toxicokinetics of methyleugenol in f344 rats and b6c3f 1 mice

293

Introduction

Methyleugenol (MEG) (Figure 1) is a natural constituent of a large number of essential oils including rose, basil, hyacinth, pimetto, citronella [Flavor and Extract Manufacture Association (FEMA) 1978], anise, nutmeg, mace, cinnamon leaves (Furia & Bellanca 1975), pixuri seeds (Carlini et al. 1983), laurel fruits and leaves (Meister 1992), blackberry essence, bananas, black pepper, and bilberries [World Health Organization (WHO) 1981]. MEG is also produced commercially by the methylation of eugenol, a common constituent of oil of cloves (Opdyke 1979). The annual production in the United States is estimated at 25,000 pounds (SRI International 1990).

MEG has been used commercially as a flavouring agent, fragrance, and insect attractant. The FEMA gave

MEG Generally Recognized As Safe (GRAS) status in 1965 and the Food and Drug Administration (FDA) has approved use of MEG in foods (Code of Federal Regulations 21 ξ 121.1164). As a flavouring agent, MEG was used at concentrations of 5–52 ppm in jellies, baked goods, nonalcoholic beverages, chewing gum, candy, puddings, relish, and ice cream (Furia & Bellanca 1975). Worldwide, MEG is used more extensively as a fra-grance and insect attractant than as a flavouring agent. Opdyke (1979) reported MEG concentrations in various fragrance and perfumes (0.3–0.8%), creams and lotions (0.01–0.05%), and soaps and detergents (0.02–0.2%). MEG was used in combination with pesticides in control and eradication programs because of its effectiveness as an insect attractant for fruit flies (Ibrahim et al. 1979).

RESEARCH ARTICLE

Toxicokinetics of methyleugenol in F344 rats and B6C3F

1 mice

S. Peter Hong1, Alfred F. Fuciarelli2, Jerry D. Johnson1, Steven W. Graves1, Derrick J. Bates3, Suramya Waidyanatha4, and Cynthia S. Smith4

1Battelle Memorial Institute, Columbus, OH, USA, 2Valdosta State University, Valdosta, GA, USA, 3Pacific Northwest National Laboratory, Richland, WA, USA, and 4National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA

Abstract1. Methyleugenol (MEG) has been used as a flavouring agent in food, as a fragrance in cosmetic products, and as an

insect attractant. MEG was carcinogenic in both rats and mice following gavage administration. In this study we investigated plasma toxicokinetics of MEG in F344 rats and B6C3F1 mice of both sexes following single gavage (37, 75, or 150 mg/kg) and intravenous (IV) (37 mg/kg) administration.

2. Following IV administration, MEG was rapidly distributed and cleared from the systemic circulation in both species and sexes. Absorption of MEG was rapid following gavage administration with secondary peaks in the plasma MEG concentration-versus-time profiles. Cmax and AUCT increased and the clearance decreased greater than proportional to the dose in rats and mice of both sexes. In general, rats had higher internal exposure to MEG than mice.

3. The results for AUCT and clearance suggest that perhaps the metabolism of MEG is saturated at higher doses tested in this study. Absolute bioavailability following gavage administration of 37 mg/kg was low in both rats (~4%) and mice (7–9%) of both sexes indicating extensive first-pass metabolism. There was no sex difference in plasma toxicokinetics of MEG following gavage administration both in rats and mice.

Keywords: Methyleugenol, toxicokinetics, bioavailability

Address for Correspondence: S. Peter Hong, Ph.D., Battelle Memorial Institute, Columbus, OH 43201, USA. Tel: 1-614-424-6354. Fax: 1-614-458-6354. E-mail: [email protected]

(Received 01 June 2012; revised 06 July 2012; accepted 09 July 2012)

Xenobiotica, 2013; 43(3): 293–302© 2013 Informa UK, Ltd.ISSN 0049-8254 print/ISSN 1366-5928 onlineDOI: 10.3109/00498254.2012.711496

Xenobiotica

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3

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01June2012

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10.3109/00498254.2012.711496

2013

Toxicokinetics of methyleugenol

S. P. Hong et al.

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294 S. P. Hong et al.

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The Council of Europe listed the acceptable daily intake of MEG as 5 mg/kg. The per capita human intake of MEG through the ingestion of standard and nonstan-dard foodstuffs was estimated at 0.073 mg/day (WHO 1981), but later reports estimated the daily per capita consumption of MEG at 0.26 µg/kg body weight (Stofberg & Grundschober 1987; National Academy of Sciences 1989). Commercial production and use of MEG has also resulted in unanticipated human exposure. The National Occupational Hazard Survey (1981–1983) estimated that 2,824 workers in 87 plants were potentially exposed to MEG annually [National Institute for Occupational Safety and Health (NIOSH) 1990]. MEG was detected in the oil (240 ppm) and juice (42 ppb) of oranges treated with abscission chemicals such as cycloheximide (MacGregor et al. 1974). MEG was also detected at a concentration of 0.02 mg/L in waste water effluent from a paper mill (Moshonas & Shaw 1978).

In general, allylbenzenes like MEG exhibit pharmaco-logic properties associated with central nervous system depressants. For example, eugenol has been reported to have antiseptic and analgesic properties, local anesthetic properties, spasmolytic activity, peripheral vasodila-tor action, and parasympathetic activity (Sticht & Smith 1971; Dallmeier & Carlini 1981). MEG exhibited anes-thetic, hypothermic, myorelaxant, and anticonvulsant properties in various species (Deininger & Wolfe 1977; Dallmeier & Carlini 1981; Jiang et al. 1982). Myristicin and elemicin, two allylphenol derivatives related to saf-role, eugenol and MEG, are primarily responsible for the psychotropic and hallucinogenic effects of nutmeg powder and have pronounced central nervous system depressant effects (Weiss 1960; Farnsworth 1968; Fras & Freidman 1969; Buchanan 1978).

MEG is moderately toxic with median lethal oral doses of 810–1560 mg/kg for rats and 540 mg/kg for mice (National Toxicology Program, NTP, 2000). Beroza et al. (1975) reported that MEG was neither an eye irritant nor a skin irritant using undiluted material of greater than 98 percent purity. However, long term exposure resulted in more severe effects. Two-year chronic toxicity/carcinoge-nicity studies following gavage administration using 0.5% methylcellulose as a vehicle in F344/N rats and B6C3F

1

mice revealed that MEG was a multi-site, multi-species carcinogen with target organs including liver and glan-dular stomach in both sexes and species, forestomach in female rats, and kidney, mammary gland, and subcutane-ous tissue in male rats (NTP 2000; Johnson et al. 2000).

MEG was not genotoxic in Salmonella typhimurium and E. Coli (Sekizawa & Shibamoto 1982; Mortelmans et al. 1986; Kettering & Torabinejad 1995). However, dose-dependent responses were reported in chromo-somal recombination studies in yeast (Schiestl et al. 1989; Brennan et al. 1996). In addition, unscheduled DNA syn-thesis in vitro, DNA binding in vitro, protein binding in mammalian hepatocytes in vitro and in liver and stom-ach in rats in vivo was observed with MEG (Phillips et al. 1984; Howes et al. 1990; Chan & Caldwell 1992; Gardner et al. 1996; Burkey et al. 2000).

The major pathways for the bioactivation of MEG include the oxidation of the allylic side-chain, the forma-tion of the hydroxyl acid via epoxidation of the double bond followed by hydration, and hydroxylation of the phenyl ring (Solheim & Scheline 1976). Approximately, 20% of the administered MEG dose led to formation of the reactive epoxide intermediate, whereas over 60% of the administered dose underwent 1′-hydroxylation (Solheim & Scheline 1976). The 1′-hydroxy metabolite is sulfated, yielding 1′-sulfooxy metabolite which decomposes spon-taneously in an aqueous environment to electrophilic carbonium ions that bind covalently to DNA and to other cellular macromolecules, including proteins and hence was proposed as the carcinogen in rodents (Miller et al. 1983), a hypothesis consistent with studies examining the tumorigenicity of 1′-hydroxysafrole (Boberg et al. 1983).

Given the toxicity and carcinogenicity of MEG observed in the 2-year chronic studies by NTP (NTP 2000; Johnson et al. 2000), a single administration toxi-cokinetic study of MEG was conducted to establish basic toxicokinetic parameters and estimate absolute bioavail-ability in F344 rats and B6C3F

1 mice of both sexes follow-

ing gavage administration. The current study used corn oil as a vehicle for gavage dosing formulation as opposed to 0.5% methylcellulose that was used in the chronic studies with MEG (NTP 2000). While 0.5% methylcellu-lose was acceptable in preparing gavage formulation for MEG, it was inadequate to use for a structurally related compound isoeugenol due to the lack of homogeneity in resulting suspension. On the other hand, corn oil was found to be an acceptable vehicle in preparing gavage formulations for both compounds. For this reason, corn oil was used in the present study to generate toxicoki-netic parameters for MEG and to facilitate appropriate comparison with toxicokinetics of isoeugenol in future investigation.

Materials and methods

ChemicalsMEG (4-allyl-1,2-dimethoyxbenzene) was supplied by Elan Chemical Company, (Newark, NJ) and stored at 20 ± 2°C. Upon receipt, the structure and purity were con-firmed by gas chromatography (GC)/flame ionization detection (FID), mass spectrometry (MS), infrared spec-troscopy (IR), and nuclear magnetic resonance (NMR) spectroscopy. A purity profile using GC/FID found no

Figure 1. Chemical structure of MEG.

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Toxicokinetics of methyleugenol 295


impurities with individual relative areas greater than 0.1% of the major peak. Unless specified, all other chemi-cals were purchased from Sigma (St Louis, MO), Aldrich Chemical Company, Inc. (Milwaukee, WI), Burdick and Jackson (Muskegon, MI), or Mallinckrodt Baker Inc. (Phillipsburg, NJ).

Animals and animal husbandryThese studies were conducted in Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited facilities and approved by Battelle’s Animal Care and Use Committee. Animal care was performed according to the Guide for the Care and Use of Laboratory Animals (National Research Council 1996). Animals were housed in humidity- (55 ± 15%) and temperature-controlled (75 ± 3°F), high-efficiency particulate air-filtered, mass air-displacement rooms maintained on a 12-h light/dark cycle (light starting at 0600). Animals had ad libitum access to NTP-2000 diet (irradiated pellets; Zeigler Bros., Gardners, PA) and city tap water.

Thirteen-week-old male and female F344 rats and B6C3F

1 mice (Charles River; Raleigh, NC) with Silastic®

catheters surgically implanted into the jugular vein by the supplier were used for IV dosing. Canulated animals were housed in solid bottom polycarbonate cages and acclimated for at least one day prior to use. Thirteen-week-old animals used for gavage dosing were housed in wire cages and acclimated for at least 13 days prior to use.

Treatment protocolEighteen non-fasted rats/sex and 36 non-fasted mice/sex were given a single IV bolus injection (2 mL/kg for rats; 4 mL/kg for mice) of MEG in Cremophor:ethanol:water (1/1/8; v/v/v), at a dose of 37 mg/kg through the Silastic® catheter. Twenty-one non-fasted rats/sex/dose group and 42 non-fasted mice/sex/dose group were given a single gavage bolus (5 mL/kg for rats; 10 mL/kg for mice) of MEG in corn oil at 37, 75, or 150 mg/kg.

Plasma collection and storageAnimals were anesthetized with ~70% CO

2 (30% O

2) and

blood was collected from rats via the retro-orbital sinus or from mice via a closed chest cardiac puncture. Following IV dosing, three rats/sex/time point were bled at each of the first 6 time points (up to 1 h post-dosing) and up to 2 mL of blood was collected. The rats were returned to their cages to recover and randomly re-assigned (3 rats/sex/time point) to the remaining 6 time points for additional sampling. For the second bleeding of each rat which occurred no sooner than 1 h following the initial bleeding, the maximum amount of blood was collected prior to sacrifice (3 rats/sex). Three mice/sex/time point were bled at each time point. Each mouse was bled only once and the maximum amount of blood was collected prior to sacrifice. Following gavage dosing, three rats/sex/time point/dose group were bled at each of the first 7 time points (up to 2 h post-dosing) and up to 2 mL of blood was

collected. The rats were returned to their cages to recover and randomly re-assigned (3 rats/sex/time point/dose group) to the remaining 7 time points for blood collec-tion. Three mice/sex/time point/dose group were bled at each time point. Each mouse was bled only once at 2 (IV 37 mg/kg, gavage 37 and 75 mg/kg only), 5, 10, 15 (gavage 150 mg/kg only), 20, 30 min, and 1, 1.5, 2, 3, 4, 5 (gavage 37 and 75 mg/kg only), 6, 8, 12 (gavage groups only), and 24 h (gavage 150 mg/kg only) post-dose, and the maximum amount of blood was collected prior to termination.

Blood samples were collected into glass tubes con-taining EDTA anticoagulant, mixed gently, and placed on wet ice. Plasma was separated from blood within 60 min of collection by centrifugation at ~1750g for 10 min in a refrigerated (~4°C) table-top centrifuge and stored in polypropylene vials at −70°C.

Determination of MEG in plasmaPlasma (100 µL) was extracted with 1.0 mL of an ethyl acetate solution containing the internal standard (2-methoxy-4-propenyl-phenol; IEG) at a target con-centration of 0.50 µg/mL. One micro litre of the organic layer was injected into a Hewlett-Packard Model 6890 gas chromatograph interfaced to a Hewlett-Packard Model 5973 mass selective detector. Temperature-programmed separations (45–230°C) were conducted on a 30 × 0.25 mm i.d., 0.25 µm film, DB-WAXETR fused-silica capillary col-umn (J&W Scientific Inc, Folsom, CA). The most intense characteristic ions in electron ionization were selected for quantitation which corresponded to the molecular ions for both MEG (m/z 178) and the internal standard (IEG, m/z 164). The limit of detection (LOD) and experimental limit of quantitation (ELOQ) were 0.00057 and 0.0051 µg/mL plasma, respectively. The analytical method was validated in rat plasma using the FDA guidelines for bio-analytical methods. Three days of validation including stability evaluation were conducted. Plasma standards at seven concentrations ranging from 0.005 to 32.1 µg/mL were analyzed in triplicate in each run. Six replicates of plasma quality control samples at four concentrations ranging from 0.05 to 23 µg/mL were analyzed in each run. Calibration curves were generated with a weighted lin-ear least-square fit, using the inverse square of the mean MEG concentration as the weighting factor. Correlation coefficients reflecting the fit of the regression line to calibration data obtained for spiked plasma standards exceeded 0.999. The accuracy of the method was excellent with average inter-day relative errors within 3% of target and average intra-day relative errors of within 2% of tar-get for the standards and average inter-day relative errors within 4% of target and average intra-day relative errors of within 3% of target for the quality control samples. The precision of the method was also acceptable with average inter-day relative standard deviations within 7% of target and average intra-day relative errors within 3% of target for the standards and average inter-day relative standard deviations within 9% of target and average intra-day relative errors within 5% of target for the quality control

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samples. The average recovery of MEG from plasma was 103%. Cross validation of the method using rat plasma calibration standards and mouse plasma quality control samples produced similar precision and accuracy.

Toxicokinetic modellingToxicokinetic parameter estimates following IV or gavage administration were derived only from plasma MEG measurements that were above the ELOQ. Toxicokinetic parameters were determined by fitting the equation below to the data collected in the IV study using a nonlin-ear least-squares fitting program (SAS PROC NLIN; SAS Institute, Inc., Cary, NC)

C t A e B eot

ot( ) = +− −α β

where C(t) is the plasma MEG concentration at any post-administration time (t), α and β are the bi-exponential disposition rate constants (t−1), and A

o and B

o are the

extrapolated intercepts on the ordinate (concentration) axis of the initial and terminal elimination phase, respec-tively. Estimates for these values, with their asymptotic standard errors were obtained directly from the model. However, the gavage data exhibited at least one second-ary peak in terminal elimination phase which prevented any compartmental modelling. Therefore, only a limited set of toxicokinetic parameters could be estimated from the gavage data using non compartmental analysis.

The elimination half-lives for the initial and terminal phases of the concentration versus time profiles (t

½α and t½β)

were calculated as ln2/α or ln2/β, respectively. The maxi-mum plasma MEG concentration (C

o) following IV dosing

was assumed to occur at t = 0 and was calculated as Ao + B

o.

The area under the curve (AUCT) was estimated to the last

sampling time point using the trapezoidal rule for data col-lected during the IV and gavage phases of the study.

AUC t tT n n

Cn Cn= × −− +∑ −12 1( )

where Cn−1

and Cn are the plasma MEG concentrations

measured at two consecutive time points, tn−1

and tn,

respectively. For the IV data, the area under the plasma concentration-versus time profile extrapolated to infinity (AUC

∞) was estimated using the equation below

AUC AUCC

T∞ = + T

β

where CT is the plasma MEG concentration measured

at the last sampling time point and β is the rate constant for the terminal elimination phase.

For the IV phase of the study, the clearance (Cal) was calculated as:

Cl Dose AUC= ∞/

and for the gavage phase of the study, the apparent clear-ance (Cl

app) was calculated as:

Cl Dose AUCapp T= /

The volume of distribution (Vd), defined only for the IV

data, was calculated as:

V Cld = /β

The concentration-time data following gavage administration were not subject to compartmental modelling due to multiple peaks observed in the elimi-nation phase. Instead, model-independent parameters (i.e. C

max, T

max, Cl

app, and AUC

T) were generated and

evaluated.Absolute bioavailability following gavage administra-

tion was expressed as the fraction (F) of the oral dose that actually reaches systemic circulation and was calculated as follows:

FDose AUC

Dose AUCIV T gavage

gavage T IV

=×

×( )

( )

Figure 2. Plasma MEG concentration-versus-time profiles for male and female F344 rats following IV administration of MEG at 37 mg/kg.

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Results

MEG toxicokinetics in rats after IV administrationMEG was measurable and above the ELOQ up to and including the last sample collection time point (8 h post-dose). Plasma MEG concentration-versus-time profiles were biphasic in both sexes, exhibiting a rapid initial phase (α) and slower terminal phase (β) (Figure 2). Toxicokinetic parameters estimated from these data are presented in Table 1. The highest plasma MEG concentrations were observed at the 2-min time point. V

d greatly exceeded the total body water volume

for a rat (0.668 L/kg) (Davies & Morris 1993) showing evidence for extensive distribution, high tissue binding, and/or plasma protein binding of MEG. Other than the initial distribution half-life (T

½α), there was no sex differ-ence in the estimated toxicokinetic parameters.

MEG toxicokinetics in rats after gavage administrationMEG was measurable and above the ELOQ up to and including the last sample collection time point (12 h post-dose for 37 and 75 mg/kg dose groups, and 24 h for 150 mg/kg dose group), except for a male rat at 8 h, all three male rats at 12 h, a female rat at 8 h, and all three female rats at 12 h in 37 mg/kg dose groups; all three male rats at 8 h, a female rat at 3 h, a female rat at 12 h in 75 mg/kg dose groups; two female rats at 24 h in 150 mg/kg dose groups. The plasma MEG concentration-versus-time profiles following gavage administration of MEG to rats demonstrated a rapid absorption phase that occurred within 15 min post dosing in both sexes (Figures 3 and 4). The concentration-versus-time profiles of MEG exhibited at least one secondary peak and these secondary peaks were observed up to ~300 min with a trend toward later times with increasing dose. As a result of the secondary peaks, limited toxicokinetic parameters were estimated using non compartmental analysis (Table 2).

Absorption of MEG was rapid in both sexes as sug-gested by T

max values within 15 min (Table 2). C

max

increased by a factor of ~6 (males) and ~14 (females) for ~4-fold increase in dose. However, because there were a limited number of data points collected during the absorption phase due to the rapid absorption of MEG,

AUC values provided a more appropriate measure for evaluation of dose proportionality in systemic expo-sure to MEG after gavage administration. AUC

T values

increased with dose but the magnitude of the increases was greater than proportional to dose (Table 2 and Figure 5). MEG was cleared rapidly in both sexes with Cl

app decreasing ~5 times with increasing dose. There was

no sex difference in toxicokinetic parameters.

MEG bioavailability in ratsAbsolute bioavailability following a gavage dose of 37 mg MEG/kg was similar in both sexes with values of 3.9 ± 0.9% for males and 4.1 ± 1.3% for females. Since the equation to estimate the bioavailability applies only if dose-proportionality is observed (Renwick 1994), the absolute bioavailability was not determined for gavage doses of 75 and 150 mg/kg in rats.

MEG toxicokinetics in mice after IV administrationMEG was measurable and above the ELOQ up to and including the last sample collection time point

Table 1. Toxicokinetic parameter estimates following IV administration of 37 mg/kg MEG to F344 rats.Parametera Males FemalesC

0 (µg/mL) 20.0 ± 2.0 28.9 ± 2.3

T½α (min) 17.1 ± 1.2 10.9 ± 0.7

T½β (min) 131 ± 19 119 ± 9

AUC∞

(µg mL−1 min) 593 ± 16 624 ± 11AUC

T (µg mL−1 min) 542 ± 15 557 ± 10

AUCT/dose (µg mL−1 min) 14.6 ± 0.4 15.0 ± 0.3

Cl (mL min−1 kg−1) 62.3 ± 1.6 59.3 ± 1.0V

d (L/kg) 11.8 ± 1.7 10.2 ± 0.8

a Estimate ± SE. Parameters were estimated using two-compartmental model.

Figure 3. Plasma MEG concentration-versus-time profiles for male F344 rats following gavage administration of MEG at 37, 75, and 150 mg/kg.

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(6 h post-dose), except for a male mouse at 6 h, two female mice at 5 h, and all three female mice at 6 h). Plasma MEG concentration-versus-time profiles in mice of both sexes were biphasic, exhibiting a rapid initial phase (α) and slower terminal phase (β) (Figure 6). Toxicokinetic parameters estimated from these data are presented in Table 3. The highest plasma MEG concentrations was observed at the 2-min time point in both sexes. The, T

½β, but not T

½α, was longer in males than in females. AUC∞ was higher and Cl was lower in females as compared to males. V

d exceeded the total body water volume in mice

(0.725 L/kg) showing evidence of extensive tissue distri-bution, high tissue binding, and/or plasma protein bind-ing (Davies & Morris 1993).

MEG toxicokinetics in mice after gavage administrationMEG was measurable and above the ELOQ up to and including the last sample collection time point (8 h post-dose for 37 and 75 mg/kg dose groups, and 16 h for 150 mg/kg dose group), except for a male mouse at 6 h, all three male mice at 8 h, a female mouse at 4 h, a female mouse at 5 h, a female mouse at 6 h, and two female mice at 8 h in 37 mg/kg dose groups; all three male mice at 8 h, a female mouse at 3 h, a female mouse at 4 h, a female mouse at 5 h, a female mouse at 6 h, and all three female mice at 8 h in 75 mg/kg dose groups; all male and female mice at 12 and 16 h in 150 mg/kg dose groups. The plasma MEG concentration-versus-time profiles fol-lowing gavage administration of MEG to mice showed a rapid absorption phase that occurred within 30 min post dosing (Figures 7 and 8). The concentration-versus-time profiles exhibited at least one secondary peak. Following the initial absorption phase, secondary peaks were observed up to ~360 min with a trend toward later times with increasing dose. As a result of the secondary peaks, limited toxicokinetic parameters were estimated using non compartmental analysis (Table 4).

Figure 5. Dose-normalized AUC∞

-versus-dose plots for male and female F344 rats following gavage administration.

Table 2. Toxicokinetic Parameter estimates following gavage administration of MEG to F344 rats.

Parametera

Males Females37 mg/kg 75 mg/kg 150 mg/kg 37 mg/kg 75 mg/kg 150 mg/kg

C(max)

(µg/mL) 0.314 ± 0.180 2.22 ± 1.27 1.93 ± 1.12 0.341 ± 0.049 0.895 ± 0.067 4.77 ± 0.81T

(max) (min) 5 5 15 10 10 5

AUCT (µg mL−1 min) 21.0 ± 2.2 122 ± 12 465 ± 31 23.0 ± 3.5 82.8 ± 6.1 498 ± 32

AUCT/Dose (µg mL−1 min) 0.567 ± 0.059 1.63 ± 0.16 3.10 ± 0.21 0.622 ± 0.096 1.10 ± 0.08 3.32 ± 0.22

Clapp

(mL min−1 kg−1) 1650 ± 160 603 ± 59 321 ± 21 1460 ± 210 885 ± 64 293 ± 19Absolute bioavailability (%) 3.9 ± 0.9 NAb NAb 4.1 ± 1.3 NAb NAb

aEstimate ± SE. Parameters were estimated using non-compartmental analysis.bNot applicable due to the lack of dose-proportionality (see text for explanation).

Figure 4. Plasma MEG concentration-versus-time profiles for female F344 rats following gavage administration of MEG at 37, 75, and 150 mg/kg.

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Absorption of MEG was rapid in both sexes as indicated by T

max values within 30 min (Table 4). C

max increased

by a factor of ~24 (males) and ~26 (females) for ~4-fold increase in dose. However, AUC

T values provided a more

appropriate measure for evaluation of dose proportion-ality. AUC

T values increased with dose but the magnitude

of the increases were greater than proportional to dose (Table 4 and Figure 9). MEG was cleared rapidly in both sexes with Cl

app decreasing ~4-fold (males) and ~3-fold

(females) with increasing dose. There was no sex differ-ence in toxicokinetic parameters.

MEG bioavailability in miceAbsolute bioavailability following gavage dose of 37 mg MEG/kg, was 9.2 ± 6.4% for males and 7.3 ± 3.7% for females. Since the equation to estimate the bioavailabil-ity applies only if the dose-proportionality is observed (Renwick 1994), the absolute bioavailability was not determined for gavage doses of 75 and 150 mg/kg.

Discussion

MEG is a low molecular weight, uncharged, highly lipo-philic molecule with a water-octanol partition coefficient of ~800 (Graves 1998). These properties characteristi-cally permit rapid diffusion across membranes of the gastrointestinal tract, blood vessels, and other tissues

favouring rapid and extensive absorption. Consistent with this, MEG was measurable in rodent plasma within 2 min following gavage administration and exhibited relatively short T

max (rats, 5–15 min; mice, 20–30 min)

Figure 6. Plasma MEG concentration-versus-time profiles for male and female B6C3F1 mice following IV administration of MEG at 37 mg/kg.

Table 3. Toxicokinetic parameter estimates following IV administration of 37 mg/kg MEG to B6C3F

1 mice.

Parametera Males FemalesC

0 (µg/mL) 10.9 ± 2.8 13.6 ± 2.4

T½α (min) 6.21 ± 0.98 7.51 ± 1.16

T½β (min) 71.4 ± 12.7 38.7 ± 3.8

AUC∞

(µg mL−1 min) 136 ± 10 203 ± 18AUC

T (µg mL−1 min) 111 ± 8 176 ± 18

AUCT/Dose (µg mL−1 min) 2.99 ± 0.23 4.76 ± 0.48

Cl (mL min−1 kg−1) 271 ± 20 182 ± 16V

d (L/kg) 28.0 ± 5.4 10.2 ± 1.4

a Estimate ± SE. Parameters were estimated using two-compartmental model.

Figure 7. Plasma MEG concentration-versus-time profiles for male B6C3F

1 mice following gavage administration of MEG at 37, 75, and

150 mg/kg.

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values suggestive of a rapid absorption. In general, Cmax

increased greater than proportional to the dose in both species and sexes following gavage administration. The relatively high volume of distribution of MEG after IV administration greatly exceeded the total body water vol-ume in rats and mice (Davies & Morris 1993), which was suggestive of extensive tissue distribution, high tissue binding, and/or plasma protein binding. In addition, the half-life for distribution following IV administration was

short for both species as consistent with the lipophilicity of MEG (Tables 1 and 3). However, MEG did not appear to be taken up into extravascular tissues for an extended period of time. The compound was rapidly cleared from systemic circulation in both species following IV as well as gavage administration despite the presence of secondary peaks in the plasma MEG concentration-versus-time profiles following gavage administration. The clearance values were greater than reported hepatic blood flow rates of ~55 and ~90 mL min−1 kg−1 for rats and mice, respectively (Davies & Morris 1993), and were much higher following gavage administration, indicat-ing significant first pass metabolism of MEG following gavage administration. In both species, following gavage administration, AUC

T increased and Cl

app decreased

greater than proportional to the dose indicating satura-tion of MEG metabolism and/or liver injury. As a matter of fact, following gavage administration of MEG in rats, hepatocellular injury and increased liver weights were observed ≥100 mg/kg. In mice, increased liver weights were observed ≥30 mg/kg (NTP 2000).

The appearance of secondary peaks in the plasma concentration-versus-time profiles following gavage administration was not entirely unexpected since such peaks have been observed in rats gavaged with carbon tetrachloride (Kim et al. 1990) and 1,2-dibromo-3-chloro-propane (Gingell et al. 1987) using corn oil as an admin-istration vehicle. Corn oil has been shown to markedly delay, but not diminish, the overall extent of absorption from the gut compared to aqueous vehicle groups and accounts for secondary peaks in the elimination phase (Kim et al. 1990). Although the C

max was much lower and

Tmax

longer, the areas under the plasma concentration-versus time profiles were very similar as a result of the prolonged elevation of test chemical in the blood of the corn oil groups (Kim et al. 1990). Kim et al. (1990) hypoth-esized that corn oil acts as a reservoir in the gut to retard the systemic absorption of chemicals. Fractional gastric emptying, which divides the administered dose into ali-quots of chemical contained within lipid globules, best explains the multiple secondary peaks observed after the initial peak in the plasma concentration-versus-time profiles. In support of this hypothesis, Chieco et al. (1981) observed more rapid gastric absorption of 1,1-dichlo-roethylene from a digestible corn oil vehicle than from poorly absorbed mineral oil.

Figure 8. Plasma MEG concentration-versus-time profiles for female B6C3F

1 mice following gavage administration of MEG at 37,

75, and 150 mg/kg.

Table 4. Toxicokinetic parameter estimates following gavage administration of MEG to B6C3F1 mice.

Parametera

Males Females37 mg/kg 75 mg/kg 150 mg/kg 37 mg/kg 75 mg/kg 150 mg/kg

C(max)

(µg/mL) 0.153 ± 0.075 0.505 ± 0.207 3.74 ± 1.34 0.132 ± 0.015 0.520 ± 0.389 3.46 ± 0.42T

(max) (min) 20 30 30 20 20 20

AUCT (µg mL−1 min) 10.2 ± 0.8 30.2 ± 2.5 205 ± 27 12.9 ± 0.9 24.9 ± 3.2 185 ± 25

AUCT/Dose (µg mL−1 min) 0.275 ± 0.021 0.402 ± 0.033 1.37 ± 0.18 0.348 ± 0.025 0.333 ± 0.08 1.23 ± 0.16

Clapp

(mL min−1 kg−1) 3050 ± 360 2160 ± 240 717 ± 93 2550 ± 160 2940 ± 370 807 ± 110Absolute Bioavailability (%) 9.2 ± 6.4 NAb NAb 7.3 ± 3.7 NAb NAb

aEstimate ± SE. Parameters were estimated using non-compartmental analysis.bNot applicable due to the lack of dose-proportionality (see text for explanation).

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There were species-related differences in the toxicoki-netic behaviour of MEG following gavage administration. The time to reach C

max was shorter in rats (5–10 min) than

in mice (20–30 min). In general rats had higher exposure to MEG than mice. Both C

max and AUC

T were higher in

rats than in mice of both sexes except for the 150 mg/kg group where male mice had higher C

max than male

rats. Consistent with this data, in a 14 day toxicity study conducted by NTP, rats were more sensitive than mice to MEG-induced toxicity (NTP 2000). There was no sex-related difference in the toxicokinetic behaviour of MEG in mice and rats following gavage administration.

The most notable observation regarding the toxicoki-netic behaviour of MEG following gavage administration was low bioavailability in both species and sexes (≤9.2%) indicating that only a small percentage of an orally administered dose of MEG reaches the systemic circula-tion as the parent. Metabolism and disposition studies using radiolabeled MEG support extensive first-pass metabolism as the underlying cause for low MEG bio-availability following gavage administration. The major excretion pathway reported was via urine with no evi-dence of the parent excreted in urine (Solheim & Scheline 1976; NTP 2000). Major urinary metabolites were sulfate and glucuronide conjugates of hydroxylated derivatives, and mercapturic acid derivatives. Extensive first-pass metabolism may also result in significant bioactivation of MEG to metabolites that may be related to carcinogenic effects. Although epoxide intermediates for compounds structurally related to MEG (i.e. safrole, estragole) were shown to contribute very little to their carcinogenicity (Miller et al. 1983), the 1′-hydroxy metabolites exhibited clear evidence of carcinogenic activity (Boberg et al. 1983).

Toxicokinetics of MEG was investigated previously following gavage administration of 25, 50, and 75 mg/kg MEG in F344 rats and B6C3F1 mice where 0.5% methyl cellulose was used as the gavage vehicle (NTP 2000). The time to reach C

max was shorter in that study (rats, 5 min;

mice, 5–15 min) compared to the current investigation

(mice, 20–30 min; rats, 5–15 min) which is likely due to the differences in the dosing vehicles (0.5% methyl cellulose vs. corn oil). Other than this difference, the toxicokinetic behaviour of MEG observed in the previous investiga-tion was very similar to the current investigation: both C

max and AUC

T increased greater than proportional to the

dose, MEG was cleared very rapidly, the bioavailability was low, rats had higher C

max and AUC

T than mice, and

there was no sex difference in plasma toxicokinetics in either rats or mice. The toxicokinetic parameters for MEG after gavage administration in corn oil will allow adequate comparisons with toxicokinetics of isoeugenol in the same vehicle for gavage administration.

Conclusions

In summary, we investigated the plasma toxicokinetics of MEG following a single IV and gavage administration of MEG to F344 rats and B6C3F

1 mice. MEG was rapidly

absorbed following gavage administration with both Cmax

and AUC

T increasing more than proportional to the dose.

MEG was cleared rapidly following both IV and gavage administration in both species and sexes. The absolute bioavailability following gavage administration was low (≤ 9.2%) in rats and mice of both sexes. Thus, the toxicoki-netic data are consistent with extensive first-pass metab-olism and saturation of metabolism at doses higher than 37 mg/kg. Our data is consistent with the physiologically based pharmacokinetic model developed where the absorption of oral doses of MEG in rats and mice was predicted to be rapid and complete, the distribution of methyleugenol to tissues is not hampered by capillary permeability, and the metabolism of methyleugenol is saturable (NTP 2000). The model predicts that there is an extrahepatic component in the mouse where MEG was cleared faster in mice than the rats.

Declaration of interest

The authors declare no conflict of interest.

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toxicokinetics of methyleugenol in f344 rats and b6c3f 1 mice

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