Experimental Animals

The National Toxicology Program (NTP) investigated the absorption, distribution, metabolism, and excretion (ADME) and toxicokinetic properties of Bmim-Cl and NBuPy-Cl in male Fischer 344 (F344) rats and female B6C3F1 mice^43,44 and of Bmpy-Cl in male F344 rats⁴⁵ following single or repeated gavage, intravenous administration, or dermal application. The doses selected in these three studies were 0.5, 5, or 50 mg/kg, representing approximately 0.1%, 1%, or 10%, respectively, of the reported oral median lethal dose (LD₅₀) of 550 mg/kg in female F344 rats.⁴⁶ In general, the disposition pattern of the three ILs were similar, with moderate to high oral absorption, low to moderate dermal absorption, rapid excretion, and low tissue retention. The ILs were poorly metabolized, despite the expectation that ILs with a butyl substitution (e.g., Bmim-Cl) would undergo side chain oxidation.⁴⁷ No other studies investigating the ADME of ILs were found in the literature.

Cheng et al.⁴³ administered [¹⁴C]NBuPy-Cl by gavage, intravenous, and dermal application. Following a single gavage dose of 50 mg/kg in male F344 rats, [¹⁴C]NBuPy-Cl was rapidly absorbed with a maximum blood concentration occurring at 1.3 hours. For all three doses administered (0.5, 5, or 50 mg/kg), excretion was rapid, with 55%–66% and 20%–25% of the dose recovered in urine and feces, respectively, after 24 hours. Urine profiles showed a single peak corresponding to the parent chemical, suggesting poor metabolism. The elimination half-life of [¹⁴C]NBuPy-Cl in blood was 2.2 hours after a single intravenous dose of 5 mg/kg, and the estimated oral bioavailability was 47% following administration of a single oral dose of 50 mg/kg [¹⁴C]NBuPy-Cl in male rats. After 72 hours, the dose recovered in tissues of male rats orally administered 50 mg/kg was low (approximately 0.01%, based on selected organs). There was no dose-related difference in the disposition of [¹⁴C]NBuPy-Cl in male rats following a single dose versus daily gavage administration for 5 days.⁴³ Following a single gavage dose of 50 mg/kg [¹⁴C]NBuPy-Cl in female B6C3F1 mice, the excretion pattern was similar to male rats (72% in urine and 10% in feces after 96 hours). Dermal absorption (estimated based on excreta and dose-site skin) of 5 mg/kg [¹⁴C]NBuPy-Cl 96 hours after application to a covered dose site was higher (35% of dose) when formulated in dimethylformamide (DMF)/water (55:45, v/v). The same dermal dose applied in other vehicles (ethanol/water [55:45 v/v] or water alone) was less well absorbed (16% or 10% of the dose, respectively). The pattern of excretion following dermal application was similar to that following oral administration.

Sipes et al.⁴⁴ examined oral and dermal ADME of Bmim-Cl in male F344 rats and female B6C3F1 mice. In male rats following a single gavage administration of 0.5, 5, or 50 mg/kg [¹⁴C]Bmim-Cl, absorption was rapid with the maximum blood concentration occurring at 67 minutes. Of the administered dose, 72%–78% was excreted in urine and 19%–28% in feces by 72 hours, with the majority eliminated in urine by 24 hours (55%–74%). The blood elimination half-life of [¹⁴C]Bmim-Cl was 77.2 minutes (approximately 1.3 hours), and the estimated oral bioavailability was 62.1% (for 50 mg/kg). After 72 hours, retention of radioactivity in tissue was negligible. The chemical analysis of urine showed only a single peak corresponding to the parent compound, although fecal profiles showed several peaks that did not coelute with the parent. No dose-related difference was found in the disposition of [¹⁴C]Bmim-Cl following a single dose. The disposition of [¹⁴C]Bmim-Cl between single and multiple (5 daily doses) gavage dosing was similar. The excretion pattern following a single oral administration of 50 mg/kg [¹⁴C]Bmim-Cl in female mice was similar to male rats, with 65% and 18% of the administered dose recovered in urine and feces, respectively, by 72 hours. Dermal absorption of 5 mg/kg [¹⁴C]Bmim-Cl 48 hours after a single application to a covered dose site was low to moderate, with 31.7%, 17.8%, or 29.6% (estimated based on excreta and dose-site skin) of the dose absorbed when formulated in DMF/water (1.8:1, v/v), ethanol/water (1.8:1, v/v), and water, respectively.

Knudsen et al.⁴⁵ examined oral and dermal ADME of Bmpy-Cl in F344 rats. Following a single gavage dose of 0.5, 5, or 50 mg/kg [¹⁴C]Bmpy-Cl in male rats, 29%–38% of the administered dose was eliminated in the urine and 51%–69% was excreted in feces by 72 hours. Maximum blood concentration of [¹⁴C]Bmpy-Cl occurred at 1.5 hours, with an elimination half-life of 5.6 hours and an estimated oral bioavailability of 43.4% following oral administration of a single 50 mg/kg dose. The urine profiles showed only a single peak corresponding to the parent chemical, suggesting poor metabolism. The pattern of excretion following repeated gavage with 50 mg/kg/day for 5 consecutive days was similar to that following a single gavage administration. Following a single intravenous administration, 86% of the dose was recovered in urine after 72 hours with approximately 8% in feces. The difference between fecal elimination following intravenous versus oral administration (8% vs. 51%–69%) suggests most of the dose excreted in feces was unabsorbed. Dermal absorption of 5 mg/kg [¹⁴C]Bmpy-Cl 96 hours after application to a covered dose site was low to moderate, with higher absorption observed when formulated in DMF/water (63:37, v/v) (34% of dose) compared to when formulated in ethanol/water (63:37, v/v) or water (≤22% of dose). The pattern of excretion following dermal application was similar to that following oral administration.

In vitro studies with these ILs highlight their function as substrates for transport-mediated secretion via human organic cation transporter 2 (hOCT2), rat OCT 1 and 2 (rOCT1/2), as well as multidrug and toxic extrusion transporters (MATEs).^43,45,48 These data are consistent with properties of other small molecular weight cation compounds including 1-paraquat, methyl-4-phenylpyridinium, tetraethylammonium, and tributylmethylammonium, which are also substrates for OCTs.^49-52 In addition, NBuPy-Cl, Bmpy-Cl, and Bmim-Cl were determined to be potent inhibitors of rOCT1/2 and hOCT2, much like other structurally similar compounds that potently inhibit hOCT2.⁵³ While many ILs behave in a similar manner, structure-activity relationship (SAR) studies suggest that slight differences in IL structure can influence their ADME, kinetics, and drug-chemical interactions. For example, Cheng and colleagues determined the OCT inhibitory effects of pyridinium-based ILs are influenced by the length of the alkyl side chain; an increase in the number of carbons on the alkyl chain significantly decreased median inhibitory concentration (IC₅₀).⁴⁸ This SAR has been observed for other chemical classes^53,54; therefore, additional research is needed to understand the relationship between IL structure and physiochemical and biological properties.

Humans

The literature contains no studies on the ADME of ILs in humans.

In Vitro

The structure of an IL is likely to influence its toxicity; the most significant factors are the associated anion and the alkyl chain length.^8,55,56 In cases in which the anion and alkyl chain length are the same, aromatic cations (e.g., pyridinium or imidazolium) tend to be more toxic than nonaromatic cations (e.g., pyrrolidinium) in inhibition respiration assays⁵⁷ and plant cytotoxicity models,^4,58 possibly due to greater hydrophobicity of the cation.⁵⁷ When only alkyl chain length differs among ILs, longer carbon chains are associated with increased lipophilicity of the IL, disruption of cellular membranes, and induction of cell death.^8,59,60

For example, assessment of IL toxicity in a Langmuir monolayer cell membrane model, rat IPC-81 leukemia cells, and bacterial bioassays confirmed ILs with alkyl chain lengths of 2–5 carbons exhibit limited toxicity, ILs with chain lengths of 6–10 carbons exhibit linear increases in toxicity with increasing chain length, and ILs with chain lengths >10 carbons exhibit plateaued toxicity.^59,61-63 Bmim-Cl (4 carbons), 1-octyl-3-methylimidazolium chloride (Omim-Cl, 8 carbons), and 1-dodecyl-3-methylimidazolium chloride (Ddmim-Cl, 12 carbons) inhibited cell growth and viability in mouse J774A.1 macrophage cells,³⁵ rat pheochromocytoma PC12 cells,⁶⁴ and human hepatocellular carcinoma HepG2 cells,⁶⁵ respectively. Omim-Cl and Ddmim-Cl also induced DNA damage, oxidative stress, apoptosis, and altered membrane permeability in PC12 and HepG2 cells. In addition, Omim-Cl induced adenosine triphosphate depletion in PC12 cells, and Ddmim-Cl altered expression of apoptosis-related genes in HepG2 cells.^64,65

These effects may be due to the surfactant-like activity observed at ≥6 carbons⁶¹ or the very low octanol:water partition coefficient (log K_ow = 1) measured at 8–10 carbons.⁶³ Molecular dynamic simulations indicated that 14-carbon alkyl chains were of similar length to the fatty acids in the lipids, resulting in straight line conformation of the alkyl chain, greater stability of the lipids, and IL aggregates in the membrane.⁶⁶ Although the anion component of an IL is not a major contributor to its toxicity, hydrophobic anions (e.g., bis(trifluoromethanesulfonyl)imide [Tf2N]) have been shown to be more toxic than hydrophilic anions (e.g., Cl, tetrafluoroborate [BF4]). This difference in toxicity is evidenced by the lower median effective concentration (EC₅₀) for Emim-Cl-Tf2N (1.69 mM) compared to Emim-Cl (4.19 mM) in inhibition respiration assays⁵⁷ and by the increased disruption of the lipid bilayer by hydrophobic anions, relative to hydrophilic anions, in a biomimetic membrane model.⁶⁷

Experimental Animals

High-dose oral exposure to ILs is reported to induce acute toxicity in rodents. Morbidity and mortality increased in adult F344 rats exposed to Bmim-Cl (≥550 mg/kg)⁴⁶ and in pregnant CD-1 mice exposed to Emim-Cl (3,000 mg/kg body weight/day; mg/kg/day) and Bmim-Cl (225 mg/kg/day).⁶⁸ Gestational exposure of CD-1 mice to Bmim-Cl (≥169 mg/kg/day) and 1-decyl-3-methylimidazolium chloride (Dmim-Cl; ≥75 mg/kg/day) resulted in decreased fetal body weight.⁶⁸

Rodent dermal exposure studies with ILs have shown toxicity to be concentration- and vehicle-dependent. In brief, dermal studies of Bmim-Cl exposure in F344 rats confirmed compound absorption was more effective when the IL was dissolved in DMF:water compared to ethanol:water or water alone.^43,44 When dissolved in DMF:water, lower concentrations of Bmim-Cl (5 mg/kg) were not well absorbed through the skin. When the concentration increased to 200–800 mg/kg, Bmim-Cl induced dermal irritation. At higher concentrations (2,000 mg/kg), Bmim-Cl in DMF:water resulted in mortality of male and female rats, likely due to an increase in the amount of Bmim-Cl absorbed.⁴⁶ Similar effects were observed in female Balb/c mice exposed dermally to Bmim-Cl in DMF. Two percent (approximately 50 mg/kg) Bmim-Cl was nontoxic. When the concentrations increased to 10%–75% Bmim-Cl (≥250 mg/kg), a dose-related effect on dermal irritation and mortality was observed following dermal application.⁴⁶

Skin sensitization potential of Emim-Cl, Bmim-Cl, Bmpy-Cl, and NBuPy-Cl in female Balb/c mice was previously evaluated by NTP using a combined local lymph node (LLNA)/primary irritancy assay.⁶⁹ In addition, these four ILs were evaluated in a suite of in chemico and in vitro assays validated by the OECD (Organisation for Economic Co-operation and Development): Direct Peptide Reactivity Assay (DRPA) (OECD 442C), KeratinoSens™ Assay (OECD 442D), and human Cell Line Activation Test (hClat) (OECD 442E).^70-72 Overall, Bmim-Cl, Bmpy-Cl, and NBuPy-Cl were more potent than Emim-Cl, which was negative in all in vivo, in chemico, and in vitro assessments.⁶⁹ NBuPy-Cl was found to be a dermal irritant, but not a sensitizer, when applied to the skin at concentrations of 3.12%–12.5%. NBuPy-Cl was classified as a sensitizer in vitro; however, false positives in these assays have been reported when a compound is also a skin irritant.^73,74 In vivo, Bmim-Cl and Bmpy-Cl were found to be skin sensitizers at lower applied concentrations of 12.5% and 6.25%, respectively, and were acutely toxic at higher concentrations of 25% and 12.5%, respectively.⁶⁹ This result for Bmim-Cl matches that reported by Landry and colleagues.⁴⁶ Although other studies suggest that aromatic cations (e.g., Bmim-Cl) are more toxic than nonaromatic cations (e.g., Bmpy-Cl).^57,58,75 Frawley et al.⁶⁹ found that Bmim-Cl and Bmpy-Cl have similar toxicity in mammalian and in vitro models of hypersensitivity.

Humans

There has been little to no research evaluating the toxicity of ILs to humans. However, the Globally Harmonized System of Classification and Labeling of Chemicals (GHS)-aggregated information for Bmim-Cl, one of the most studied ILs, suggests that human exposure can cause irritation to the eye, skin, and respiratory system (specifically, the mucous membranes in the mouth, pharynx, esophagus, and gastrointestinal tract following swallowing). Inhalation of the compound may result in irritation of the mucous membranes, coughing, and dyspnea.^35,76