Comparative Pharmacokinetic and Disposition Studies of [1-c]1- Eicosanylcyclohexane, a Surrogate Mineral Hydrocarbon, in Female Fischer-344 and Sprague-dawley Rats

White oils or waxes [mineral hydrocarbons (MHCs)] with substantial levels of saturated hydrocarbons in the range of C18 to C32 have produced hepatic microgranulomas and lymph node microgranulomas (also referred to as histiocytosis) after repeated administration to female Fischer-344 (F-344) rats. Female SpragueDawley (S-D) rats are less sensitive to these MHC-induced hepatic and lymph node effects. Studies reported herein characterized the pharmacokinetics and disposition of a representative C-26 MHC, [1-C]1-eicosanylcyclohexane ([C]EICO), in these two rat strains. Female F-344 and S-D rats were administered by oral gavage either a high (1.80 g/kg) or a low (0.18 g/kg) dose of MHC in olive oil (1:4, v/v) containing [C]EICO as a tracer. Blood, urine, feces, liver, and mesenteric lymph nodes (MLNs) were analyzed for [C]EICO and C-metabolites. After the high dose, F-344 rats had a higher blood Cmax of [ C]EICO, a longer time to Cmax, and a greater area under the systemic blood concentration-time curve from zero to time infinity compared with S-D rats. After the low dose, F-344 rats displayed a unique triphasic blood concentrationtime profile, meaning two distinct Cmax values were observed. Fecal excretion was the major route of [C]EICO elimination for both rat strains (70–92% of the dose). S-D rats eliminated the majority of [C]EICO metabolites recovered in the urine by 16 h (8–17% of the dose), whereas F-344 rats did not excrete the same amount until 72 to 96 h. Beyond 24 h, a greater level of [C]EICO was recovered in livers of F-344 rats; at 96 h, 3 and 0.1% of the dose was retained in livers of F-344 and S-D rats, respectively. The major urinary metabolites of EICO in both rat strains were identified as 12-cyclohexyldodecanoic acid and 10-cyclohexyldecanoic acid. Based on the pharmacokinetic parameters and disposition profiles, the data indicate inherent strain differences in the total systemic exposure, rate of metabolism, and hepatic and lymph node retention of [C]EICO, which may be associated with the different strain sensitivities to the formation of liver granulomas and MLN histiocytosis. Mineral hydrocarbons (MHCs), 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 LongEvans 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 strainand 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 This research was supported by the American Petroleum Institute White Oil and Wax Technical Workgroup, the National Institute of Environmental Health Sciences-sponsored Southwest Environmental Health Sciences Center (P30-ES06694), and the Flinn Foundation. 1 Abbreviations used are: MHC, mineral hydrocarbon; S-D, Sprague-Dawley; F-344, Fischer-344; EICO, [1-C]1-eicosanylcyclohexane; JVC, jugular vein cannula; MLN, mesenteric lymph node; HPLC, high-performance liquid chromatography; LC-MS/MS, liquid chromatography with tandem mass spectrometry; AUC, area under the systemic blood concentration-time curve from zero to time infinity; APCI, atmospheric pressure chemical ionization; CID, collision-induced dissociation; RT, retention time; ABT, 1-aminobenzotriazole. Address correspondence to: Dr. I. Glenn Sipes, Department of Pharmacology, College of Medicine, P.O. Box 210207, The University of Arizona, Tucson, AZ 85721-0207. E-mail: [email protected] 0090-9556/02/3012-1470–1477$7.00 DRUG METABOLISM AND DISPOSITION Vol. 30, No. 12 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 722/1027040 DMD 30:1470–1477, 2002 Printed in U.S.A. 1470 at A PE T Jornals on Jne 2, 2017 dm d.aspurnals.org D ow nladed from 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 [C]octadecane (2 g/kg), exhaled greater amounts of CO2 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-C]1-Eicosanylcyclohexane ([C]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). [C]EICO was labeled with C in the cyclohexane ring, which minimizes the loss of C signal due to exhalation of CO2 and C-organics after metabolism. Materials and Methods Chemicals. The following chemicals were purchased from the vendors indicated: [1-C]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, highpurity 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 [C]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 l (400 Ci) of [C]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/[C]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 [C]EICO (2.14 mg/kg; 100 Ci/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 [C]EICO) of 1.80 g/kg. In the low-dose study, each rat received 0.2 ml/kg of the dosing solution described above. Therefore, the dose of [C]EICO was 0.21 mg/kg ( 10 Ci/kg), the dose of MHC was 34 mg/kg, and that of olive oil was 0.15 g/kg. This represents a total oil dose of approximately 0.18 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 Ci/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 C-organics or CarboSorb E and ethylene glycol (2:1, v/v) for collection of expired CO2. 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 CO2 (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 [C]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 [C]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 l) 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 C equivalents by direct liquid scintillation counting (3 50 l 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 C 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 C. 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 [C]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 [C]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 FIG. 1. Chemical structure and molecular weight of [1-C]1eicosanylcyclohexane, a representative MHC. Location of the radiolabel is indicated by . 1471 COMPARATIVE STUDIES OF EICO at A PE T Jornals on Jne 2, 2017 dm d.aspurnals.org D ow nladed from