mean ± standard error was calculated by sex, dose group, and time point using tables and graphic illustrations. Graphic illustrations include semilog plots of concentration versus time and area under the curve (AUC) versus dose. Values for AUC were calculated for each concentration-versus-time profile using the trapezoidal method. A software program (Sigma Plot, Version 5.0) was used to calculate the AUC values. Reported toxicokinetic parameters, i.e., Cmax, Tmax, and t½, are observed values only.

Details on absorption:

Absorption from oral doses was rapid, with peak plasma levels achieved within the first 5 minutes for all doses in males and females.

Details on distribution in tissues:

Methyleugenol and its metabolites were distributed preferentially to the ovaries, stomach, fat, spleen, and liver 72 hours after oral administration of [14C]-methyleugenol to males. Tissue:blood ratios of methyleugenol-derived radioactivity were approximately 5 in the liver, 5 to 9 in the stomach, 7 in the fat and the spleen, and over 100 in the ovaries after 72 hours. Many other tissues had elevated ratios; this may represent residual binding of metabolites rather than tissue solubility.

Details on excretion:

Elimination of methyleugenol from the bloodstream was rapid and multiphasic, with terminal half-lives on the order of 15 to 30 minutes. No difference in the elimination of methyleugenol between naive males and females was apparent with either young or aged animals. Aged females exhibited a significantly higher AUC; this may be due to differences in the amount of body fat or an age-related decrease in metabolic capability. Core study animals eliminated methyleugenol with AUCs similar to those of the naive animals. Exceptions were for the low-dose females. The AUCs increased linearly with dose in females at 12 months and sublinearly in males at 12 months. The latter finding is indicative of metabolic saturation at the higher doses in males at this time point.

Metabolites identified:

yes

Details on metabolites:

Approximately 85% of methyleugenol orally administered to females was eliminated in urine as parent or metabolites by 72 hours after dosing. Bioavailability of methyleugenol was low, with 3% to 5% bioavailability at 25 mg/kg. This increased to approximately 12% at 50 mg/kg and 13% to 19% at 75 mg/kg. These findings suggest a strong, but saturable, first-pass metabolic effect, leading to a nonlinear relationship between dose and parent chemical dosimetry. No unchanged methyleugenol was found in urine from females dosed with methyleugenol orally. Hydroxylated, sulfated, and glucuronidated metabolites constituted a minority of the metabolites detected in urine, with the majority unknown.

Toxicokinetic Parameters in Mice at the 12-Month Interim Evaluation in the 2-Year Gavage Study of Methyleugenol:

	Dose (mg/kg)	Cmax (μg/mL)	Tmax (minutes)	T1/2 (minutes)	AUC (μg/mL*min)
Male	37	0.79	5	49.3	15.1
	75	2.53	5	99.3	45.0
	150	4.03	5	174.1	167.9
Female	37	2.03	5	45.3	28.3
	75	3.03	5	87.8	56.6
	150	4.69	5	120.7	123.2

Cmax = maximum mean concentration;

Tmax = time of maximum mean concentration;

AUC = area under the curve calculated using the trapezoidal rule

Toxicokinetic Parameters in Aged Mice after a Single Gavage Dose of 75 mg/kg Methyleugenol:

	Cmax (μg/mL)	Tmax (minutes)	T1/2 (minutes)	AUC (μg/mL*min)
Male	1.54	5	74.4	48.4
Female	3.30	5	76.3	119.4

Cmax = maximum mean concentration;

Tmax = time of maximum mean concentration;

AUC = area under the curve calculated using the trapezoidal rule

Summary of Toxicokinetic Data from a Single-Dose Intravenous and Oral Gavage Methyleugenol Study in B6C3F1 Mice:

Route	Dose (mg/kg)	Cmax (μg/mL)	Tmax (minutes)	T1/2 (minutes)	AUC (μg/mL*min)	Absolute bioavailability (%)
Male
Intravenous	25	18.2	2	15	116.4	-
Gavage	25	0.382	5	30	4.91	4.2
Gavage	50	1.40	5	30	27.4	11.8
Gavage	75	3.10	5	30	48.4	13.9
Female
Intravenous	25	9.34	2	15	106.5	-
Gavage	25	0.123	15	30	3.27	3.1
Gavage	50	1.01	5	30	25.0	11.7
Gavage	75	4.39	5	30	60.5	18.9

Cmax = maximum mean concentration;

Tmax = time of maximum mean concentration;

t½ = elimination half-life;

AUC = area under the curve calculated using the trapezoidal rule

Bioavailability calculated as AUCoral/AUCIV × DoseIV/Doseoral × 100

- :Not applicable to intravenous dosing

Oral Study of Methyleugenol in the Female B6C3F1 Mouse:

Approximately 85% of the total administered radioactivity was excreted in the urine by 72 hours. Approximately 6% was recovered in feces, and less than 0.1% was recovered as [14C]-CO2 or expired [14C]-organics. [14C]-Equivalents determined in tissues accounted for less than 0.3% of the administered dose. Blood collected at 72 hours contained less than 0.02% of the administered dose.

Urinary Metabolic Profiles for Methyleugenol:

With either route of administration, the majority of the metabolites were excreted in the urine within 24 hours of dosing.

Methyleugenol Metabolism:

Due to low amounts of radioactivity in the urine beginning at 24 hours, samples from 24 hours and later were not analyzed by HPLC. The metabolites in rats and mice suggest that methyleugenol can undergo demethylation, ring or side chain hydroxylation, and that these hydroxylated metabolites are subjected to sulfation or glucuronidation.

Conclusions:

Methyleugenol is rapidly absorbed following oral administration to rats and mice. The kinetic data are consistent with rapid clearance from the blood, metabolism in the liver, and excretion of the parent and various metabolites in the urine.

Executive summary:

Toxicokinetics studies of methyleugenol in male and female B6C3F1 Mice were conducted by National Toxicology Program (US Deptartment of Health and Human Serviced). Bioavailability studies, including intravenous administration, were also performed to determine blood levels of parent and major metabolites in the B6C3F1 Micea. Tissue levels of radiolabeled compound following oral administration were determined. During the two years gavage study of methyleugenol, special study groups of 10 male and 10 female mice administered 37, 75 and 150 mg/kg bw/day were designated for toxicokinetic studies. Toxicokinetcis parameters were evaluated at 12 months of exposure. Moreover, single-dose intravenous and oral gavage toxicokinetic studies of methyleugenol were performed in male and female mice. Groups of 24 male and 24 female rats were administered a single intravenous injection of 25 mg/kg bw; or a single dose of 25, 50, or 75 mg/kg bw by gavage. Blood was collected from three males and three females per time point. In a parallel, the absorption, distribution, metabolism, and excretion of methyleugenol following oral exposure in female mice were determined. A single dose of [14C]-methyleugenol (118 mg/kg, 50 μCi/kg) in corn oil was administered orally to three female mice. Urine and feces were collected and measured for radioactivity. Urine was also analyzed for the presence of parent and metabolites by HPLC. Major metabolites eliminated in the urine following oral administration were determined. Fractions corresponding to the major metabolites were then collected, lyophilized, and subjected to LC-MS analysis. Absorption from oral doses was rapid, with peak plasma levels achieved within the first 5 minutes for all doses in males and females. Methyleugenol and its metabolites were distributed preferentially to the ovaries, stomach, fat, spleen, and liver 72 hours after oral administration of [14C]-methyleugenol to males. Tissue:blood ratios of methyleugenol-derived radioactivity were approximately 5 in the liver, 5 to 9 in the stomach, 7 in the fat and the spleen, and over 100 in the ovaries after 72 hours. Many other tissues had elevated ratios; this may represent residual binding of metabolites rather than tissue solubility. Elimination of methyleugenol from the bloodstream was rapid and multiphasic, with terminal half-lives on the order of 15 to 30 minutes. No difference in the elimination of methyleugenol between naive males and females was apparent with either young or aged animals. Aged females exhibited a significantly higher AUC; this may be due to differences in the amount of body fat or an age-related decrease in metabolic capability. Core study animals eliminated methyleugenol with AUCs similar to those of the naive animals. Exceptions were for the low-dose females. The AUCs increased linearly with dose in females at 12 months and sublinearly in males at 12 months. The latter finding is indicative of metabolic saturation at the higher doses in males at this time point. Approximately 85% of methyleugenol orally administered to females was eliminated in urine as parent or metabolites by 72 hours after dosing. Bioavailability of methyleugenol was low, with 3% to 5% bioavailability at 25 mg/kg. This increased to approximately 12% at 50 mg/kg and 13% to 19% at 75 mg/kg. These findings suggest a strong, but saturable, first-pass metabolic effect, leading to a nonlinear relationship between dose and parent chemical dosimetry. No unchanged methyleugenol was found in urine from females dosed with methyleugenol orally. Hydroxylated, sulfated, and glucuronidated metabolites constituted a minority of the metabolites detected in urine, with the majority unknown.

Reference 3

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Objective of study:

metabolism

Principles of method if other than guideline:

The objective of this study was to compare the kinetics of phase I and phase II metabolism in the bioactivation and detoxification of methyleugenol in different species. The metabolic routes of [14C]-methyleugenol were investigated in human, rat, and mouse, using in vitro hepatic and pulmonary pooled subcellular fractions. The formation of the 1′-hydroxy proximate carcinogen and the cytotoxic quinone methide were quantified and kinetic parameters (appKm, appVmax, and CLint) were calculated.

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Charles River Laboratories (Edinburgh, UK).

Radiolabelling:

yes

Details on species / strain selection:

Human (NS), Mouse (ICR/CD-1) and Rat (sprague dawley) liver and lung tissues.

Sex:

male/female

Metabolites identified:

yes

Details on metabolites:

The 1′-hydroxy metabolite (RT: 16.6 min) and phenoxy-glucuronide conjugates (RT: 18.1) of [14C]-methyleugenol were identified. The identities of the other soluble metabolites in the profile were not investigated for the purpose of this study which essentially focused on the 1′-hydroxy proximate carcinogen and the quinone methide.

Kinetic comparison of 1′-hydroxylation and covalent binding in hepatic microsomes of three species:

appKm is the substrate concentration at which the reaction rate (appVmax) reaches half its maximum value. A smaller

appKm value indicates that a reaction will approach its maximum rate (appVmax) at lower substrate concentration. A high appVmax will correspond to a rapid conversion of a substrate into its metabolite. Thus, a low appKm and a high appVmax are characteristic of an efficient reaction. The relative percentage of 1′-hydroxy metabolite formation for [14C]-methyleugenol (52.4 ± 13.3%) was highest in mouse. The mouse pooled fractions showed both, a low appKm (33.2 μM) and a moderate appVmax (0.45 nmol/mg prot/min) compared to the other two species, which is indicative of a comparatively increased metabolic turnover. A higher appKm (109.9 μM) was observed in human for the formation of 1′-hydroxy metabolites while the appVmax was the lowest in rat, both negatively affecting the reaction yield. The formation of the quinone methide was limited in all species (2.5%–7.8%), the reaction being characterized by a low appVmax and a high appKm.

Kinetic comparison of phase II conjugation in hepatic S9 of three species:

[14C]-methyleugenol glucuronidation was limited and was only observed in the presence of NADPH. The addition of

uridine diphosphate-glucuronic acid (UDPGA) while it had a minor impact on the metabolic profile. Although [14C]-methyleugenol is not directly glucuronidated, dealkylation of the methoxy groups can yield a conjugable phenolic group. This reaction does not appear to be favorable and metabolic turnover was sufficient to establish the kinetic parameters only in

human and mouse. The kinetic of formation of the methyleugenol-derived phenoxy-glucuronide could only be determined in human due to the poor yield of the reaction in other species.

Comparative metabolism of methyleugenol in lung and liver fractions:

Given that inhalation is one possible route for, the authors compared the formation of the 1′-hydroxy and covalent binding of [14C]-methyleugenol derived metabolites in the lung and liver microsomes from human, mouse, and rat.

The metabolism of [14C]-methyleugenol is limited in the lung compared to liver. 1′-hydroxylation in lung microsomes is only a fraction of what is observed in liver. Covalent binding was also reduced in lung in comparison to liver. Overall, [14C]-methyleugenol oxidation in the lung is approximately 20% to 30% to what is observed in the liver. Because of the low metabolic turnover in lung fractions, it was not possible to establish appKm and appVmax for oxidation and conjugation of [14C]-methyleugenol.

Relative percentage and kinetic values (appKm, appVmax, CLint) for 1′-hydroxylation (A), covalent binding (CB; B), and phenolic glucuronidation (C, D) of [14C]-methyleugenol in human, mouse, and rat hepatic microsomes + NADPH, and S9 fractions ±NADPH+UDPGA:

Species	Microsomes + NADPH
	1′-hydroxylation	appKm (μM)	appVmax (nmol/min/ mg prot)	CLint (μl/min/mg prot)
Human	22.2% ± 0.2	127.8	0.95	7.4
Mouse	52.4% ± 13.3	78.49	2.49	31.8
Rat	24.4% ± 5.7	67.4	1.2	17.8

 

Species	Microsomes + NADPH
	CB	appKm (μM)	appVmax (nmol/min/ mg prot)	CLint (μl/min/mg prot)
Human	2.7% ± 0.5	NC	NC	NC
Mouse	7.8% ± 3.1	241.1	0.1	0.4
Rat	2.5% ± 0.5	424.6	0.2	0.5

 

Species	S9 + UDPGA
	Glucuronination	appKm (μM)	appVmax (nmol/min/ mg prot)	CLint (μl/min/mg prot)
Human	No peak	NC	NC	NC
Mouse	No peak	NC	NC	NC
Rat	No peak	NC	NC	NC

 

Species	S9 + NADPH + UDPGA
	Glucuronination	appKm (μM)	appVmax (nmol/min/ mg prot)	CLint (μl/min/mg prot)
Human	12.2% ± 0.4	52.9	0.1	1.9
Mouse	4.0% ± 0.4	NC	NC	NC
Rat	3.9% ± 0.8	NC	NC	NC

Results are expressed as relative percentage (%) of starting substrate dose ± SD for independent triplicates. NC = not calculated due to insufficient turnover for this metabolite.

Relative percentage of 1′-hydroxy (1′-OH) and covalently bound (CB) [14C]-methyleugenol metabolites following incubation with human, mouse, and rat hepatic S9 fractions with NADPH ± UDPGA:

Species	Cofactors	1′-OHmethyleugenol	Methyleugenol CB
Human	NADPH	34.7% ± 0.6	2.0% ± 0.2
	NADPH + UDPGA	34.3% ± 0.9	1.2% ± 0.2
Mouse	NADPH	54.7% ± 3.2	1.06% ± 0.1
	NADPH + UDPGA	64.1% ± 1.7	0.3% ± 0.06
Rat	NADPH	39.0% ± 2.0	0.5% ± 0.1
	NADPH + UDPGA	40.3% ± 0.1	0.5% ± 0.2

Results are expressed as relative percentage (%) of starting substrate dose ± SD for independent triplicates. NC = not calculated due to insufficient turnover for this metabolite.

Conclusions:

In conclusion and in the context of toxicological assessment, the kinetic data suggested a higher potential for the formation of the 1′-hydroxy proximate carcinogen in mouse compared to other species. The study points out, a greater in vitro hepatic clearance similarities between mouse and human, with a moderate potential for 1′-hydroxylation and oxidation to a quinone methide, both being suppressed by extensive phenoxy-glucuronidation. Although the overall clearance rate for oxidation and conjugation are similar in human and mouse, significant inter-species variations in appKm and appVmax were recorded, indicating species-specific differences in enzymatic affinities and reaction rates. Finally, it seems that limited dealkylation of methyleugenol in all tested species is not sufficient to prevent the extensive formation of the 1′-hydroxymethyleugenol proximate carcinogen.

Executive summary:

In this study, the formation kinetics of the 1′-hydroxy proximate carcinogen and the cytotoxic quinone methide metabolites for the known carcinogen methyleugenol were analysed. The metabolic route was evaluated in vitro using hepatic and pulmonary test systems (microsomes and S9 fractions) from rodent and human. The impact of detoxification pathways was also assessed. 1′-hydroxilation of methyleugenol was particularly favorable in rodents compared to humans. Overall, hepatic CLint indicated that the 1′-hydroxilation of the allylic chain is most efficient in mouse. The proposed mechanism for covalent binding involves electrophilic attack of protein sulfhydryl groups. In the present study, quinone methide formation was estimated by quantification of protein-bound [14C] material and calculated the reaction kinetics. It was anticipated that formation of the quinone methide would be limited since dealkylation of the 1-methoxy group is a necessary step. Low CLint values (3.8–4.2 μl/min/mg prot) in human and mouse confirmed that dealkylation is not a favorable reaction. CLint could not be calculated in rat due to the limited reaction turnover. [14C]-methyleugenol does not appear to be directly glucuronidated which is consistent with the presence of a dimethoxybenzene group in place of a phenoxy. Glucuronidation is possible following dealkylation of the dimethoxybenzene group. As indicated previously, this reaction is limited with a CLint of 3.75 μl/min/mg prot in human and CLint of 4.2 μl/min/mg prot in mouse which is between 2 and 7.5 fold lower than the CLint for 1′-hydroxilation of methyleugenol in each respective species. As a result of the limited dealkylation reaction, 1′-hydroxylation remains the major metabolic pathway for [14C]-methyleugenol in liver in vitro systems in all three species. The lung does not appear to be an active metabolic site for methyleugenol in all tested species. Bioactivation was low in mouse lung microsomes obtained from the ICR/CD-1 strain. Given the low metabolic turnover, it was not possible to establish Km and Vmax in this tissue. In conclusion and in the context of toxicological assessment, the kinetic data suggested a higher potential for the formation of the 1′-hydroxy proximate carcinogen in mouse compared to other species. The study points out, a greater in vitro hepatic clearance similarities between mouse and human, with a moderate potential for 1′-hydroxylation and oxidation to a quinone methide, both being suppressed by extensive phenoxy-glucuronidation. Although the overall clearance rate for oxidation and conjugation are similar in human and mouse, significant inter-species variations in appKm and appVmax were recorded, indicating species-specific differences in enzymatic affinities and reaction rates. Finally, it seems that limited dealkylation of methyleugenol in all tested species is not sufficient to prevent the extensive formation of the 1′-hydroxymethyleugenol proximate carcinogen.

Reference 4

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Objective of study:

metabolism

Principles of method if other than guideline:

The entire phase I metabolism of methyleugenol was investigated in liver microsomes of human, rat, and bovine origin with a special emphasis on potentially reactive metabolites distinct from the products of the well-described 1'-side-chain hydroxylation-sulfation pathway. Furthermore, the pattern of metabolites released from intact rat hepatocytes and identify DNA base adducts in the same cell type were analysed.

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Sigma Aldrich/ Fluka (Taufenkirchen, Germany).

Details on test animals or test system and environmental conditions:

Liver microsomes of human, rat, and bovine.

Metabolites identified:

yes

Details on metabolites:

See below.

Oxidative Metabolism of 1 in Liver Microsomes:

By far the widest spectrum and the highest yield of metabolites were detected in incubations with Aroclor 1254-treated rat liver microsomes (ARLM). As early as 2 h after starting the incubation, the level of methyl eugenol had decreased by about 90%, the residual concentration remaining almost constant afterwards. In addition to nonmetabolized methyleugenol, 10 metabolite-derived peaks were detected eluting between 6 and 26 min: a mixture of the side-chain-hydroxylated metabolites 1'-hydroxymethyleugenol and 3'-hydroxymethylisoeugenol (the largest to peaks, tR = 16.4 -16.8 min), 6-hydroxymethyleugenol ( ring-hydroxylated metabolite, tR = 20.3 min), (RS)2',3'-dihydroxy-2',3'-dihydromethyleugenol (dihydrodiol, tR = 11.0 min), demethylation products eugenol (tR = 24.3 min) and chavibetol (tR = 24.7 min). The twice demethylated compound 3,4-dihydroxyallylbenzene was not observed. The side-chain aldehyde (E)-3'-oxomethylisoeugenol became observable 1h after starting the incubation as a "negative" peak at tR = 18.7 min.. This was due to the reference wavelength of 340 nm used. Other minor metabolites were observed: 1'-oxomethyleugenol (tR = 19.3 min)), 6-hydroxymethylisoeugenol (tR = 21.3 min) and (E)-3,4-dimethoxycinnamic acid (tR = 6.3 min). The major metabolites became rapidly detectable almost reaching steady state after 3 h. The diol was detectable from start of the incubation and increased constantly over the observed time period, whereas the probable precursor (R/S)-methyleugenol 2',3'-epoxidewas not found in any incubation.

Incubations of methyleugenol with liver microsomes from humans (HLM), untreated rats (RLM), and of bovine origin (BLM) resulted in a lower number and amount of metabolites. The substrate methyleugenol and the metabolites 1'-hydroxymethyleugenol, 3'-hydroxymethylisoeugenol, 6-hydroxymethyleugenol, (E)-3'-oxomethylisoeugenol, and the sum of eugenol and chavibetol, could be quantified. Hydroxylations in 1' and 3'-position were dominating in all microsomal incubations. Although 1'-hydroxymethyleugenol and 3'-hydroxymethylisoeugenol were found to be present at nearly equal concentrations in incubations with RLM and HLM at both substrate concentrations, the level of 1'-hydroxymethyleugenol prevailed in incubations with ARLM (500 µM) and BLM (100 and 500 µM). 6-hydroxymethyleugenol was a major metabolite found in incubations with ARLM (500 µM) and at lower concentrations also in BLM. However, it was not detected in incubations with RLM and could not be quantified in incubations with HLM, both after 2 h. After 21 h, however, minor amounts of 6-hydroxymethyleugenol and 6-hydroxymethylisoeugenol were found in HLM.

Incubation of ARLM With Synthesized Metabolites:

In order to elucidate the metabolic pathways and for a more precise specification of minor metabolites generated during incubations with methyleugenol, ARLM were incubated with synthesized compounds 1'-hydroxymethyleugenol, 3'-hydroxymethylisoeugenol, 6-hydroxymethyleugenol, (R/S)-methyleugenol 2',3'-epoxide and eugenol. After work-up, the supernatants of these incubations were applied to HPLC analysis.

See the proposed metabolic pathway attached.

Cytotoxicity:

Methyleugenol was found to have an EC50 value of 307 ± 52 µM in the resazurin reduction assay (RRA). Higher cytotoxicity was found for ketone 1'-oxomethyleugenol and the suggested proximate carcinogen 1'-hydroxymethyleugenol. The other investigated metabolites showed slightly lower ( 6-hydroxymethyleugenol, (E)-3´-oxomethylisoeugenol, and (R/S)-methyleugenol 2',3'-epoxide) or clearly lower ( 3'-hydroxymethylisoeugenol) hepato-cytotoxicity compared with methyleugenol. In the dehydrogenase (LDH)-leakage assay (LLA), EC50 values were generally higher than in the RRA with the exception of 3'-hydroxymethylisoeugenol.

DNA Adduct Formation in pRH:

Methyleugenol and several metabolites ( 1'-hydroxymethyleugenol, 3'-hydroxymethylisoeugenol, (E)-3'-oxomethylisoeugenol) formed N2-MIE-dG in rat hepatocytes in primary culture. Samples showing high levels of these adducts also contained the corresponding dA adduct, N6-MIE-dA. However, levels of dA adducts were approximately 50 times lower than those of the dG adduct. Extension of treatment with methyleugenol from 2 to 12 h led to a nearly 10-fold increase in the level of adducts detected. Although 1'-hydroxymethyleugenol was used at only one-tenth of the concentration of methyleugenol, it formed substantially higher levels of adducts. Moreover, high levels were already observed after 2 h, indicating that the activation (probably via sulfation) was relatively fast. 3 was much less efficient in forming DNA adducts than its isomer 2. Moreover, adducts were only detected with 3'-hydroxymethylisoeugenol after the short treatment period (2 h) but not after 12 h. (E)-3'-oxomethylisoeugenol, used at higher concentration than 3'-hydroxymethylisoeugenol, showed formation of modest levels of adducts at both time points. The other compounds tested (methylisoeugenol, 6-hydroxymethyleugenol,1'-oxomethyleugenol and (R/S)-methyleugenol 2',3'-epoxide) did not produce detectable levels of N6-MIE-dA or N2-MIE-dG adducts under the used analytical conditions.

Conclusions:

The study determined that microsomes from human, rat and bovine liver are capable of forming a broad spectrum of phase I metabolites from the genotoxic compound methyleugenol. 1'-Hydroxylation, considered to form the proximate genotoxic and carcinogenic metabolite 1'-hydroxymethyleugenol, was the major pathway of methyleugenol in microsomes from all species tested. DNA adduct formation confirmed the outstanding role of 1'-hydroxylation in the genotoxicity of methyleugenol. However, both (E)-3'-oxomethylisoeugenol and its possible precursor 3'-hydroxymethylisoeugenol were also able to form DNA adducts, suggesting that metabolic pathways other than 1'-hydroxylation may also contribute to the carcinogenicity of methyleugenolThe species comparison of the microsomal metabolite patterns did not reveal any fundamental differences in the amounts of reactive metabolites formed from methyleugenol.

Executive summary:

The entire phase I metabolism of methyleugenol was investigated in liver microsomes of human, rat, and bovine origin with a special emphasis on potentially reactive metabolites distinct from the products of the well-described 1'-side-chain hydroxylation-sulfation pathway. Furthermore, the pattern of metabolites released from intact rat hepatocytes and identify DNA base adducts in the same cell type were analysed. Human, bovine, and rat (Aroclor 1254-induced and non-induced) liver microsomes were incubated with methyleugenol in the presence of an NADPH-generating system to encompass metabolites formed by metabolic phase I reactions. After workup, incubation supernatants were analyzed by RP-HPLC/ DAD. Peaks of metabolites were separated and examined using LC/MS and 1H-NMR spectroscopy. Concentrations of the main metabolites and decrease in substrate concentration were monitored in microsomal incubations from different species (human, rat, and bovine) at various concentrations using quantitative HPLC/DAD analyses. Cytotoxicity of the metabolites using the RRA as well as the LLA was measured in pRHs in culture. Levels of phase I metabolites and phase II conjugates were monitored in pRH. The formation of DNA-adducts with dA and dG after incubation of pRH with the metabolites at noncytotoxic concentrations was determined. The study determined that microsomes from human, rat and bovine liver are capable of forming a broad spectrum of phase I metabolites from the genotoxic compound methyleugenol. 1'-Hydroxylation, considered to form the proximate genotoxic and carcinogenic metabolite 1'-hydroxymethyleugenol, was the major pathway of methyleugenol in microsomes from all species tested. The second major metabolic step of methyleugenol in bovine and ARLM, ring hydroxylation at position 6 leading to 6-hydroxymethyleugenol, was not or hardly detectable in microsomes from humans and RLM. HLMs also differed markedly from the other microsomal preparations in other respects because they showed considerable amounts of the side-chain dihydrodiol (RS)2',3'-dihydroxy-2',3'-dihydromethyleugenol and metabolite 3'-hydroxymethylisoeugenol, the 3'-hydroxylated product. The latter was shown to be the precursor of the potentially reactive carbonyl intermediate (E)-3'-oxomethylisoeugenol, whereas the dihydrodiol is derived from the epoxide (R/S)-methyleugenol 2',3'-epoxide. Both, the epoxide and the 3'-aldehyde have the capacity to bind to proteins and other hepatic macromolecules being probably involved in liver toxicity and hapten formation. DNA adduct formation confirmed the outstanding role of 1'-hydroxylation in the genotoxicity of methyleugenol. However, both (E)-3'-oxomethylisoeugenol and its possible precursor 3'-hydroxymethylisoeugenol were also able to form DNA adducts, suggesting that metabolic pathways other than 1'-hydroxylation may also contribute to the carcinogenicity of methyleugenol. Cytotoxic metabolites possibly also involved in hapten formation were the 1'-derivatives 1'-hydroxymethyleugenol and 1'-oxomethyleugenol (alcohol and aldehyde), whereas the others including (R/S)-methyleugenol 2',3'-epoxide are obviously detoxified more efficiently, at least when added to hepatocyte cultures.

Reference 5

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Objective of study:

other: Binding to DNA

Principles of method if other than guideline:

In vitro binding of methyleugenol to purified DNA was evaluated. Moreover, in-vivo protein binding in rat Liver and glandular stomach by methyleugenol was assessed.

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Elan Chemical Company (Newark, NJ) / Lot: 9224705

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The bulk chemical was stored at room temperature, protected from light in amber glass bottles with Teflon®-lined caps.
- Stability under test conditions: Accelerated stability test: Methyleugenol is stable as bulk chemical for 2 weeks when stored protected from light at temperatures up to 60 ºC.

Radiolabelling:

yes

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

F344/N

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 5 to 6 weeks old
- Housing: Individually in polycarbonate cages containing hardwood bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Between 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24 ºC,
- Humidity (%): 40-65%
- Air changes (per hr): minimum of 10/hour
- Photoperiod (hrs dark / hrs light): 12:12 hour light/dark cycle

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared by mixing [14C]-methyleugenol with corn oil.

DOSE VOLUME: 5 mL/kg by gavage.

Homogeneity and stability were confirmed.

Duration and frequency of treatment / exposure:

Single treatment

Dose / conc.:

118 other: mg/kg (50 μCi/kg)

Remarks:

(gavage [14C]-methyleugenol)

No. of animals per sex per dose / concentration:

9 male rats

Control animals:

no

Details on dosing and sampling:

Nine male Fischer 344 rats received a single oral dose of 118 mg/kg of [14C]-methyleugenol (50 µCi/kg) in corn oil (5 mL/kg). Three of the animals were killed by carbon dioxide asphyxiation at 1, 3.5, and 24 hours after dosing. The liver (at 1, 3.5, and 24 hours) and glandular stomach (at 1 and 3.5 hours) were removed and frozen at -80 ºC until processing. In processing the organs, tissue was removed from the freezer, frozen in liquid nitrogen, and then pulverized. Protein was extracted using trichloroacetic acid; extractions were repeated until no radioactivity could be detected in the wash. The protein was then washed with ethanol to remove any noncovalently bound methyleugenol. Radioactivity associated with the protein was determined by scintillation counting. Binding to protein was also determined for three rats 12 hours following a 11.8 mg/kg intravenous dose of methyleugenol (See endpoint record "Basic toxicokinetics.001_NTP 491 (rat)" for further information on the treatment).

In vitro Binding of Methyleugenol to Purified DNA:

The binding of methyleugenol to calf thymus DNA was dependent on hepatic biotransforming enzymes.  Incubations that did not contain S9 did not elicit binding to DNA. The binding of methyleugenol to DNA was dependent on hepatic biotransforming enzymes. Incubations that did not contain S9 did not elicit binding to DNA. When S9 was obtained from the livers of rats and mice pretreated with Arochlor 1254, methyleugenol binding to DNA occurred at higher levels than in the presence of S9 from non-induced animals, significantly so in the mouse. There were no major differences between rat, mouse, and human DNA binding of methyleugenol in the presence of non-induced S9. Human S9 tended to cause less binding of methyleugenol overall as compared to S9 from rats and mice.

Unscheduled DNA Synthesis in Rat, Mouse, and Human Hepatocytes:

Methyleugenol showed levels of UDS that were approximately half the value of the positive control, and the level of UDS was similar in hepatocytes from rats and mice. In both species, the dose response curve for methyleugenol was atypical; DNA damage appeared to be greater at lower methyleugenol concentrations and appeared to return to control levels at higher concentrations. Often, an inverse dose response relationship is observed for cytotoxic chemicals; however, methyleugenol did not cause extensive cytotoxicity at these concentration ranges. In human hepatocytes, methyleugenol caused significantly less UDS than that seen in rats and mice.

UDS Modulation in Rat and Mouse Hepatocytes:

UDS was similarly affected by modulators in hepatocytes from rats and mice. The addition of cyclohexane oxide did not significantly alter the level of genotoxicity thereby suggesting that the metabolic pathway does not involve epoxidation.  The elevated UDS levels seen with methyleugenol were decreased significantly in the presence of pentachlorophenol, suggesting the sulfation pathway may play a part in the genotoxicity of methyleugenol.

Protein Binding in Rat Liver and Glandular Stomach by Methyleugenol:

Binding to protein in liver and stomach was similar, with slightly more binding seen in the stomach than in the liver following oral dosing and more in liver than stomach following intravenous dosing.  Protein binding by methyleugenol after oral dosing in both liver and stomach was at the same level regardless of time after dosing.

Conclusions:

The study determined that methyleugenol causes macromolecular binding (to DNA, protein, and lipid) in Fischer 344 rats, B6C3F1 mice, and humans.

Executive summary:

The DNA binding of methyleugenol was evaluated in a combination of in-vitro and in-vivo tests. In a firs step, the in-vitro binding of the substance to purified DNA was evaluated, after the incubation of methyleugenol with calf thymus DNA (with or without S9, 1 hour, 37 ºC) and an NADPH recycling system. There were no major differences between rat, mouse, and human DNA binding of methyleugenol in the presence of non-induced S9. Human S9 tended to cause less binding of methyleugenol overall as compared to S9 from rats and mice. In a second step, after the incubation of rat, mice and human hepatocytes with various concentrations of methyleugenol for 18 hours, unscheduled DNA synthesis (UDS) in hepatocytes was estimated by the mean number of silver grains in the nucleus relative to grains in the cytosol. Methyleugenol showed levels of UDS that were approximately half the value of the positive control, and the level of UDS was similar in hepatocytes from rats and mice. It should be pointed out that the doser response curve was atypical althoug no cytotoxicity was osberved. In human hepatocytes, methyleugenol caused significantly less UDS than that seen in rats and mice. Moreover, UDS modulation in rat and mouse hypatocytes was evaluated. UDS was similarly affected by modulators in hepatocytes from rats and mice. The addition of cyclohexane oxide did not significantly alter the level of genotoxicity thereby suggesting that the metabolic pathway does not involve epoxidation.  The elevated UDS levels seen with methyleugenol were decreased significantly in the presence of pentachlorophenol, suggesting the sulfation pathway may play a part in the genotoxicity of methyleugenol. At last, protein binding in rat liver and glandular stomach by methyleugenol was evaluated. Nine male rats received a single oral dose of 118 mg/kg of [14C]-methyleugenol (50 µCi/kg) in corn oil (5 mL/kg). The liver (at 1, 3.5, and 24 hours) and glandular stomach (at 1 and 3.5 hours) were removed, processed for protein extraction. Radioactivity associated with the protein was determined by scintillation counting. Binding to protein in liver and stomach was similar, with slightly more binding seen in the stomach than in the liver following oral dosing and more in liver than stomach following intravenous dosing.  Protein binding by methyleugenol after oral dosing in both liver and stomach was at the same level regardless of time after dosing.

Description of key information

The NTP study on toxicokinetics, absorption, distribution, metabolism and elimination (NTP TR 491, 2000) concluded that methyleugenol is rapidly absorbed following oral administration to rats and mice. The kinetic data are consistent with rapid clearance from the blood, metabolism in the liver, and excretion of the parent and various metabolites in the urine. Moreover, the binding to DNA was evaluted and it was determined that methyleugenol causes macromolecular binding (to DNA, protein, and lipid) in Fischer 344 rats, B6C3F1 mice, and humans.

The objective of Minet et al. (2012) was to compare the kinetics of phase I and phase II metabolism in the bioactivation and detoxification of methyleugenol in different species. The metabolic routes of [14C]-methyleugenol were investigated in human, rat, and mouse, using in vitro hepatic and pulmonary pooled subcellular fractions. The formation of the 1′-hydroxy proximate carcinogen and the cytotoxic quinone methide were quantified and kinetic parameters (appKm, appVmax, and CLint) were calculated. suggested a higher potential for the formation of the 1′-hydroxy proximate carcinogen in mouse compared to other species. The study points out, a greater in vitro hepatic clearance similarities between mouse and human, with a moderate potential for 1′-hydroxylation and oxidation to a quinone methide, both being suppressed by extensive phenoxy-glucuronidation. Although the overall clearance rate for oxidation and conjugation are similar in human and mouse, significant inter-species variations in appKm and appVmax were recorded, indicating species-specific differences in enzymatic affinities and reaction rates. Finally, it seems that limited dealkylation of methyleugenol in all tested species is not sufficient to prevent the extensive formation of the 1′-hydroxymethyleugenol proximate carcinogen.

In the study by Cartus et al. (2012), the entire phase I metabolism of methyleugenol was investigated in liver microsomes of human, rat, and bovine origin with a special emphasis on potentially reactive metabolites distinct from the products of the well-described 1'-side-chain hydroxylation-sulfation pathway. Furthermore, the pattern of metabolites released from intact rat hepatocytes and identify DNA base adducts in the same cell type were analysed. The study determined that microsomes from human, rat and bovine liver are capable of forming a broad spectrum of phase I metabolites from the genotoxic compound methyleugenol. 1'-Hydroxylation, considered to form the proximate genotoxic and carcinogenic metabolite 1'-hydroxymethyleugenol, was the major pathway of methyleugenol in microsomes from all species tested. DNA adduct formation confirmed the outstanding role of 1'-hydroxylation in the genotoxicity of methyleugenol. However, both (E)-3'-oxomethylisoeugenol and its possible precursor 3'-hydroxymethylisoeugenol were also able to form DNA adducts, suggesting that metabolic pathways other than 1'-hydroxylation may also contribute to the carcinogenicity of methyleugenolThe species comparison of the microsomal metabolite patterns did not reveal any fundamental differences in the amounts of reactive metabolites formed from methyleugenol.