4-allylveratrole

Basic toxicokinetics

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

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

Test material

Test material information

Constituent 1

Reference substance name:

4-allylveratrole

EC Number:

202-223-0

EC Name:

4-allylveratrole

Cas Number:

93-15-2

Molecular formula:

C11H14O2

IUPAC Name:

1,2-dimethoxy-4-(prop-2-en-1-yl)benzene

Test material form:

liquid

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

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

37 mg/kg bw/day (nominal)

Remarks:

(intravenous injection or gavage)

Doses / concentrations 2

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

(gavage)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

(gavage)

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

(gavage)

Doses / concentrations 5

Dose / conc.:

118 other: mg/kg (50 μCi/kg)

Remarks:

(gavage [14C]-methyleugenol)

Doses / concentrations 6

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.

Results and discussion

Toxicokinetic / pharmacokinetic studies

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.

Metabolite characterisation studies

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.