Abstract
These studies characterize the effect of dose and route of administration on the disposition and elimination of the ionic liquid, 1-butyl-3-methylimidazolium chloride (Bmim-Cl). After i.v. (5 mg/kg) or oral (50 mg/kg) administration to male F-344 rats [14C]Bmim-Cl detected in blood decreased rapidly. Clearance rates from the blood after i.v. and oral administration were similar (7.4 and 11.9 ml/min, respectively). Systemic bioavailability was determined to be 62.1% of a 50 mg/kg dose in rats. Urinary excretion of the parent compound by rats was the major route of elimination (i.v.: 91% in 24 h; oral: 55–74% in 24 h). The rates and routes of elimination were not affected by escalation of dose (0.5–50 mg/kg) or repeated oral administration (five daily administrations, 50 mg/kg) and were similar in male rats and B6C3F1 female mice (86–95% of dose eliminated in 24 h). Apparent systemic exposure to Bmim-Cl after dermal administration was dependent upon vehicle, as assessed by the percentage of dose eliminated in urine after application in a particular vehicle (water: 1%; ethanol/water: 3%; and dimethylformamide/water: 13% of dose). Regardless of gender, species, dose, route, or number of exposures, high-pressure liquid chromatography-UV/visible-radiometric analyses of urine samples showed a single peak that coeluted with the Bmim-Cl standard. These studies illustrate that systemic bioavailability of Bmim-Cl is high, tissue disposition and metabolism are negligible, and absorbed compound is extensively extracted by the kidney and eliminated in the urine as the parent compound.
Ionic liquids (ILs) comprise a class of organic salts that are distinguished by a range of useful properties such as negligible vapor pressure, thermal stability, nonflammability, high ionic conductivity, and variable solubility properties (Welton, 1999). The structural characteristics of ILs feature an organic ammonium cation and an exchangeable anion. This anion can range from a simple halide to complex polyatomic ions (Sheldon, 2001). Commonly used ILs are based on imidazolium, pyridinium or pyrrolidinium cations, a number of which are commercially available (Freemantle, 2003). Because they are generally nonvolatile and stable at standard temperature and pressure, ILs may supplant or reduce the reliance on classic volatile organic solvents currently used in industrial chemistry (Integrated Laboratory Systems, 2004).
Room temperature ionic liquids possess a number of physical and chemical properties that explain their potential for industrial and laboratory applications. There is growing interest in developing cleaner technologies across the chemical industry, for both economic and environmental reasons, with the search for alternatives to conventional solvents being foremost (Seddon, 1997). A major reason is that although organic solvents are used in massive quantities, their use is generally restricted to a single use, as the high vapor pressure of these solvents promotes loss into the environment as air pollutants. The low vapor pressures of ILs would result in limited evaporative and environmental losses (Huddleston et al., 1998). High-volume use of solvents for large-scale synthesis is a standard procedure for industry, but tailored physical properties for specific processes and reactions coupled with improved recapture and reuse of IL-based solvents could reduce these volumes and decrease waste (Marsh et al., 2002). Also, intrinsically the low vapor pressures of ILs allow for use in high-vacuum reaction systems without solvent loss. Ionic liquids can be designed for both organic and inorganic reactions, a unique property that could be exploited to bring particular chemicals together in the same phase. Furthermore, ILs can be adapted to be immiscible with particular solvents, resulting in nonaqueous alternatives for two-phase reaction systems (Welton, 1999).
Rogers and Seddon (2003) noted that althouh some ILs are toxic, the theoretical structural versatility of ILs could result in the design of numerous nontoxic and chemically useful ILs. The environmental impact of these compounds has not yet been determined, and few data on the mammalian toxicity, metabolism, and disposition of these chemicals are available. The studies reported here represent the first comprehensive studies on the absorption, distribution, metabolism and elimination of a model IL, 1-butyl-3-methylimidazolium chloride (Bmim-Cl). We describe the toxicokinetics of Bmim-Cl after i.v. or oral dosing and the extent of its systemic bioavailability after oral or dermal administration and assess its metabolism. Studies were also designed to characterize the elimination profile after dermal application and repeated oral administration.
Materials and Methods
Chemicals. [14C]Labeled Bmim-Cl in water (lot no. 9719-75) was received from RTI International (Research Triangle Park, NC) and stored at 8°C. The 14C label was located on the α-carbon of the butyl side chain and is indicated by an asterisk in Fig. 1. An unlabeled Bmim-Cl reference standard (98% Bmim-Cl) was obtained from EMD Chemicals Inc. (Gibbstown, NJ). The radiochemical purity of [14C] Bmim-Cl was 97.5%, as determined by reversed-phase HPLC-radiometric detection. The specific activity was reported to be 27.5 mCi/mmol. Soluene-350 and solvable tissue solubilization solvents and Pico-Flour 40 scintillation cocktail solution were obtained from PerkinElmer (Torrance, Ca). Dimethylformamide (DMF) was obtained from J. T. Baker (Phillipsburg, NJ). Hydrogen peroxide (30%) was obtained from VWR (West Chester, PA). All reagents used in these experiments were HPLC or analytical grade.
Animal Studies.Animals. Male Fischer-344 (F-344) rats (8–9 weeks of age; 161–200 g) without surgical alteration (conventional) or with in-dwelling jugular vein cannulas (JVCs) were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). Female B6C3F1 mice (8–10 weeks old; 17–20 g) were obtained from Harlan (Indianapolis, IN). Animals were maintained in the University of Arizona Animal Care facility, which is accredited by the Association for Assessment and Accreditation for Laboratory Animal Care. The animals were maintained in a temperature-controlled (25°C) room under a 12-h light/dark cycle. Conventional animals were acclimated for 5 to 7 days after receipt. Those animals with indwelling JVCs were acclimated for only 1 day after arrival to ensure the patency of the cannulas. The rats and mice were allowed food and water ad libitum except as noted. Animals used in oral- and i.v.-dose administered disposition and toxicokinetic studies were fasted for 12 h before administration of Bmim-Cl. Food was returned 2 h after dosing. Animals used in the dermal and repeated oral administration studies were not fasted. Food (NTP 2000; Harlan Teklad, Madison, WI) was provided as a powder except to the animals in dermal administration studies. Food particles were separated from feces during collection in these studies.
Dose selection. The selection of doses was based on the acute oral LD50 of 550 mg/kg b.wt. in female F-344 rats reported by Landry et al. (2005). In the studies reported herein, subtoxic doses of 50 mg/kg (0.1 × LD50), 5 mg/kg (0.01 × LD50), and 0.5 mg/kg (0.001 × LD50) were chosen to assess the effect of dose on the rate and route of excretion after oral administration. For repeated doses studies 50 mg/kg was administered daily by oral gavage for 5 days. The dose of 5 mg/kg was selected for the i.v. route of administration. Landry et al. (2005) reported that a dermal application of 2000 mg/kg Bmim-Cl (95% in water) was not toxic to F-344 rats but did induce dermal irritation at the site of application. However, 2000 mg/kg Bmim-Cl (75% in DMF) was toxic to B6C3F1 mice. A preliminary dermal study in rats with a 50 mg/kg dose of [14C]Bmim-Cl [1900 μg/cm2 in methanol/water (1.8:1)] also resulted in irritation at the site of application; therefore, a dose of 5 mg/kg (190 μg/cm2) was used for the dermal application studies. All doses provided 50 μCi/kg [14C]Bmim-Cl unless otherwise indicated.
Sample collection and preparation. After dosing, the animals were maintained in Nalgene metabolism cages for collection of urine and feces. In single dose studies, urine was collected at 6, 12, 24, 36, 48, and 72 h; feces were collected at 12, 24, 36, 48, and 72 h. In the repeated dose studies, urine was collected at 6, 12, and 24 h after each dose, and feces were collected at 12 and 24 h after each administration. The metabolism cages were rinsed with deionized water (approximately 15 ml) after the collection of urine. Radioactivity recovered in cage rinses was added to that determined for urine.
After collections, aliquots of urine and cage rinse (3 × 100 μl) were directly analyzed for 14C radioactivity content by liquid scintillation counting. In addition, 100 μl of urine was diluted with 100 μl of water (1:1 ratio), vortexed, and filtered through a 0.45-μm Whatman nylon syringe filter. The filtered sample was transferred into an insert in a HPLC vial for HPLC-UV/Vis-radiometric analysis. Samples of urine for LC/MS analysis came from animals that were administered nonradiolabeled Bmim-Cl. These samples were prepared using the same methods as for HPLC-UV/Vis-radiometric analysis.
After collection, feces were mixed with water to form a homogeneous mixture. To determine total 14C equivalent content, fecal samples were solubilized with Soluene-350 (Thompson and Burns, 1996). In addition, aliquots of feces (250 mg) were extracted with 1.25 ml of methanol (three times) and pooled. Pooled supernatants were evaporated to dryness and reconstituted in methanol. Reconstituted samples (250 μl) were transferred into an insert in a HPLC vial for HPLC-UV/Vis-radiometric analysis.
After solubilization and addition of Pico-Fluor (15 ml), all samples were stored in the dark for 48 h to control for chemiluminescence and were corrected for background. Total radioactivity in all samples was determined by LSC.
At the termination of each study, animals were euthanized by CO2 inhalation. Blood was collected from the posterior vena cava, and the animals were subjected to necropsy. All samples collected (adipose tissue, blood, cecum, cecum contents, heart, intestine, intestinal contents, kidneys, lung, liver, muscle, spleen, stomach, stomach contents, skin, and testes) were analyzed immediately or stored at –80°C until analysis. Body composition estimates of 50% for muscle, 8% for blood, 11% for adipose tissue, and 16% for skin were used to estimate total masses of these tissues (Birnbaum et al., 1980). Triplicate aliquots from collected tissues (∼100 mg) were solubilized using Solvable (Thompson and Burns, 1996).
Dermal Application Studies. Male F-344 rats were prepared for nonoccluded dermal application of Bmim-Cl as described previously (Winter and Sipes, 1993). Rats were topically dosed with [14C]Bmim-Cl (5 mg/kg, 100 μCi/kg, 400 μl/kg) in water, ethanol-water (1.8:1), or DMF-water (1.8:1). Additionally, Bmim-Cl [5 mg/kg in DMF/water (1.8:1)] was applied for five consecutive days to determine the irritancy potential of repeated dermal exposure to Bmim-Cl. Feces, urine, and cage rinses were collected and analyzed as described above. Animals dosed with aqueous vehicle were euthanized by CO2 inhalation after 72 h, whereas animals dosed with [14C]Bmim-Cl in DMF-water or ethanol-water were euthanized at 48 h after application. Skin from the application area was treated in accordance with the Organization for the Economic Cooperation and Development guideline (OCDE 427) for the testing of chemicals (OECD/OCDE, 2004). Briefly, the skin was swabbed 5 times using filter paper soaked with a 10% soap solution and then tape stripped five times using clear sticky-tape to maximize recovery of the dose remaining on the surface of the skin. Skin and tape strips were solubilized using Soluene as described above and were analyzed by LSC. Skin washes were analyzed directly by LSC.
Blood toxicokinetics of Bmim-Cl: dosing regimen. [14C]Bmim-Cl (5 mg/kg, 50 μCi/kg, 1 ml/kg) was administered i.v. through the jugular vein cannula in saline for determination of i.v. blood kinetics. Blood (100 μl) was then drawn into the cannula and returned to the circulation, and the cannula was flushed with normal saline (1 ml/kg). For oral blood kinetic studies, [14C]Bmim-Cl (50 mg/kg, 100 μCi/kg, 2 ml/kg), in saline was administered by oral gavage to male F-344 rats with JVCs (n = 4).
For toxicokinetic studies, blood samples (300 μl) were collected via the JVC at 0.125, 0.25, 0.5, 0.75, 1, 1.5, 3, 6, 9, 12, 24, and 36 h into heparinized syringes. Aliquots of blood removed via the JVC were replaced with an equal volume of saline. The strategy for blood sampling was designed to reduce interanimal variability by obtaining blood time points from the same animal, instead of obtaining time points from different animals. The decision to obtain 300 μl per time point was based on recommendations made in the report by Diehl et al. (2001) who demonstrated that in short-term toxicokinetic studies, the removal of 20% of the blood volume over 24 h produces minimal disturbance of normal physiological function. Aliquots of blood (two 50-μl aliquots) were solubilized for quantification of 14C radioactivity by LSC. For HPLC-radiometric analysis of 14C radioactivity in blood, aliquots of blood (150 μl) were mixed with acetonitrile (450 μl), vortexed, and centrifuged. Extractions were performed three times. Supernatants from each sample were collected and pooled, and the solvent was evaporated to dryness under vacuum (MiVac; Genevac, Valley Cottage, NY). Extracts were reconstituted in 150 μl of a water-acetonitrile mixture (93%:7%, v/v) and placed in HPLC vials for analysis.
Blood toxicokinetics of Bmim-Cl: toxicokinetic analyses. The blood concentration-time data after oral or i.v. administration were analyzed using a one- or two-compartment toxicokinetic model, respectively. A modeling program (WinNonlin Professional, version 5.1; Pharsight, Mountain View, CA) was used to fit the data by nonlinear regression analyses, assuming first-order kinetics for all processes. The parameters of the model were used to calculate values for the area under the blood concentration-time curve from time 0 to infinity (AUC[0–∞]), distribution half-life (t1/2α), terminal elimination half-life (t1/2β), maximum concentration of Bmim-Cl in the blood (Cmax), rate of clearance from blood (CLb), and volume of distribution under steady-state conditions (Vss). Only samples containing quantities of compound above the limit of quantification were used in toxicokinetic analyses. For this reason data points after 6 h were not used for toxicokinetic analyses. Kinetic parameters were not adjusted for sample loss due to blood withdrawal because total loss was only 0.05 and 0.19% for the oral and i.v. doses, respectively.
Analytical Methods.HPLC analysis of Bmim-Cl in blood, urine, and feces. Aliquots of the reconstituted samples (100 μl) were subjected to HPLC separation (Agilent Technologies, Palo Alto, CA). The samples were injected onto a C18 guard column (SecurityGuard, 4.0 × 3.0 mm, AJO-4287; Phenomenex, Torrance, CA) followed by a C18 reversed-phase column (Luna Prodigy ODS3, 5 μm, 250 × 4.6 mm; Phenomenex). The mobile phases consisted of acidified water (0.1 M trifluoroacetic acid) and acidified acetonitrile at a flow rate of 1 ml/min with a total run time of 35 min. The mobile phase gradient was run from 93% water and 7% acetonitrile for the first 8 min, then up to 9% acetonitrile over 7 min, then to 15% acetonitrile over 10 min, and finally to 95% acetonitrile for the last 10 min. The mobile phase was brought back to initial conditions, and the column was allowed to reequilibrate for 10 min between injections. Using this gradient, Bmim-Cl eluted at 18 min. The eluent from the HPLC column was monitored with a diode array detector for Bmim-Cl (λmax = 220 nm) and a flow-through beta ram detector for 14C radioactivity (IN-US, Tampa, FL). Fractions were collected at 1-min intervals using an Agilent 220 fraction sampler. Data were acquired with data acquisition software (ChemStation for LC 3D, Rev B 01.01 [164]; Agilent Technologies). Calibration standards and quality control samples were prepared from concentrated stock solutions. The limit of detection using HPLC-UV/Vis detection at 220 nm was 4.83 μg/ml and the limit of quantification (LOQ) was 14.63 μg/ml. The limit of detection and LOQ using LSC were 0.1 and 0.35 ng, respectively, as determined using the methods described by Zhu et al. (2005).
LC-MS analysis of Bmim-Cl in urine. HPLC separation of urine samples was performed as described above. The HPLC system was coupled to an MSD-Trap SL ion trap mass spectrometer (Agilent Technologies). Analytes were ionized using an electrospray ionization source in the positive ion mode over a scan range of m/z = 50 to 1000. Samples were dried with nitrogen at a flow rate of 10 ml/min, drying temperature of 350°C, nebulizer pressure of 50 psi, and capillary and end-plate currents of 17 and 650 nA, respectively. MS/MS fragmentation was performed at m/z = 4.0 isolation width at 1 V fragmentation amplitude.
Results
Toxicokinetics of Bmim-Cl. Blood data after i.v. administration of [14C]Bmim-Cl (5 mg/kg) indicated that the radiolabel was rapidly removed from the systemic circulation. HPLC-radiometric analysis of blood extracts showed a single radioactive peak that coeluted with Bmim-Cl (Rt = 17.1 min; Fig. 2, A and B). This peak decreased with time after dosing, and new peaks were not observed. As no evidence of metabolism was obtained, the blood toxicokinetics of Bmim-Cl were determined from total radioactivity detected in whole blood. The effect of time on the concentration of Bmim-Cl in blood was described by a biexponential equation consistent with a two-compartment model (Fig. 3A). Toxicokinetic analysis showed that Bmim-Cl was rapidly (t1/2α = 13 min) and widely distributed to the tissues (Vss = 618.2 ml) and readily eliminated (t1/2β = 85.4 min). The AUC[0-∞] and CLb values were 141.3 μg · min/ml and 7.4 ml/min, respectively.
The effect of time on the blood concentration of [14C]Bmim-Cl after oral administration of [14C]Bmim-Cl (50 mg/kg, 100 μCi/kg) is shown in Fig. 3B. HPLC analysis of extracts showed a single peak that coeluted with the [14C]Bmim-Cl standard (Fig. 2, A and C). Toxicokinetic analysis of Bmim-Cl present in blood revealed that it was readily absorbed after oral dosing; Cmax (3.6 μg/ml) was reached 67 min after administration. After Cmax, [14C]Bmim-Cl disappeared from the blood with a t1/2β of 77.2 min. Oral systemic bioavailability (F) was based on the ratio of AUC[0–∞] calculated for oral administration (733.3 μg · min/ml) and AUC[0-∞] calculated for i.v. administration (141.3 μg*min/ml) and adjusted for dose (doseoral = 8756 μg/animal; dosei.v. = 1047 μg/animal). F was determined to be 62.1%. A summary of toxicokinetic parameters is given in Table 1.
Disposition and Elimination of Bmim-Cl.Intravenously administered Bmim-Cl. The route of elimination of Bmim-Cl was almost exclusively urinary after i.v. administration (Table 2). HPLC-radio-metric analyses of the urine detected a single radioactive peak that coeluted with the [14C]Bmim-Cl standard. This single peak accounted for 96% of the radioactivity detected in the urine. The remaining 4% was due to contaminants present in the dosing solution. Within the first 12 h, 84 ± 10% of the administered radioactivity was eliminated in the urine. An additional 7% was eliminated by this route in the next 12 h. By 48 h, 92% of the dose had been recovered in urine (Fig. 4).
Orally administered Bmim-Cl. After oral administration of [14C]Bmim-Cl (50, 5, or 0.5 mg/kg) as a single oral bolus dose to male F-344 rats 72 to 77% of the total administered radioactivity was eliminated in the urine over the 72-h collection period. Administration of [14C]Bmim-Cl to female B6C3F1 mice (50 mg/kg) resulted in an elimination profile similar to that of male F-344 rats (Table 3). The more variable recoveries of radioactivity in murine studies were attributed to the probability of the mice urinating and defecating in the feed hoppers. This presumption was based on observations of feces found in the ground feed and notable amounts of time spent in the enclosures. The peak excretion for all doses and in both species occurred between 6 and 12 h after administration (Fig. 5). The elimination of radioactivity in feces accounted for less than 30% of the administered dose in both species. Disposition of radioactivity in tissues after oral administration of Bmim-Cl was negligible. Regardless of species or dose level, routes of elimination were the same, and recovery was >80% of the administered dose.
Urine was analyzed to ascertain the chemical identity of radioactivity present in the samples over time. Figure 6 shows the HPLC-radiometric profile of rat urine samples obtained 6 and 12 h after a single oral dose of 5 or 0.5 mg/kg [14C]Bmim-Cl. In all samples, one major peak was detected, which coeluted with the Bmim-Cl standard (Rt = 18 min). This peak accounted for >96% of 14C radioactivity detected in urine after oral administration of [14C]Bmim-Cl. Two minor peaks that were observed coeluted with impurities (Rt = 4.4 and 5.4 min) present in the [14C]Bmim-Cl dosing solution. These two peaks accounted for 3% of the total 14C radioactivity detected in urine. Similar HPLC profiles were observed from urine obtained from rats dosed with 50 mg/kg (oral) or 5 mg i.v. and mice orally administered 50 mg/kg (data not shown).
Urine samples from rats dosed with unlabeled Bmim-Cl (50 mg/kg) were analyzed by LC-MS to confirm the identity of the major chemical present. Mass spectrometry confirmed the presence of the molecular mass of Bmim+ (m/z = 139.2). Collision-induced fragmentation of this ion resulted in the loss of 56 mass units (butyl group) and the formation of a single ion (m/z = 83.2). This mass corresponded to the 3-methylimidazolium ion (Fig. 7).
Analysis of fecal extracts obtained from rats administered [14C]Bmim-Cl orally (50 mg/kg, 12-h time point) detected a number of peaks that did not coelute with [14C]Bmim-Cl. These peaks accounted for 10 to 20% of the administered dose. Further analysis of these was not attempted. A peak that coeluted with the [14C]Bmim-Cl standard accounted for 3 to 7% of the dose.
After each daily oral dose of [14C]Bmim-Cl to male F-344 rats, elimination of Bmim-Cl was rapid, with the majority of administered radioactivity being eliminated via the urine in each 24-h dosing period. The effect of repeated oral administrations on the excretion of Bmim-Cl is shown in Fig. 8. Elimination patterns over sequential 24-h periods were similar; with 86 to 92% of each dose eliminated in a 24-h period. Of this 51- to 68% of the dose was eliminated via the urine per 24 h. No notable differences in elimination were observed between the group dosed with a single administration of [14C]Bmim-Cl and the group receiving serial administrations. Summaries of radioactivity recovered from tissues are shown in Table 4.
Topically applied Bmim-Cl. After dermal application of [14C]Bmim-Cl, (5 mg/kg, 190 μg/cm2), small amounts of [14C]Bmim-Cl were detected in the urine. By 48 h, 12% of the applied dose was found in the urine when it was applied in DMF-water (Fig. 9). When Bmim-Cl was applied in water or in ethanol-water, 1.1 and 2.4% of the applied dose was recovered in the urine, respectively. Excretion in feces accounted for 0.3 to 0.8% of the dose (Table 5). At termination, the majority (71–85%) of the applied dose remained on the skin surface. A major portion of this was removed by washing and tape stripping. However, 14 to 28% of the dose that was present at the site of application was not removed by the washing/stripping procedure. At the termination of the experiments (48 or 72 h), Bmim-Cl was not detectable in the blood, liver, and kidney. No gross abnormalities were noted at the site of application or in tissues that were analyzed. The dosing vehicles were nonirritating (data not shown).
No skin irritation or discomfort was observed at any time after application of this dose (5 mg/kg) to rats in any of the above-mentioned vehicles. Also, when Bmim-Cl (5 mg/kg) was topically administered for 5 consecutive days, no irritation or discomfort was noted at the site of application. Hair growth at the site of application precluded further applications.
Discussion
Because they possess low vapor pressures, ionic liquids have been referred to as “green solvents.” They are expected to decrease the contribution of industrial solvents to air pollution, particularly indoor air pollution, compared with conventionally used volatile organic solvents. However, less polluting does not mean that ionic liquids may be without negative biological effect or hazard. In fact, some studies have reported ecological as well as mammalian toxicity of ionic liquids. Bernot et al. (2005a,b) showed that nonlethal concentrations of a series of imidazolium and pyridinium-based ILs negatively affected freshwater invertebrates, indicating potential hazards to exposed ecosystems and watersheds. Landry et al. (2005) reported that topical exposure to Bmim-Cl (2000 mg/kg) resulted in dermal irritation in rats and systemic toxicity in mice.
The results of the studies reported here describe the absorption, distribution, metabolism, and excretion of a specific 1,3-dialkylimidazolium cation, Bmim-Cl. After oral administration to rats, Bmim-Cl was extensively absorbed into the systemic circulation with Cmax achieved at 67 min. Comparison of the blood AUC[0–∞] values obtained after oral administration with that obtained after i.v. administration resulted in an oral systemic bioavailability of 62% for male F-344 rats. This value closely approximates the percentage of dose eliminated in the urine at 24 h after a single oral administration of Bmim-Cl as well as at 24 h after each of five repeated daily doses of Bmim-Cl. The volume of distribution after i.v. administration exceeded 600 ml, indicating that systemically available Bmim-Cl is widely distributed outside the plasma compartment. Therefore, recoveries of Bmim-Cl in the urine at later time points most likely represent elimination of Bmim-Cl that is distributed to fluids associated with extravascular tissues. Recoveries of 14C radioactivity in the feces after oral administration ranged from 20 to 30%, most likely representing elimination of unabsorbed Bmim-Cl rather than Bmim-Cl that was extracted by the liver and eliminated in the bile. This conclusion is supported by the results of the i.v. study, which indicated that systemically available Bmim-Cl was almost exclusively eliminated in the urine as unchanged parent compound.
Parallel studies with female B6C3F1 mice showed that apparent rates and route of elimination of Bmim-Cl by mice were similar to those seen in male F-344 rats. Slightly higher urinary recoveries were observed at 72 h after oral administration of a single dose of Bmim-Cl to male F-344 rats than to female B6C3F1 mice. However, these differences in total recovery were not reflected in differences in disposition in tissues.
On the basis of toxicological data presented by Landry et al. (2005), Bmim-Cl can be absorbed after dermal exposure. However, the extent of and rate of absorption are greatly influenced by vehicle used for application. Landry et al. (2005) reported that high doses of Bmim-Cl caused severe systemic toxicity when it was applied in DMF but not in water. The results obtained in the dermal absorption studies reported here provide an explanation for those observations. Urine levels of Bmim-Cl recovered in 48 h were 10-fold higher when applied in DMF-water compared with application of Bmim-Cl in water. Also, skin irritation as a result of the large dose of Bmim-Cl applied topically by Landry et al. (2005) could have promoted additional percutaneous absorption. Urinary levels of Bmim-Cl suggest that although only 10 to 14% of the dose was systemically available, an additional 16 to 22% remained in the skin after washing and tape-stripping the site of application. This remaining dose has the potential for subsequent systemic exposure. The ethanol-water and water vehicle groups showed similar levels of Bmim-Cl remaining at the site of application after washing and stripping. Additionally, the urinary recoveries of Bmim-Cl in the ethanol-water and water vehicle groups did not appear to plateau, suggesting that extended exposure to Bmim-Cl in water-based vehicles could lead to continued systemic exposure.
As indicated above, no evidence was obtained for the metabolism of systemically available Bmim-Cl. After oral or i.v. administration of Bmim-Cl, >97% of 14C equivalents eliminated in the urine were associated with Bmim-Cl. HPLC-radiometric analysis of extracts of fecal samples from animals that had received an oral administration of Bmim-Cl provided some evidence of biotransformation. A number of small peaks that did not coelute with the parent compound or known contaminants were observed. The peaks may represent products of biotransformation of Bmim-Cl mediated by gut microflora. However, it is also possible that at the 10-fold higher oral dose of Bmim-Cl, the liver may form a spectrum of metabolites that are eliminated via the bile. Although these metabolites (microflora and/or mammalian) have not been characterized, theoretical metabolic schemes for Bmim-Cl have been published (Jastorff et al., 2003; Stepnowski and Storoniak, 2005).
A number of small molecular weight organic cations are known to be substrates for organic cation transporters (OCT). OCTs promote both the intestinal uptake of such cations into the systemic circulation and facilitate their secretion in the nephron (Zhang et al., 1998; Slitt et al., 2001). The prototypical organic cations tetraethylammonium and tributylmethylammonium are actively transported across the intestinal mucosa by a sodium-independent cation transporter (OCT) system (Bowman and Hook, 1972; Koepsell, 1998; Kim et al., 2005). As Bmim-Cl has structural characteristics similar to these water-soluble compounds, it is likely that its uptake from the intestine into the systemic circulation is mediated by these transporters present in the intestinal mucosa.
The estimated systemic clearance rate of Bmim-Cl determined in the studies reported here suggests that Bmim-Cl may also be secreted from the nephron by OCTs. The estimated clearance rates of Bmim-Cl (CLb = 7.4–11.9 ml/min) approached reported values for renal blood flow (12 ml/min) (Corley et al., 2005). Because glomerular filtration is approximately equivalent to 12% of renal blood flow (Shargel and Yu, 1992; Caron and Kramp, 1999), glomerular filtration of Bmim-Cl cannot explain the efficient systemic clearance and rapid urinary elimination of Bmim-Cl. Based on its structural characteristics, rapid urinary excretion as parent compound, and high systemic clearance, we suggest that Bmim-Cl is a substrate for OCTs and that these transporters play a major role in the renal secretion of this and other ionic liquids.
In summary, after i.v., oral or dermal administration, Bmim-Cl is rapidly cleared from the systemic circulation and is excreted unchanged in the urine. Efficient oral absorption suggests that this highly water-soluble compound may be a substrate for intestinal OCTs. Similarly, the rapid clearance by the kidney suggests that it may be transported and secreted by OCTs present on the basolateral membrane of proximal tubule of the nephron. Studies are underway to assess the role of OCTs in the disposition of Bmim-Cl and other structurally similar ionic liquids.
Acknowledgments
The authors thank Dr. M. L. Cunningham (National Toxicology Program and National Center for Toxicogenomics, National Institute of Environmental Health Sciences) for advice and support as well as Leigh Jacobs, Aniko Solyom, Veronica Rodriguez, and Ryan Miller for assistance in this project.
Footnotes
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This research was supported by the National Toxicology Program/National Institute Environmental Health Science (NIEHS) Contract N01-ES-45529. The authors acknowledge the support of the analytical core of the NIEHS-funded Southwest Environmental Health Science Center (P30-ES 06694) and National Cancer Institute Grant (CA023074).
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.018515.
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ABBREVIATIONS: IL, ionic liquid; Bmim-Cl, 1-butyl-3-methylimidazolium chloride; HPLC, high-pressure liquid chromatography; DMF, dimethylformamide; JVC, jugular vein cannula; LC, liquid chromatography; MS, mass spectrometry; UV/Vis, UV-visible; AUC[0–∞], concentration-time curve from time zero to infinity; LOQ, limit of quantification; OCT, organic cation transporters.
- Received August 27, 2007.
- Accepted October 25, 2007.
- The American Society for Pharmacology and Experimental Therapeutics