Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Mineral hydrocarbons (MHCs¹), as defined in recent health studies and regulatory reviews, are a class of highly refined petroleum products that include white mineral oils (liquid paraffins), petrolatums, and petroleum waxes. These products are complex mixtures, which consist of almost entirely of saturated hydrocarbons, predominately naphthenic and isoparaffinic hydrocarbons of carbon chains ranging from C15 to C85. The white mineral oils generally range from C15 to C50, with a peak at C25 to C26 in many of the commercial grades (CONCAWE, 1984). MHCs are used in many consumer items, including foods, plastics, cosmetics, pharmaceuticals, and agricultural products.

Numerous subchronic and chronic toxicity studies on MHCs conducted in mice, beagle dogs, and Sprague-Dawley (S-D) and Long-Evans rats have demonstrated that these products are safe, because few indications of toxicity have developed (Shubik et al., 1962; McKee et al., 1987; Firriolo et al., 1995; Smith et al., 1995; Miller et al., 1996). Similarly, human exposure to MHCs has not been associated with any significant adverse effects, although human exposure to MHC can result in oil deposits (lipogranulomas) in the liver and other lymphatic tissues (Cruickshank, 1984; Wanless and Geddie, 1985).

In spite of prior safety studies and evaluations, MHCs have come under further regulatory review primarily due to toxicological findings obtained after a 90-day feeding studies in F-344 rats (Baldwin et al., 1992; CONCAWE, 1993; Firriolo et al., 1995; Smith et al., 1996). These studies showed that large doses of white mineral oils and waxes produced some inflammatory cell accumulation and pathology in livers (granulomas) of female F-344 rats, as well as in the mesenteric lymph nodes. These responses have been shown in various feeding studies to be strain- and species-dependent, and the F-344 rat responds to the greatest degree. The underlying mechanism(s) for strain and species-specific responses to MHCs remains unknown but may relate to more extensive accumulation/retention of MHCs in the livers of F-344 rats.

Lonardo et al. (1998) evaluated rat strain differences in the pharmacokinetics and disposition of n-octadecane (C18), a representative lower molecular weight hydrocarbon in white oils and waxes (Lonardo et al., 1998). Female S-D rats, after a single oral gavage dose of [¹⁴C]octadecane (2 g/kg), exhaled greater amounts of ¹⁴CO₂ and had lower levels of radioactivity in liver 48 h postexposure than did similarly treated F-344 rats. These data suggested that S-D rats may be more efficient at metabolizing and clearing MHCs compared with F-344 rats after a single oral gavage dose. Thus, strain differences in the absorption, distribution, metabolism, and/or elimination of MHCs may play an important role in the observed differential toxicological responses observed in the MHC feeding studies.

Additional pharmacokinetic and disposition studies were conducted to test this hypothesis further. These studies were designed to better define inherent strain differences in the pharmacokinetics, disposition, and metabolism of MHCs. [1-¹⁴C]1-Eicosanylcyclohexane ([¹⁴C]EICO), illustrated in Fig. 1, was chosen as the surrogate MHC in these studies because it contains 26 carbons, the peak or average carbon number in many of the complex mixtures of MHCs (CONCAWE, 1984). [¹⁴C]EICO was labeled with ¹⁴C in the cyclohexane ring, which minimizes the loss of ¹⁴C signal due to exhalation of ¹⁴CO₂ and ¹⁴C-organics after metabolism.

View larger version (4K): [in this window] [in a new window]   Fig. 1.   Chemical structure and molecular weight of [1-14C]1-eicosanylcyclohexane, a representative MHC. Location of the radiolabel is indicated by .

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Chemicals. The following chemicals were purchased from the vendors indicated: [1-¹⁴C]1-eicosanylcyclohexane, specific activity 17 mCi/mmol (99.9% pure; Moravek Biochemicals, Brea, CA); 1-eicosanylcyclohexane and food-grade white mineral oil with a viscosity 15 centistokes at 40°C, an average molecular weight 350, and specific gravity approximately 0.85 g/ml (American Petroleum Institute, Washington, DC); olive oil, reagent grade, specific gravity approximately 0.91 g/ml (ICN Pharmaceuticals Biochemicals Division, Aurora, OH); ethylene glycol and 2-methoxyethyl ether (Mallinckrodt, Paris, KY); CarboSorb E and Flo-Scint III (Packard Instrument Company, Inc., Meriden, CT); tissue solubilizer TS-2 (Research Products International Corp., Mount Prospect, IL); toluene, high-purity solvent (Burdick and Jackson, Inc., Muskegon, MI); and Universol Cocktail (ICN Pharmaceuticals, Irvine, CA).

Animal Studies.

Animals Female F-344 and S-D rats and male S-D rats in a weight range of 175 to 199 g were purchased from Hilltop Laboratory Animals, Inc. (Scottdale, PA). Upon arrival, the animals were acclimated for 5 to 7 days in a temperature-controlled room (20-22°C) with a 12:12-h light/dark cycle before any treatment. Food (Teklad 4% Mouse-Rat Diet; Harlan Teklad, Madison, WI) and water were provided ad libitum. Some of these animals were fitted with an indwelling jugular vein cannula (JVC).

Dosing Solutions. Radiolabeled [¹⁴C]EICO, a representative MHC with 26 carbons (Fig. 1), was incorporated as a tracer in the dosing solutions to characterize the pharmacokinetics and disposition of MHCs. The dosing solutions were prepared in a stepwise procedure. Approximately 400 µl (400 µCi) of [¹⁴C]EICO in hexane was added to 1.6 ml of white oil and warmed slightly to remove the hexane volume and facilitate dissolution. Once dissolved into solution, 6.4 ml of olive oil was added to the white oil/[¹⁴C]EICO mixture (1.6 ml) without further warming and thoroughly mixed. The resulting solution (8.0 ml) was allowed to cool to room temperature before dosing. In the high-dose study, each rat was administered a single oral gavage dose (dosing volume 2 ml/kg) of MHC in olive oil (1:4, v/v) containing [¹⁴C]EICO (2.14 mg/kg; 100 µCi/kg). Using the densities of 0.85 g/ml (white oil) and 0.91 g/ml (olive oil), the dose of MHC was 340 mg/kg and that of olive oil was 1.46 g/kg. This represents a total oil dose (MHC + olive oil + [¹⁴C]EICO) of 1.80 g/kg.

Routes of Elimination Study. All rats were fasted for 18 h before oral administration of MHCs, but continued to receive water during this time. After dosing (1.80 g/kg; 100 µCi/kg), rats were immediately placed individually into sealed glass metabolism cages maintained with a constant inflow of ambient air. Two hours after dosing, food was reintroduced ad libitum. Urine, feces, and exhaled organics and CO₂ were collected at 8, 16, 24, 48, 72, and 96 h. Total airflow through each cage was passed through a series of traps containing either 2-methoxyethyl ether for collection of expired ¹⁴C-organics or CarboSorb E and ethylene glycol (2:1, v/v) for collection of expired ¹⁴CO₂. All trapping solvents were collected and changed at selected times and measured for total radioactivity by direct liquid scintillation counting (LS5000TD liquid scintillation counter; Beckman Coulter, Inc., Fullerton, CA). Rats were killed at 96 h by inhalation of carbon dioxide. Blood was immediately collected from the inferior vena cava into a heparinized syringe, and liver, mesenteric lymph nodes (MLNs), kidneys, lung, heart, spleen, and subcutaneous fat were excised. All samples were weighed and stored immediately at 80°C until analyzed. Direct liquid scintillation counting was used to determine total radioactivity associated with urine and exhaled products. Samples of homogenized feces and selected tissues were oxidized to ¹⁴CO₂ (Oxidizer 306; Packard Instrument Company, Inc., Downers Grove, IL) (Winter et al., 1992). Each oxidized product was collected into scintillation fluid and then counted directly for total radioactivity by direct liquid scintillation counting. A body composition estimate of 9% of body weight for subcutaneous fat was used (Mathews and Anderson, 1975).

Oral Pharmacokinetic and Disposition Studies. Rats with indwelling JVCs were orally administered either a high dose (1.80 g/kg) or a low dose (0.18 g/kg) of MHC, olive oil, and [¹⁴C]EICO tracer as described above. In another study, eicosanylcyclohexane was substituted for MHC. Female rats of both strains were administered a single high oral dose of eicosanylcyclohexane (340 mg/kg + 2.14 mg/kg [¹⁴C]EICO) in olive oil (total oil dose 1.80 g/kg). After administration, rats were immediately placed individually into Nalgene metabolism cages to allow collection of urine and feces at selected times. Two hours after dosing, food was reintroduced ad libitum. Serial blood samples (200 µl) were collected through the JVC at selected times (0.5-96 h), and an equal volume of saline was injected to replace the blood volume. Blood samples were measured directly for ¹⁴C equivalents by direct liquid scintillation counting (3 × 50 µl of whole blood). At least three rats per strain were killed at 4, 8, 16, 24, 48, 72, and 96 h by carbon dioxide asphyxiation in the high-dose study, whereas only six rats per strain were killed at 96 h in the low-dose study. At these times, blood, liver, and MLNs were collected. Direct liquid scintillation counting was used to determine the total ¹⁴C associated with urine. Homogenized fecal and selected tissue samples were prepared for radioactive counting by the addition of 1 ml of the tissue solubilizer TS-2. Once the samples were completely solubilized, glacial acetic acid was added to eliminate chemiluminescence and, together with scintillation cocktail, made possible accurate liquid scintillation counting for total ¹⁴C.

Metabolite Identification Study. Male S-D rats were orally administered a high dose (1.80 g/kg) of eicosanylcyclohexane (unlabeled material), olive oil, and the [¹⁴C]EICO tracer. After administration, rats were immediately placed individually into Nalgene metabolism cages to allow collection of urine at 16 h. Urine samples were analyzed by high-performance liquid chromatography (HPLC) to determine the metabolite profile and by liquid chromatography with tandem mass spectrometry (LC-MS/MS) to identify the major urinary metabolites.

Data Analysis. The blood concentration-time data after a single oral gavage dose of MHC, olive oil, and [¹⁴C]EICO to female JVC F-344 and S-D rats were analyzed by compartmental methods using nonlinear regression analysis (WinNonlin; Scientific Consulting, Inc., 1995). F-344 rats administered the low MHC dose exhibited an unexpected and unique systemic blood concentration-time curve at early time points (0.5 to 24 h). Therefore, these data were analyzed using noncompartmental methods as described by Rowland and Tozer (1995). For all other data, several models were analyzed statistically to determine the appropriate model to fit the blood curves. A one-compartment model with apparent first-order input adequately described the systemic blood concentration-time profiles of female F-344 rats after the high dose, whereas a two-compartment model described the profiles of S-D rats after both high and low doses. Pharmacokinetic parameter values best describing a linear compartmental model, and assuming first-order kinetics for all processes, were used to calculate values for the first-order terminal phase rate constant (K), terminal phase half-life (t_1/2), area under the systemic blood concentration-time curve from zero to time infinity (AUC), maximum systemic blood concentrations of [¹⁴C]EICO (C_max), and times to maximum systemic blood concentrations (T_max). The mean systemic blood concentration-time data were fit to the appropriate model to provide a graphical display of the data, except for the early times of F-344 rats after the low dose. These data were graphically displayed by connecting the data points.

Analytical Methods.

HPLC Analysis of EICO and Its Metabolites Processed blood, feces, liver, and MLN samples were analyzed via a reverse-phase HPLC method for [¹⁴C]EICO and ¹⁴C-metabolites at varying times. Whole blood samples were collected from each rat and centrifuged to separate the plasma. Plasma was then removed and concentrated by vacuum centrifugation to approximately 200 µl. Solubilized fecal, liver, and MLN samples that were counted for total radioactivity (described above) were also analyzed by HPLC. All samples were recentrifuged to remove any precipitate before being injected (100 µl) onto a Luna C₁₈ analytical column (250 × 4.6 mm; Phenomenex, Torrance, CA) and eluted with a mobile phase of water, methanol, and toluene; a flow rate of 1 ml/min; and a total run time of 210 min. The mobile phase gradient was run from 100% water to 100% methanol over a 177.5-min time period. After 5 min of 100% methanol, the gradient then proceeded to 100% toluene, which was held for 5 min. The column was brought back to initial conditions over 25 min. The HPLC system used throughout was composed of a SP8800 ternary HPLC pump and SP8775 autosampler (Spectra Physics, San Jose, CA). The column effluent was monitored radiochemically with a -Ram Flow-Through Monitor System (IN/US Systems, Inc., Tampa, FL). The retention time (R_T) of [¹⁴C]EICO was 195 min under these conditions.

Isolation of Urinary Metabolites. The two major urinary metabolites, labeled as peaks J and K, were isolated using solid phase extraction. Approximately 400 µl of urine was loaded onto a hydrophilic/lipophilic balance-solid phase extraction cartridge (3 ml; Waters, Milford, MA) and washed with ~1 ml of water. After washing, the metabolites were eluted with 1 ml of methanol. The methanol extract was analyzed by HPLC (same conditions described above) to confirm that only the two urinary metabolites were extracted and analyzed by LC-MS/MS to identify the urinary metabolites.

Metabolite Identification Using Mass Spectrometry and MS/MS Analysis. LC-MS was carried out using a Finnigan TSQ 7000 triple quadrupole mass spectrometer (Finnigan MAT, San Jose, CA) equipped with a Hewlett Packard HPLC system (Series 1050) consisting of an HPLC pump, automatic solvent degasser, and an atmospheric pressure chemical ionization (APCI) ion source. Extracted urinary metabolites were injected onto a Luna C₁₈ analytical column (250 × 4.6 mm; Phenomenex) and eluted with a mobile phase of water and methanol, a flow rate of 0.5 ml/min, and a total run time of 45 min. The HPLC mobile phase gradient was run from 90% water and 10% methanol containing 0.1% trifluoroacetic acid for 5 min and then to 100% methanol over 15 min. These conditions were held for 20 min before being brought back to initial conditions over 5 min.

Statistical Analysis. Statistical evaluations between F-344 and S-D rats were conducted using parametric and nonparametric procedures at the 5% level of significance. For the parametric procedures, a one-way analysis of variance program to assess significance was used (SigmaStat, 1994). If significant differences among the means were indicated, Student-Newman-Keuls method was used. For nonparametric procedures, the Kruskal-Wallis one-way analysis of variance on ranks was applied (Holland and Wolf, 1973). Statistical analyses of the fecal and urinary excretion data (Figs. 3 and 4, respectively) between the two rat strains were based on the individual amounts of ¹⁴C equivalents recovered during the sample collection periods (0-16, 16-24, 24-48, 48-72, and 72-96 h) and not based on the cumulative percentage of dose values graphically displayed in the figures. For example, the amount of ¹⁴C equivalents recovered in the fecal samples of F-344 rats during the 24- to 48-h collection period was significantly greater compared with the amount recovered from S-D rats, even though the cumulative data points overlap at 48 h in Fig. 3.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Blood Kinetics. Fig. 2 displays the concentration-time data of [¹⁴C]EICO from blood of rats after either a high oral dose (1.80 g/kg) or a low dose (0.18 g/kg) of MHC in olive oil. After administration of the high dose to F-344 rats, the C_max of 110 ng/ml was obtained at 16 h (T_max). The blood concentration then declined in a log-linear manner until the end of the study at 96 h. In contrast, the C_max in S-D rats was nearly 3-fold less (41 ng/ml) and occurred at an earlier T_max of 6 h. After the low dose, S-D rats had even lower C_max and T_max values (27 ng/ml and 5 h, respectively), whereas F-344 rats displayed a triphasic blood concentration-time profile. An initial peak concentration of 44 ng/ml was reached at 2 h, which has been labeled the first phase in the triphasic curve. The concentration in blood then decreased until 6 h but then increased again to 34 ng/ml at 16 h (second and third phases, respectively). After this time, the blood concentration of [¹⁴C]EICO declined log linearly until the end of the study at 96 h. After peak concentrations were achieved, a relatively slow elimination of [¹⁴C]EICO from blood was apparent in both rat strains at the two doses. Pharmacokinetic parameters are presented in Table 1.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Semilogarithmic plots of systemic blood concentrations of EICO, expressed as nanograms per millilter, as a function of time after a high dose of MHC (1.80 g/kg) to female F-344 () and S-D rats () (A), or a low dose of MHC (0.18 g/kg) to female F-344 and S-D rats () (B). Data represent mean systemic blood concentrations of EICO ± S.D. (n = 3-25/time point for the high-dose studies and n = 6/time point for the low-dose studies). *, significantly different between F-344 and S-D rats at P < 0.05.

Excretion and Distribution Data. Fecal excretion of total radioactivity was the major route of elimination for both rat strains in the high- and low-dose studies (Fig. 3). After the high dose, 92% (F-344) and 88% (S-D) of the administered radioactivity was recovered in the feces by 96 h, whereas 76% (F-344) and 70% (S-D) was recovered after the low dose. Although comparable amounts of [¹⁴C]EICO were excreted in the feces by 96 h within each study, fecal elimination was more rapid in S-D rats. At 16 h the majority of the administered radioactivity was present in the intestinal contents of F-344 rats (data not shown), whereas the bulk of [¹⁴C]EICO had been excreted in the feces by S-D rats.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Cumulative recovery of [14C]EICO eliminated in the feces as a function of time after a high dose of MHC (1.80 g/kg) to female F-344 () and S-D () rats (A), or a low dose of MHC (0.18 g/kg) to female F-344 () rats and S-D () rats (B). Data expressed as mean percentage of administered radioactivity ± S.D. (n = 3-23/time point for the high-dose studies and n = 6/time point for the low-dose studies). The solid lines are the nonlinear regression fits of the data. *, significantly different between F-344 and S-D rats at P < 0.05. See "Statistical Analysis" under Materials and Methods for further explanation.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Cumulative recovery of total radioactivity excreted in the urine as a function of time after a high dose of MHC (1.80 g/kg) to female F-34 () and S-D () rats (A), or a low dose of MHC (0.18 g/kg) to female F-344 () rats and S-D () rats (B). Data expressed as mean percentage of administered radioactivity ± S.D. (n = 3-23/time point for the high-dose studies and n = 6/time point for the low-dose studies). The solid lines are the nonlinear regression fits of the data. *, significantly different between F-344 and S-D rats at P < 0.05. See "Statistical Analysis" under Materials and Methods for further explanation.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Recovery of [14C]EICO in the livers and MLNs of female F-344 () and S-D () rats after oral administration of MHC. A, liver samples after high dose of MHC (1.80 g/kg). B, Liver samples after low dose of MHC (0.18 g/kg). C, MLN samples after high dose of MHC. D, MLN samples after low dose of MHC. Data expressed as mean percentage of administered radioactivity ± S.D. (n = 3-16/time point for the high-dose studies and n = 6/time point for the low-dose studies). a Recovery of [14C]EICO in the liver and MLN samples only determined at 96 h in the low-dose studies. *, significantly different between F-344 and S-D rats at P < 0.05.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Percentage of administered radioactivity per gram liver or MLN at 96 h after a high oral dose (1.80 g/kg) or a low oral dose (0.18 g/kg) of MHC to female F-344 () and S-D () rats. Data expressed as mean percentage of administered radioactivity per gram of tissue ± S.D. (n = 16/time point for the high-dose studies and n = 6/time point for the low-dose studies).

Determination of Urinary Metabolites. HPLC analysis of pooled urine collected from rats treated orally with the high dose of MHC containing [¹⁴C]EICO revealed that only metabolites of EICO were excreted. A large number of metabolites were present, as evidenced by the HPLC radiochromatograms (Fig. 7). Interesting strain differences were observed with respect to the elimination of [¹⁴C]EICO metabolites in the urine. Although the profiles of ¹⁴C-containing peaks were the same for both rat strains, S-D rats eliminated the great bulk of these metabolites within the first 16 h. Only minute amounts were present in urine collected at 24 or 48 h, and none were present at 72 or 96 h. On the other hand, F-344 rats excreted metabolites much more slowly. Similar amounts of each metabolite appeared in the urine at all times.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   HPLC radiochromatograms of pooled urine samples (16-96 h) collected from female F-344 (A) and S-D rats (B) after a high oral dose (1.80 g/kg) of MHC in olive oil (1:4, v/v) containing [14C]EICO as a tracer. Peaks J and K are major urinary metabolites detected at RT of 150 min and 163 min, respectively, in both rat strains.

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Representative APCI-MS/MS spectra of the major urinary metabolite 12-cyclohexyldodecanoic acid (HPLC peak K; RT of 163 min) (A) and the urinary metabolite 10-cyclohexyldecanoic acid (HPLC peak J; RT of 150 min) (B).

Determination of [¹⁴C] in Blood, Feces, Liver, and MLNs. Only one major radioactive peak at R_T of 195 min was detected by HPLC analysis in prepared plasma samples and solubilized fecal, liver, and MLN samples regardless of dose (radiochromatograms not shown). This peak had a retention time identical to that of [¹⁴C]EICO.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Previous 90-day feeding studies have shown that female F-344 rats are more susceptible to the development of liver and MLN microgranulomas after exposure to white mineral hydrocarbons and waxes than other species and/or rat strains (Firriolo et al., 1995; Smith et al., 1995; Miller et al., 1996). The pathological responses observed in F-344 rats include hepatic granulomas, mesenteric lymph node microgranuloma, and inflammation of the mitral valve (only with paraffin wax), as well as an increase in tissue weights and MHC tissue concentrations. The mechanism(s) for the reported sex, species, and strain-dependent responses to MHC is unknown, but the effects may be due, in part, to differences in the disposition and metabolism of MHC. To test this hypothesis further, studies were performed using a representative tracer MHC, [¹⁴C]EICO), dissolved in MHC to examine possible inherent rat strain differences in pharmacokinetic parameters and disposition profiles after oral administration of a single dose. The data obtained demonstrate strain differences in pharmacokinetic and disposition profiles for this representative MHC. In general, the results show higher systemic exposure and enhanced hepatic retention of [¹⁴C]EICO in F-344 rats because of greater absorption and/or decreased ability to metabolize [¹⁴C]EICO and by extrapolation MHC.

Albro and Fishbein (1970) reported that MHCs (and/or their metabolites) are mainly absorbed within the small intestine and are subsequently transported via the lymphatic and portal systems into the systemic circulation. Although not directly assessed, one or both of these processes may be more efficient in F-344 rats compared with S-D rats, thus resulting in the greater systemic blood exposure of [¹⁴C]EICO. Possible strain differences in the absorption processes may have been observed in the low dose studies. After administration of the low dose of MHC, a unique triphasic blood concentration-time profile (two distinct C_max values) was seen in F-344 rats, but not in S-D rats. All F-344 rats (n = 6) displayed this type of profile, whereas all six S-D rats did not. The two C_max values may be the result of two separate routes of MHC/[¹⁴C]EICO absorption. As reviewed by Porter and Charman (1997), the process of intestinal lymphatic drug transport often continues over time periods longer than typically observed for drug absorption via the portal vein. Based on this information, the initial rise in blood concentration (first phase) may be absorption via the hepatic portal system, and the second increase in blood concentration may be the result of absorption through the lymphatic system. This two-phase absorption profile was not detected in F-344 rats after the high dose, where the larger amount of MHC/[¹⁴C]EICO administered may have masked these processes. Interestingly, this type of profile was not detected in S-D rats regardless of the dose. This suggests that differences could exist between the two rat strains in the uptake of MHC/[¹⁴C]EICO into the systemic circulation.

Compared with small molecular weight, lipophilic agents, the absorption of [¹⁴C]EICO into the systemic blood circulation was a slow process. After administration of the high dose of MHC, T_max did not occur until 6 and 16 h after oral dosing in the S-D and F-344 rats, respectively. This slow absorption process may relate to the extremely lipophilic nature of [¹⁴C]EICO (estimated logP of 12.9), as well as the complex process of chylomicron formation before systemic absorption (Levy, 1992). Also, the more extensive, but slower absorption of [¹⁴C]EICO in F-344 rats suggests a prolonged gastrointestinal transit time compared with S-D rats, which would allow more contact time for increased MHC absorption. However, when gastrointestinal transit times of female F-344 and S-D rats were experimentally determined by two different methods, no differences were observed (data not shown). The delay in fecal elimination of [¹⁴C]EICO observed in F-344 rats most likely relates to the large variations in the amount of feces eliminated between individual F-344 rats. In general, the less stool that was eliminated at these early times (16-24 h), the less radioactivity that was recovered. S-D rats showed considerably less animal-to-animal variation at these times.

Along with strain differences in systemic exposure and fecal excretion profiles of [¹⁴C]EICO, the urinary excretion rates were also distinctly different. S-D rats excreted the majority of the absorbed ¹⁴C equivalents by 16 h, a rate significantly faster than that observed for F-344 rats. The F-344 rats eliminated ¹⁴C-metabolites of [¹⁴C]EICO at a relatively constant rate through 96 h. The time-dependent rate of urinary excretion is indicative of a depot effect, where a rate-limiting process is involved before urinary excretion. This rate-limiting step in F-344 female rats could include tissue retention of [¹⁴C]EICO and/or impaired capacity to biotransform [¹⁴C]EICO into excretable metabolites.

Previous studies have shown that F-344 rats retain greater amounts of MHC in target tissues compared with other rat strains and species after 60- and 90-day dietary exposure (2% in diet) to MHCs (Firriolo et al., 1995; Hoglen et al., 1998). This observation is consistent with the data presented herein. Although peak concentrations of [¹⁴C]EICO in livers of both rat strains were reached at the same time after administration, S-D rats clear [¹⁴C]EICO more efficiently from livers compared with F-344 rats. As a result, higher hepatic levels of [¹⁴C]EICO are maintained in F-344 rats.

The greater AUC, the relatively long terminal t_1/2 value, the time-dependent urinary excretion profile, and the observed hepatic retention of [¹⁴C]EICO may all result from a reduced capacity of F-344 rats to metabolize [¹⁴C]EICO. Initial metabolic steps include oxidative metabolism of the saturated hydrocarbon in the liver (and intestine) to the corresponding alcohols and fatty acids. Subsequently, the oxidized metabolites can undergo the same metabolic fate as endogenous fatty acids and saturated fats (Lester, 1979; Le Bon et al., 1988; Barrowman et al., 1989). This metabolic process of saturated hydrocarbons was further confirmed in these studies by the identification of 12-cyclohexyldodecanoic acid and 10-cyclohexyldecanoic acid, C18- and C16-carboxylic acids, respectively, in the urine of rats after [¹⁴C]EICO administration. The initial oxidative step of the saturated hydrocarbon chain could be the rate-limiting step in female F-344 rats, as described for the metabolism of phytane (2,6,10,14-tetramethylhexadecane) before urinary elimination by rats (Albro and Thomas, 1974). Evidence for this is that only parent [¹⁴C]EICO is retained by the liver of F-344 rats. Therefore, the retention of parent [¹⁴C]EICO occurs because of the lack of hepatic clearance due to impaired metabolism.

The initial oxidative step of [¹⁴C]EICO, and most likely of other MHCs, involves cytochrome P450 enzymes (McCarthy, 1964; Nicolaides and Kellum, 1966; Bartley et al., 1971; Frommer et al., 1972; Toftgard and Nilsen, 1982; Perdu-Durand and Tulliez, 1985). Results from preliminary in vivo studies using 1-aminobenzotriazole (ABT) to inhibit P450 enzymes support this view. The hepatic retention of [¹⁴C]EICO was increased 3- to 4-fold in ABT-pretreated F-344 rats compared with non-ABT-pretreated rats at 96 h after [¹⁴C]EICO dosing (data not shown).

In summary, the results reported herein demonstrate that inherent differences in the pharmacokinetic and disposition of [¹⁴C]EICO are present between female F-344 and S-D rats. F-344 rats are systemically exposed to higher blood concentrations of [¹⁴C]EICO; excrete metabolites of [¹⁴C]EICO in urine in a slower, time-dependent manner; and retain a significant amount of [¹⁴C]EICO in livers at 96 h. Based on the hepatic retention of parent [¹⁴C]EICO in F-344 rats, a difference in the ability of the livers to metabolize [¹⁴C]EICO most likely explains the pharmacokinetic/dispositional differences observed between the two rat strains. How this lack of metabolism and retention of MHC by livers of F-344 female rats relates to the development of hepatic granulomas after repeated dosing remains to be established.