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Comparative metabolism and pharmacokinetics of diisobutyl ketone and diisobutyl carbinol in male SD rats
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TOXLET 8928 1–7 Toxicology Letters xxx (2014) xxx–xxx
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Comparative metabolism and pharmacokinetics of diisobutyl ketone and diisobutyl carbinol in male SD rats Fagen Zhang * , Michael J. Bartels, Amy J. Clark, Jen L. Staley, Tom S. Lardie, Dan A. Markham, Brian J. Hughes, Nicholas S. Ball Toxicology and Environmental Research and Consulting, The Dow Chemical Company, 1803 Building, Midland, MI 48674, USA
H I G H L I G H T S
Comparative metabolism and pharmacokinetics of diisobutyl ketone (DIBK) and diisobutyl carbinol (DIBC) in male rats were explored. DIBC and DIBK are both well absorbed following oral gavage with substantial evidence of enterohepatic recirculation of DIBK, DIBC, DIBK-alcohol, and DIBC-alcohol. DIBK and DIBC are interconverted metabolically in rats. Higher systemic exposure was found for DIBK-alcohol than DIBC-alcohol, implying that DIBC-alcohol may be more easily conjugated and eliminated in bile. The toxicological data on DIBK could be used to characterize the hazards of DIBC.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 July 2014 Received in revised form 21 October 2014 Accepted 22 October 2014 Available online xxx
Diisobutyl ketone (DIBK) and diisobutyl carbinol (DIBC) are important organic solvents widely used as industrial intermediates. It was hypothesized that DIBC and DIBK have common metabolic pathways and metabolites, and as such, toxicological data on DIBK could be used to characterize the hazards of DIBC. To confirm or refute this hypothesis a comparative metabolism and pharmacokinetics assessment of DIBK and DIBC was conducted. Dosing was via single oral gavage dosing in male SD rats, followed by blood collection, metabolite identification, major biomarker quantitation, and pharmacokinetics analysis. Overall, the major metabolites of both DIBC and DIBK in blood were their corresponding monohydroxylated metabolites (DIBC alcohol and DIBK alcohol) with the site of hydroxylation at the s and s-1 positions, respectively. Quantitative analysis of DIBC, DIBK, DIBC-alcohol, and DIBK-alcohol in blood samples collected from 5 min to 120 h after single dosing indicated the following: (1) DIBC and DIBK are both well absorbed following oral gavage with substantial evidence of enterohepatic recirculation of DIBK, DIBC, DIBK-alcohol, and DIBC-alcohol; (2) DIBK and DIBC are interconverted metabolically in rats; (3) DIBC and DIBK have similar bioavailability after oral administration; (4) higher systemic exposure was found for DIBK-alcohol than DIBC-alcohol, implying that DIBC-alcohol may be more easily conjugated and eliminated in bile. In summary, the metabolic similarities and the difference in systemic exposure to metabolites between these substances observed in the current study support the hypothesis that DIBC might have a lower potential toxicity than that of DIBK. The current study results support that toxicological data on DIBK could be used to characterize the hazards of DIBC ã 2014 Elsevier Ireland Ltd. All rights reserved.
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1. Introduction
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DIBK (diisobutylketone) and DIBC (diisobutylcarbinol) are highproduction volume chemicals that are widely used in the chemical
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* Corresponding author. Tel.: +1 989 638 4172; fax: +1 989638 9305. E-mail address: [email protected] (F. Zhang).
industry as industrial intermediates.Sufficient DIBK toxicity data have been generated (Carpenter et al., 1953; Chataigner et al., 1992; De Ceaurriz et al., 1983; De Ceaurriz et al., 1984; Dodd et al., 1987) and have been successfully used to support DIBK registration (ECHA REACH and OCED SIDS) (ECHA 2014; SIDS, 1998). In contrast, only limited toxicity data are available for DIBC. To address this potential data gap, a read-across approach was proposed, wherein the DIBK toxicity data, in conjunction with new pharmacokinetic
http://dx.doi.org/10.1016/j.toxlet.2014.10.027 0378-4274/ ã 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Zhang, F., et al., Comparative metabolism and pharmacokinetics of diisobutyl ketone and diisobutyl carbinol in male SD rats. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.10.027
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1995; Granvil et al., 1994), and some differences in overall ADME fate of these two test materials may occur, the final result from Gingell’s study showed that systemic equivalence of MIBC with MIBK support the conclusion that MIBC will have a similar toxicological profile to MIBK, and reduce the need for additional animal studies (Gingell et al., 2003). DIBK and DIBC are quite comparable in structure to the related MIBK and MIBC compounds. It would therefore be expected that DIBK and DIBC would undergo similar interconversion in mammals, and also form a common HMP-like metabolite, which would be 6-hydroxy-2,6-dimethyl-4-heptanone (HDH) (DIBK alcohol, Scheme 2). Based on this background information, a bridging limited ADME study was proposed to confirm this hypothesis via a similar approach to that reported by Gingell et al., (2003).
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2. Materials and methods
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2.1. Reagents, solvents and materials
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Acetonitrile (HPLC grade), methanol (HPLC grade), water (HPLC grade), and acetic acid (HPLC grade) were obtained from Fisher Scientific (Itasca, IL, USA). 13C-DIBC, DIBK and their metabolites (Scheme 3) were obtained from Chemdepot (California, USA). All other reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA), unless otherwise stated.
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2.2. Animals
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In accordance with the U.S. Department of Agriculture animal welfare regulations, 9 CFR, subchapter A, Parts 1–4, the animal care and use activities required for conduct of this study were reviewed and approved by the Institution Animal Care and Use Committee (IACUC). Male NTac:SD rats (both cannulated and uncannulated) were obtained from Taconic (Germantown, New York) 250 g, approximately 9–10 weeks of age).
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2.3. Animal treatment procedures
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Two animal treatment procedures were used in the current study:
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2.3.1. Probe experiment for metabolite identification in blood Groups (Group 1 and 2) of 2 male rats/per group (uncannulated) were administered either DIBK or DIBC, via oral gavage, at a dose level of 700 mg/kg in corn oil. Animals were sacrificed at the time of the estimated Cmax based on the previous study on MIBK and
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Scheme 1. Proposed metabolism of MIBC and MIBK.
data showing systemic bioequivalence between DIBK and DIBCwould comprise the DIBC registration dossier. The proposed bioequivalence study was based on the hypothesis that these alcohol-ketone isomers undergo interconversion in vivo, as has been shown for the analogous isomer compounds, methylisobutylcarbinol (MIBC) and methylisobutylketone (MIBK) (Gingell et al., 2003). In that study, Gingell et al. showed that MIBC and MIBK are essentially bioequivalent in rats after single oral gavage administration of MIBC or MIBK, with MIBC converting first to MIBK enzymatically, followed by metabolism to the common HMP metabolite (4-hydroxy-4-methyl-2-pentanone) (Scheme 1). Pharmacokinetic analysis of blood samples, following single oral doses of either MIBK or MIBC, showed that the total systemic exposure of the two test materials (plus the major HMP metabolite) were comparable, as measured by the individual and total plasma AUC values. Although no mass balance of administered radiolabel and urinary metabolite profiling were conducted in Gingell’s study or other prior metabolism studies with these compounds (DiVincenzo et al., 1976; Duguay and Plaa,
Scheme 2. Chemical structures of DIBC and DIBK and their potential metabolites.
Scheme 3. Proposed metabolism of DIBC and DIBK.
Please cite this article in press as: Zhang, F., et al., Comparative metabolism and pharmacokinetics of diisobutyl ketone and diisobutyl carbinol in male SD rats. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.10.027
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MIBC (Gingell et al., 2003) and blood samples were collected and used for preparation of plasma samples. These probe experiment plasma (estimated Cmax plasma) samples were analyzed for identification of the major biomarkers (DIBK, DIBC, and their corresponding metabolites such as DIBK alcohol and DIBK alcohol) (Scheme 3). 2.3.2. Definitive pharmacokinetic experiment Based on the metabolite identification results of the probe experiment (Group 1 and 2), the definitive pharmacokinetic experiment was conducted to determine kinetics of the major biomarkers of DIBK, DIBC, and their major identified hydroxylated metabolites (such as DIBK alcohol, Scheme 2) in blood. Groups (Group 3 and 4) of 4 male rats, with cannula implanted in the jugular vein, were administered either DIBK or DIBC, via oral gavage, at a dose level of 700 mg/kg in corn oil. Repetitive blood samples were taken from each rat at 0.08, 0.17, 0.25, 0.5, 1, 2, 3, 6, 12, 24, 48, 72, 96 and 120 h post-dosing. Chemical analysis was then conducted on individual blood samples to quantify the major biomarkers of DIBC and DIBK and their corresponding major metabolites. Limited pharmacokinetic analysis (such as AUC (areaunder-the-curve)) was conducted based on the quantitative analysis results of major biomarkers by using the PK_AUCt Excel function of PK Solver (Zhang et al., 2010)
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2.5. Calibration standard preparation
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Stock solutions of DIBK, DIBC and DIBK alcohol were prepared at a nominal concentration of 10 mg/mL in acetonitrile (ACN). A series of standard working solutions in the range of 2.5 mg/mL–2500 mg/ mL were prepared by further dilution of the corresponding standard stock solution(s) with ACN. The stock solutions of internal standards (IS, 13C-labeled DIBC and 13C-Labeled BIBK (Scheme 2)) were prepared at a concentration of 4000 mg/mL in ACN. The IS working solution (50 mg/mL) was prepared in ethyl acetate. All solutions were stored at 20 C. The calibration standards of DIBK, DIBC, and DIBK alcohol at concentrations of 0.125, 0.313, 1.250, 3.125, 12.5, 31.25, and 125 mg/mL (all containing 300 mL of IS solution, nominal concentration of 30 mg/mL), were prepared by diluting the working standard stock solutions with blood collected from control rats.
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2.6. Sample preparation for biomarker quantitation
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Heparinized blood samples from the Group 3 and 4 rats were maintained at 80 C prior to analysis. Samples were removed from the freezer and allowed to warm at a room temperature. Weighed aliquots (approximately 200 mL) of each blood sample were transferred to 4 mL glass vials and the appropriate amount of IS working solution (300 mL) was added. After mixing, 100 mL of ethyl acetate was added. The resulting mixture was vortex-mixed and then centrifuged at 863 g for 10 min. The organic phase was
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transferred to a clean GC vial for GC/MS analysis. The calibration standards were processed in a similar manner.
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2.7. GC/MS methods
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DIBC and DIBK and their potential metabolites in blood or in probe experiment plasma were analyzed by gas chromatographymass spectrometry (GC/MS). GC/MS analyses were performed by an Agilent 6890 GC equipped with an Agilent 5973 (EI) MS detector (Agilent Technologies, Palo Alto, CA, USA), using the MSD Chemstation Software for data acquisition and processing. A 30 m 0.32 mm 0.25 mm film thickness Phenomenex ZB-Wax plus, w/Guardian (Phenomenex, Torrance, CA, USA) was used for the gas chromatographic analysis. The injector was a Gerstel MultiPurpose Sampler (MPS) injector (Gerstel, GmbH, Mülheim an der Ruhr, Germany). The injector was coupled by a capillary transfer line with the external thermal extraction unit, containing a removable glass tube for the sample desorption. The GC conditions were as follows: injection mode: splitless; injector port temperature: 230 C; carrier gas: helium with flow rate: 2.1 mL/min; oven thermal program: from 50 to 240 C at a rate of 20 C/min with initial hold time 1 min and final hold time 5 min; ion source temperature: 230 C; interface temperature: 230 C.
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2.4. Plasma or blood extraction from probe animal treatment procedure Individual probe experiment plasma samples (Group 1 and 2), control plasma samples, or pooled blood samples (Group 3 and 4) were maintained at 80 C prior to analysis. Samples were removed from the freezer and allowed to warm to room temperature. Aliquots (approximately 100 mL) of each sample were transferred to 4 mL vials and 200 ml of ethyl acetate was added. The resulting mixture was vortex-mixed and then centrifuged at 863 g for 10 min. The organic layer was transferred to a clean GC vial for full scan GC/EI-MS analysis for major biomarker (metabolite) identification.
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Fig. 1. Chromatograms of GC/EI-MS analysis of ethyl acetate extracts of DIBC and DIBK probe experiment plasma and 3 h DIBC blood: (A) DIBC Cmax plasma (this chromatogram is the same as the chromatogram from control plasma ethyl acetate extract, data not shown); (B) DIBK probe experiment Plasma; (C) 3 h DIBC blood.
Please cite this article in press as: Zhang, F., et al., Comparative metabolism and pharmacokinetics of diisobutyl ketone and diisobutyl carbinol in male SD rats. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.10.027
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Preliminary mass spectra of parent and metabolites were obtained in full scan mode after electron ionization (EI, 70 ev) from m/z 50 to 500 amu (scan rate 0.50 scan/s, solvent delay 2 min), while selective ion monitoring (SIM) mode was selected for the quantification of parent compunds and metabolites.
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3. Results and discussion
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3.1. Metabolite profiling and identification from probe experiment plasma and blood samples
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In order to identify the potential metabolites (biomarkers) in probe experiment plasma (Group 1 and 2) or pooled blood samples (Group 3 and 4), a mixture of standards containing DIBK, DIBC, DIBC-alcohol, DIBK-alcohol (Scheme 2) in ethylacetate was analyzed by GC/EI-MS mass spectrometer via a full scan mode. As shown in Fig. 1, good GC/MS chromatograms with separation and MS spectra from the corresponding peaks were obtained (data not shown). Using the same GC/EI-MS method described above, ethylacetate extracts of probe experiment plasma samples from Group 1 to Group 2 were also analyzed to tentatively identify major biomarkers (parent and metabolites). As shown in Fig. 1A, only peaks from control plasma were detected and no DIBC or DIBC metabolites were detected in the estimated probe experiment plasma of rats dosed with DIBC (Group 2; Fig. 1A). However, two major peaks (DIBK alcohol and DIBC alcohol), and two minor peaks (DIBK and HMP) were present in DIBK probe experiment plasma (Group 1; Fig. 1B). Further GC/MS analysis with Wiley EI/MS database showed that peak HMP is 4-hydroxy-4-methyl-2pentanone, a major metabolite of methyl isobutyl ketone (MIBK) which was contained in the original DIBK test material. It was postulated that the major biomarkers of DIBC may not be at peak levels at the proposed probe experiment sampling time of 1.5 h. Further GC/MS analysis was therefore conducted with selected whole-blood extracts (1, 2, 3 and 6 h) from Group 4 rats (DIBC-dosed) to attempt to identify circulating biomarkers of DIBC and the result is shown in Fig. 1C. As shown in Fig. 1C, DIBC, DIBK alcohol and DIBC alcohol were detected as DIBC metabolites in 3 h pooled whole blood samples. The quantitative blood results below support that the 1.5 h sampling time was, in fact, Cmax between two DIBC blood concentration peaks, most probably due to enterohepatic recirculation of conjugated forms of DIBC metabolites (discussed below). Synthetic standards of novel metabolites of either test material were required to confirm chemical structures. Based on the known metabolism of MIBC and MIBK to HMP (Gingell et al., 2003), via s-1 hydroxylation, the proposed compounds DIBC alcohol A (2, 6dimethylheptane-2,4-diol) and DIBK alcohol (6-hydroxy-2,6-dimethyl-4-heptanone; Scheme 2) were custom synthesized. The synthesized DIBK-alcohol standard (Scheme 2) was found to have the same retention time and MS spectra (data not shown) as the DIBK alcohol metabolite (Fig. 1) observed in probe experiment plasma from Group 1 animals, thereby confirming the structure of the DIBK alcohol metabolite as 6-hydroxy-2,6-dimethyl-4-heptanone. In contrast, the retention time and mass spectra (data not shown) of the synthesized DIBC alcohol A (Scheme 2) did not Table 1 Retention time and typical ions of DIBC and DIBK and their major metabolites. Analyte
Retention time (min)
Typical ions (m/z)
DIBK DIBC DIBK alcohol DIBC alcohol
6.9 13.1 18.9 23.6
41, 57, 85, 142 43, 69, 87 43, 59, 85, 100, 143 43, 59, 83, 101, 141
Fig. 2. A typical linear response of analytes by GC/EI/SIM-IS-MS: (A) DIBC (0.1–120 mg/mL); (B) DIBK (0.002–120.0 mg/mL).
match those of the DIBC alcohol metabolite (Fig. 1) found in probe experiment plasma from Group 1 animals. Based on the GC/MS spectral analysis, the DIBC alcohol metabolite was identified as 2,6-dimethylheptane-1,4-diol, arising from hydroxylation at the s position of DIBC (Scheme 3). In order to identify any potential conjugated metabolites of DIBC, DIBK, the collected estimated probe experiment plasma samples from both DIBC and DIBK were also analyzed by LC/MSMS with neutral loss scan (for glucuronide conjugate characterization) (Beaudry et al., 1999; Fabregat et al., 2013; Poon et al., 1999; Qu et al., 2001). However, no detectable conjugates or other novel metabolites of either test material were found (data not shown).
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3.2. Metabolite quantitation and metabolism
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After DIBC, DIBK, DIBC-alcohol and DIBK-alcohol were identified from plasma or blood samples, a more sensitive SIM-GC/EI-MS method based on the abundant ions of major biomarkers (Table 1) was developed for the definitive quantitation of these biomarkers in Group 3 and 4 whole blood samples. Ions of stable-isotope labeled internal standards of DIBK, DIBC (DIBC-IS, DIBK-IS, Scheme 2, Table 1) were also incorporated into the quantitation method. A typical standard curve is shown in Fig. 2. The final quantitation results are shown in Table 2. Overall, DIBK, DIBK alcohol and DIBC alcohol were quantifiable in most blood samples from rats administered DIBK (Group 3). In a similar manner, DIBC, DIBK alcohol, and DIBC alcohol were also quantifiable in blood samples from rats administered DIBC (Group 4). Based on the metabolite identification conducted with probe experiment plasma and selected Group 4 whole blood samples (described above) and the final quantitation results (Table 2), the
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Table 2 Final biomarker concentrations in blood samples from oral administration of DIBC and DIBK. Concentration of biomarker (mg/g blood) from DIBC oral administration Biomarker name
DIBC
Time point (h)
Average
STDEV
Average
STDEV
Average
STDEV
0.08 0.17 0.25 0.50 1.0 2.0 3.0 6.0 12.0 24.0 48.0 72.0 96.0 120
0.548 1.21 1.51 2.33 1.43 3.54 5.04 2.01 0.418 0.414 0.404 0.432 0.454 0.288
0.192 0.618 0.727 1.19 1.36 1.78 2.18 1.90 0.00423 0.0318 0.00458 0.0427 0.0741 0.0360
0.189 0.182 0.198 0.179 0.202 0.269 0.365 0.311 0.196 0.194 0.189 0.202 0.213 0.135
0.0423 0.0140 0.0104 0.0201 0.0196 0.0607 0.119 0.140 0.00198 0.0149 0.00215 0.0200 0.0347 0.0168
0.189 0.182 0.198 0.179 0.192 0.209 0.340 0.590 1.03 0.534 0.189 0.202 0.213 0.135
0.0423 0.0140 0.0104 0.0205 0.0146 0.0309 0.0509 0.263 0.965 0.618 0.00215 0.0200 0.0347 0.0168
BIBK alcohol
DIBC alcohol
Concentration of biomarker (mg/g blood) from DIBC oral administration
278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304
Biomarker name
DIBK
Time point (h)
Average
STDEV
Average
STDEV
Average
STDEV
0.08 0.17 0.25 0.50 1.0 2.0 3.0 6.0 12.0 24.0 48.0 72.0 96.0 120
0.566 2.05 3.18 2.21 2.49 4.01 4.27 1.27 0.181 0.173 0.159 0.183 0.169 0.173
0.415 0.960 1.79 1.45 0.543 2.61 2.46 1.21 0.0132 0.0107 0.00353 0.00441 0.0134 0.0283
1.17 4.40 7.20 9.19 10.7 15.0 16.0 9.22 0.533 0.201 0.184 0.212 0.196 0.201
0.915 1.71 2.96 4.24 2.43 7.49 7.93 6.22 0.371 0.0124 0.00408 0.00510 0.0155 0.0327
0.302 1.05 1.69 4.02 8.11 16.0 22.4 33.0 24.6 2.77 0.205 0.212 0.196 0.201
0.169 0.363 0.401 0.993 1.76 2.49 5.20 12.7 19.1 1.21 0.0277 0.00510 0.0155 0.0327
BIBK alcohol
metabolic pathways of DIBK and DIBC in rats can be proposed as described in Scheme 3. DIBC and DIBK are initially interconverted by alcohol dehydrogenase and ketone reductase. DIBK and DIBC are further oxidized to their corresponding metabolites (DIBK alcohol and DIBC alcohol, respectively) by CYP 450-based hydroxylation at the s-1 or s positions, respectively. Note that with these unique sites of hydroxylation for DIBK and DIBC, there is no possibility for initial hydroxylation of either test material, followed by redox interconversion. 3.3. Time-course concentration and pharmacokinetics of DIBC, DIBK and metabolites in blood The time-course blood concentrations of the four quantifiable major biomarkers in Group 3 (DIBK) and Group 4 (DIBC) animals are shown in Fig. 3. Overall, the Cmax for the highest concentration of DIBC, DIBK, and DIBK-alcohol was about 3 h, while the Cmax for DIBC-alcohol was about 10 h (Fig. 3). These results showed that DIBC and DIBK are both readily absorbed after oral gavage. The pattern of an initial, lower Cmax, at about 30 min, was also observed for both DIBC (from DIBC administration) and for DIBK (from DIBK administration). This dual-peak kinetic pattern possibly reflects enterohepatic recirculation of DIBC and DIBK, possibly through biliary elimination of yet-unidentified Phase II conjugates of the test materials and/or their metabolites. Due to this apparent enterohepatic recirculation, some of the pharmacokinetic parameters (such as elimination half-life) were not calculated. Therefore only AUC (area under the curve) values were calculated and summarized in Table 3. The average AUC
DIBC alcohol
values for both major test material (DIBK and DIBC) were similar (26.8 and 35.3 ug/g h, respectively), indicating that DIBK and DIBC have a similar bioavailability after excluding the contribution from their potential metabolites. The total AUC of all biomarkers from administration of DIBK is about 10 times of the total AUC from administration of DIBC (Table 3). There are potentially two factors contributing to this difference in systemic bioavailability. One factor may be a higher rate of phase II conjugation DIBC (Fig. 3) and its metabolites than that of DIBK or DIBK metabolites. Another factor may be a lower oral absorption of DIBC than DIBK. Regardless of these quantitative differences, these toxicokinetics analysis results show a high degree of metabolic similarity between DIBC and DIBK, with only lower bioavailability seen for the DIBC compound. Similar systemic metabolic bioequivalence arguments have been previously made to predict certain aspects of systemic toxicity of alcohol isomers based on the available toxicity data of the corresponding ketone isomer (such as methyl ethyl ketone and sec-butanol (OECD, 2000); MIBK and MIBC (Gingell et al., 2003)). MIBC and MIBK had similar total AUC (similar systemic metabolic bioequivalence) (Gingell et al., 2003). The 6-week inhalation toxicity study in rats with MIBC showed that LOAEL (low observed adverse effect level) was 900 ppm (Gingell et al., 2003); in the similar study conditions, the LOAEL for MIBK is 1000 ppm. These inhalation toxicity results for MIBC and MIBK are correlated very well with the similar bioequivalence of MIBC and MIBK. Therefore, it can be predicted that the systemic toxicity of DIBC may be lower than that from DIBK based on the current study results.
Please cite this article in press as: Zhang, F., et al., Comparative metabolism and pharmacokinetics of diisobutyl ketone and diisobutyl carbinol in male SD rats. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.10.027
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A)
B)
Fig. 3. Kinetics of various biomarkers in blood from rat orally dosed with DIBK and DIBC: (A) DIBC; (B) DIBK.
Table 3 Area under the curve (AUC) of DIBK, DIBC, DIBK alcohol and DIBC alcohol. Test material
DIBK
Major quantifiable biomarkers in blood AUC (mg/g h) Total AUC (mg/g h)
DIBK 22.5 599
DIBC DIBK alcohol 106
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4. Conclusions
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The current study showed that DIBC and DIBK are well interconverted metabolically in rats. Both DIBC and DIBK have similar metabolic pathways to form the major common metabolites (DIBC-alcohol and DIBK-alcohol). Both DIBC and DIBK are rapidly absorbed following oral administration, with substantial evidence of enterohepatic recirculation. Quantitatively, DIBK was shown to have higher bioavailability (AUC) than DIBC after oral administration. This higher bioavailability may be due to lower absorption of DIBC or a higher rate of Phase II conjugation of DIBC and metabolites, followed by biliary elimination. The lower total systemic bioavailability of DIBC than that of DIBK, and the common major metabolite formation from DIBC and DIBK, imply that DIBC may have lower toxicity than DIBK. The current study results support that toxicological data on DIBK could be used to characterize the hazards of DIBC.
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DIBC alcohol 493
DIBC 24.5 48.5
DIBK alcohol 2.8
DIBC alcohol 21.9
Conflict of interest The authors declare no conflict of interest.
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Transparency document
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The Transparency document associated with this article can be found in the online version.
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De Ceaurriz, J., Desiles, P., Bonnet, B., Marignac, J., Muller, J.P., 1983. Concentrationdependent behavioral changes in mice following short-term inhalation exposure to various industrial solvents. Toxicol. Appl. Pharmacol. 67, 383–389. De Ceaurriz, J., Micillino, B., Marignac, P., Bonnet, J., JP, Guenier, 1984. Quantitative evaluation of sensory irritating and neurobehavioural properties of aliphatic ketones in mice. Food Chem. Toxicol. 22, 545–549. DiVincenzo, G.D., Kaplan, C.J., Dedinas, J., 1976. Characterization of the metabolites of methyl n-butyl ketone, methyl iso-butyl ketone, and methyl ethyl ketone in guinea pig serum and their clearance. Toxicol. Appl Pharmacol. 36, 511–522. Dodd, D.E., Losco, P.E., Troup, C.M., Tyler, T.R., 1987. Hyalin droplet nephrosis in male Fischer-344 rats following inhalation of diisobutyl ketone. Toxicol. Ind. Health 3, 443–457. Duguay, A.B., Plaa, G.L., 1995. Tissue concentrations of methyl isobutyl ketone, methyl n-butyl ketone and their metabolites after oral or inhalation exposure. Toxicol. Lett. 75, 51–58. ECHA, 2014. 2,6-dimethylheptan-4-one complete REACH Registration. http://echa. europa.eu/web/guest/information-on-chemicals/registered-substances? p_p_id=registeredsubstances_WAR_regsubsportlet&_registeredsubstances_ WAR_regsubsportlet_name-sc=&_registeredsubstances_WAR_regsubsportlet_ec-number-sc=108-83-8&_registeredsubstances_WAR_regsubsportlet_casnumber-sc=108-83-8&_registeredsubstances_WAR_regsubsportlet_sc= true&_registeredsubstances_WAR_regsubsportlet_do-search=.
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Fabregat, A., Pozo, O.J., Marcos, J., Segura, J., Ventura, R., 2013. Use of LC-MS/MS for the open detection of steroid metabolites conjugated with glucuronic acid. Anal. Chem. 85, 5005–5014. Gingell, R., Regnier, J.F., Wilson, D.M., Guillaumat, P.O., Appelqvist, T., 2003. Comparative metabolism of methyl isobutyl carbinol and methyl isobutyl ketone in male rats. Toxicol. Lett. 199–204. Granvil, C.P., Sharkawi, M., Plaa, G.L., 1994. Metabolic fate of methyl n-butyl ketone, methyl isobutyl ketone and their metabolites in mice. Toxicol.Lett. 70, 263–267. OECD, 2000. Proceedings of the SIDS Initial Assessment Meeting (SIAM), 14, March 2002, Paris, France. Poon, G.K., Kwei, G., Wang, R., Lyons, K., Chen, Q., Didolkar, V., Hop, C.E., 1999. Integrating qualitative and quantitative liquid chromatography/tandem mass spectrometric analysis to support drug discovery. Rapid Commun. Mass Spectrom. 13, 1943–1950. Qu, J., Wang, Y., Luo, G., Wu, Z., 2001. Identification and determination of glucuronides and their aglycones in Erigeron breviscapus by liquid chromatography–tandem mass spectrometry. J. Chromatogr. A 928, 155–162. SIDS, O., 1998. DI-ISO-BUTYLKETONE. http://webnet.oecd.org/HPV/UI/handler.axd? id=6165f80b-008c-4cf7-ab6f-284ec31cf213. Zhang, Y., Huo, M., Zhou, J., Xie, S., 2010. PKSolver: an add-in program for pharmacokinetic and pharmacodynamic data analysis in microsoft excel. Comput. Methods Programs Biomed. 314.
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Contents lists available at ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
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Comparative metabolism and pharmacokinetics of diisobutyl ketone and diisobutyl carbinol in male SD rats Fagen Zhang * , Michael J. Bartels, Amy J. Clark, Jen L. Staley, Tom S. Lardie, Dan A. Markham, Brian J. Hughes, Nicholas S. Ball Toxicology and Environmental Research and Consulting, The Dow Chemical Company, 1803 Building, Midland, MI 48674, USA
H I G H L I G H T S
Comparative metabolism and pharmacokinetics of diisobutyl ketone (DIBK) and diisobutyl carbinol (DIBC) in male rats were explored. DIBC and DIBK are both well absorbed following oral gavage with substantial evidence of enterohepatic recirculation of DIBK, DIBC, DIBK-alcohol, and DIBC-alcohol. DIBK and DIBC are interconverted metabolically in rats. Higher systemic exposure was found for DIBK-alcohol than DIBC-alcohol, implying that DIBC-alcohol may be more easily conjugated and eliminated in bile. The toxicological data on DIBK could be used to characterize the hazards of DIBC.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 July 2014 Received in revised form 21 October 2014 Accepted 22 October 2014 Available online xxx
Diisobutyl ketone (DIBK) and diisobutyl carbinol (DIBC) are important organic solvents widely used as industrial intermediates. It was hypothesized that DIBC and DIBK have common metabolic pathways and metabolites, and as such, toxicological data on DIBK could be used to characterize the hazards of DIBC. To confirm or refute this hypothesis a comparative metabolism and pharmacokinetics assessment of DIBK and DIBC was conducted. Dosing was via single oral gavage dosing in male SD rats, followed by blood collection, metabolite identification, major biomarker quantitation, and pharmacokinetics analysis. Overall, the major metabolites of both DIBC and DIBK in blood were their corresponding monohydroxylated metabolites (DIBC alcohol and DIBK alcohol) with the site of hydroxylation at the s and s-1 positions, respectively. Quantitative analysis of DIBC, DIBK, DIBC-alcohol, and DIBK-alcohol in blood samples collected from 5 min to 120 h after single dosing indicated the following: (1) DIBC and DIBK are both well absorbed following oral gavage with substantial evidence of enterohepatic recirculation of DIBK, DIBC, DIBK-alcohol, and DIBC-alcohol; (2) DIBK and DIBC are interconverted metabolically in rats; (3) DIBC and DIBK have similar bioavailability after oral administration; (4) higher systemic exposure was found for DIBK-alcohol than DIBC-alcohol, implying that DIBC-alcohol may be more easily conjugated and eliminated in bile. In summary, the metabolic similarities and the difference in systemic exposure to metabolites between these substances observed in the current study support the hypothesis that DIBC might have a lower potential toxicity than that of DIBK. The current study results support that toxicological data on DIBK could be used to characterize the hazards of DIBC ã 2014 Elsevier Ireland Ltd. All rights reserved.
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1. Introduction
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DIBK (diisobutylketone) and DIBC (diisobutylcarbinol) are highproduction volume chemicals that are widely used in the chemical
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* Corresponding author. Tel.: +1 989 638 4172; fax: +1 989638 9305. E-mail address: [email protected] (F. Zhang).
industry as industrial intermediates.Sufficient DIBK toxicity data have been generated (Carpenter et al., 1953; Chataigner et al., 1992; De Ceaurriz et al., 1983; De Ceaurriz et al., 1984; Dodd et al., 1987) and have been successfully used to support DIBK registration (ECHA REACH and OCED SIDS) (ECHA 2014; SIDS, 1998). In contrast, only limited toxicity data are available for DIBC. To address this potential data gap, a read-across approach was proposed, wherein the DIBK toxicity data, in conjunction with new pharmacokinetic
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Please cite this article in press as: Zhang, F., et al., Comparative metabolism and pharmacokinetics of diisobutyl ketone and diisobutyl carbinol in male SD rats. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.10.027
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1995; Granvil et al., 1994), and some differences in overall ADME fate of these two test materials may occur, the final result from Gingell’s study showed that systemic equivalence of MIBC with MIBK support the conclusion that MIBC will have a similar toxicological profile to MIBK, and reduce the need for additional animal studies (Gingell et al., 2003). DIBK and DIBC are quite comparable in structure to the related MIBK and MIBC compounds. It would therefore be expected that DIBK and DIBC would undergo similar interconversion in mammals, and also form a common HMP-like metabolite, which would be 6-hydroxy-2,6-dimethyl-4-heptanone (HDH) (DIBK alcohol, Scheme 2). Based on this background information, a bridging limited ADME study was proposed to confirm this hypothesis via a similar approach to that reported by Gingell et al., (2003).
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2. Materials and methods
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2.1. Reagents, solvents and materials
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Acetonitrile (HPLC grade), methanol (HPLC grade), water (HPLC grade), and acetic acid (HPLC grade) were obtained from Fisher Scientific (Itasca, IL, USA). 13C-DIBC, DIBK and their metabolites (Scheme 3) were obtained from Chemdepot (California, USA). All other reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA), unless otherwise stated.
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2.2. Animals
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In accordance with the U.S. Department of Agriculture animal welfare regulations, 9 CFR, subchapter A, Parts 1–4, the animal care and use activities required for conduct of this study were reviewed and approved by the Institution Animal Care and Use Committee (IACUC). Male NTac:SD rats (both cannulated and uncannulated) were obtained from Taconic (Germantown, New York) 250 g, approximately 9–10 weeks of age).
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2.3. Animal treatment procedures
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Two animal treatment procedures were used in the current study:
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2.3.1. Probe experiment for metabolite identification in blood Groups (Group 1 and 2) of 2 male rats/per group (uncannulated) were administered either DIBK or DIBC, via oral gavage, at a dose level of 700 mg/kg in corn oil. Animals were sacrificed at the time of the estimated Cmax based on the previous study on MIBK and
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Scheme 1. Proposed metabolism of MIBC and MIBK.
data showing systemic bioequivalence between DIBK and DIBCwould comprise the DIBC registration dossier. The proposed bioequivalence study was based on the hypothesis that these alcohol-ketone isomers undergo interconversion in vivo, as has been shown for the analogous isomer compounds, methylisobutylcarbinol (MIBC) and methylisobutylketone (MIBK) (Gingell et al., 2003). In that study, Gingell et al. showed that MIBC and MIBK are essentially bioequivalent in rats after single oral gavage administration of MIBC or MIBK, with MIBC converting first to MIBK enzymatically, followed by metabolism to the common HMP metabolite (4-hydroxy-4-methyl-2-pentanone) (Scheme 1). Pharmacokinetic analysis of blood samples, following single oral doses of either MIBK or MIBC, showed that the total systemic exposure of the two test materials (plus the major HMP metabolite) were comparable, as measured by the individual and total plasma AUC values. Although no mass balance of administered radiolabel and urinary metabolite profiling were conducted in Gingell’s study or other prior metabolism studies with these compounds (DiVincenzo et al., 1976; Duguay and Plaa,
Scheme 2. Chemical structures of DIBC and DIBK and their potential metabolites.
Scheme 3. Proposed metabolism of DIBC and DIBK.
Please cite this article in press as: Zhang, F., et al., Comparative metabolism and pharmacokinetics of diisobutyl ketone and diisobutyl carbinol in male SD rats. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.10.027
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MIBC (Gingell et al., 2003) and blood samples were collected and used for preparation of plasma samples. These probe experiment plasma (estimated Cmax plasma) samples were analyzed for identification of the major biomarkers (DIBK, DIBC, and their corresponding metabolites such as DIBK alcohol and DIBK alcohol) (Scheme 3). 2.3.2. Definitive pharmacokinetic experiment Based on the metabolite identification results of the probe experiment (Group 1 and 2), the definitive pharmacokinetic experiment was conducted to determine kinetics of the major biomarkers of DIBK, DIBC, and their major identified hydroxylated metabolites (such as DIBK alcohol, Scheme 2) in blood. Groups (Group 3 and 4) of 4 male rats, with cannula implanted in the jugular vein, were administered either DIBK or DIBC, via oral gavage, at a dose level of 700 mg/kg in corn oil. Repetitive blood samples were taken from each rat at 0.08, 0.17, 0.25, 0.5, 1, 2, 3, 6, 12, 24, 48, 72, 96 and 120 h post-dosing. Chemical analysis was then conducted on individual blood samples to quantify the major biomarkers of DIBC and DIBK and their corresponding major metabolites. Limited pharmacokinetic analysis (such as AUC (areaunder-the-curve)) was conducted based on the quantitative analysis results of major biomarkers by using the PK_AUCt Excel function of PK Solver (Zhang et al., 2010)
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Stock solutions of DIBK, DIBC and DIBK alcohol were prepared at a nominal concentration of 10 mg/mL in acetonitrile (ACN). A series of standard working solutions in the range of 2.5 mg/mL–2500 mg/ mL were prepared by further dilution of the corresponding standard stock solution(s) with ACN. The stock solutions of internal standards (IS, 13C-labeled DIBC and 13C-Labeled BIBK (Scheme 2)) were prepared at a concentration of 4000 mg/mL in ACN. The IS working solution (50 mg/mL) was prepared in ethyl acetate. All solutions were stored at 20 C. The calibration standards of DIBK, DIBC, and DIBK alcohol at concentrations of 0.125, 0.313, 1.250, 3.125, 12.5, 31.25, and 125 mg/mL (all containing 300 mL of IS solution, nominal concentration of 30 mg/mL), were prepared by diluting the working standard stock solutions with blood collected from control rats.
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2.6. Sample preparation for biomarker quantitation
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Heparinized blood samples from the Group 3 and 4 rats were maintained at 80 C prior to analysis. Samples were removed from the freezer and allowed to warm at a room temperature. Weighed aliquots (approximately 200 mL) of each blood sample were transferred to 4 mL glass vials and the appropriate amount of IS working solution (300 mL) was added. After mixing, 100 mL of ethyl acetate was added. The resulting mixture was vortex-mixed and then centrifuged at 863 g for 10 min. The organic phase was
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transferred to a clean GC vial for GC/MS analysis. The calibration standards were processed in a similar manner.
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2.7. GC/MS methods
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DIBC and DIBK and their potential metabolites in blood or in probe experiment plasma were analyzed by gas chromatographymass spectrometry (GC/MS). GC/MS analyses were performed by an Agilent 6890 GC equipped with an Agilent 5973 (EI) MS detector (Agilent Technologies, Palo Alto, CA, USA), using the MSD Chemstation Software for data acquisition and processing. A 30 m 0.32 mm 0.25 mm film thickness Phenomenex ZB-Wax plus, w/Guardian (Phenomenex, Torrance, CA, USA) was used for the gas chromatographic analysis. The injector was a Gerstel MultiPurpose Sampler (MPS) injector (Gerstel, GmbH, Mülheim an der Ruhr, Germany). The injector was coupled by a capillary transfer line with the external thermal extraction unit, containing a removable glass tube for the sample desorption. The GC conditions were as follows: injection mode: splitless; injector port temperature: 230 C; carrier gas: helium with flow rate: 2.1 mL/min; oven thermal program: from 50 to 240 C at a rate of 20 C/min with initial hold time 1 min and final hold time 5 min; ion source temperature: 230 C; interface temperature: 230 C.
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2.4. Plasma or blood extraction from probe animal treatment procedure Individual probe experiment plasma samples (Group 1 and 2), control plasma samples, or pooled blood samples (Group 3 and 4) were maintained at 80 C prior to analysis. Samples were removed from the freezer and allowed to warm to room temperature. Aliquots (approximately 100 mL) of each sample were transferred to 4 mL vials and 200 ml of ethyl acetate was added. The resulting mixture was vortex-mixed and then centrifuged at 863 g for 10 min. The organic layer was transferred to a clean GC vial for full scan GC/EI-MS analysis for major biomarker (metabolite) identification.
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Fig. 1. Chromatograms of GC/EI-MS analysis of ethyl acetate extracts of DIBC and DIBK probe experiment plasma and 3 h DIBC blood: (A) DIBC Cmax plasma (this chromatogram is the same as the chromatogram from control plasma ethyl acetate extract, data not shown); (B) DIBK probe experiment Plasma; (C) 3 h DIBC blood.
Please cite this article in press as: Zhang, F., et al., Comparative metabolism and pharmacokinetics of diisobutyl ketone and diisobutyl carbinol in male SD rats. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.10.027
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Preliminary mass spectra of parent and metabolites were obtained in full scan mode after electron ionization (EI, 70 ev) from m/z 50 to 500 amu (scan rate 0.50 scan/s, solvent delay 2 min), while selective ion monitoring (SIM) mode was selected for the quantification of parent compunds and metabolites.
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3. Results and discussion
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3.1. Metabolite profiling and identification from probe experiment plasma and blood samples
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In order to identify the potential metabolites (biomarkers) in probe experiment plasma (Group 1 and 2) or pooled blood samples (Group 3 and 4), a mixture of standards containing DIBK, DIBC, DIBC-alcohol, DIBK-alcohol (Scheme 2) in ethylacetate was analyzed by GC/EI-MS mass spectrometer via a full scan mode. As shown in Fig. 1, good GC/MS chromatograms with separation and MS spectra from the corresponding peaks were obtained (data not shown). Using the same GC/EI-MS method described above, ethylacetate extracts of probe experiment plasma samples from Group 1 to Group 2 were also analyzed to tentatively identify major biomarkers (parent and metabolites). As shown in Fig. 1A, only peaks from control plasma were detected and no DIBC or DIBC metabolites were detected in the estimated probe experiment plasma of rats dosed with DIBC (Group 2; Fig. 1A). However, two major peaks (DIBK alcohol and DIBC alcohol), and two minor peaks (DIBK and HMP) were present in DIBK probe experiment plasma (Group 1; Fig. 1B). Further GC/MS analysis with Wiley EI/MS database showed that peak HMP is 4-hydroxy-4-methyl-2pentanone, a major metabolite of methyl isobutyl ketone (MIBK) which was contained in the original DIBK test material. It was postulated that the major biomarkers of DIBC may not be at peak levels at the proposed probe experiment sampling time of 1.5 h. Further GC/MS analysis was therefore conducted with selected whole-blood extracts (1, 2, 3 and 6 h) from Group 4 rats (DIBC-dosed) to attempt to identify circulating biomarkers of DIBC and the result is shown in Fig. 1C. As shown in Fig. 1C, DIBC, DIBK alcohol and DIBC alcohol were detected as DIBC metabolites in 3 h pooled whole blood samples. The quantitative blood results below support that the 1.5 h sampling time was, in fact, Cmax between two DIBC blood concentration peaks, most probably due to enterohepatic recirculation of conjugated forms of DIBC metabolites (discussed below). Synthetic standards of novel metabolites of either test material were required to confirm chemical structures. Based on the known metabolism of MIBC and MIBK to HMP (Gingell et al., 2003), via s-1 hydroxylation, the proposed compounds DIBC alcohol A (2, 6dimethylheptane-2,4-diol) and DIBK alcohol (6-hydroxy-2,6-dimethyl-4-heptanone; Scheme 2) were custom synthesized. The synthesized DIBK-alcohol standard (Scheme 2) was found to have the same retention time and MS spectra (data not shown) as the DIBK alcohol metabolite (Fig. 1) observed in probe experiment plasma from Group 1 animals, thereby confirming the structure of the DIBK alcohol metabolite as 6-hydroxy-2,6-dimethyl-4-heptanone. In contrast, the retention time and mass spectra (data not shown) of the synthesized DIBC alcohol A (Scheme 2) did not Table 1 Retention time and typical ions of DIBC and DIBK and their major metabolites. Analyte
Retention time (min)
Typical ions (m/z)
DIBK DIBC DIBK alcohol DIBC alcohol
6.9 13.1 18.9 23.6
41, 57, 85, 142 43, 69, 87 43, 59, 85, 100, 143 43, 59, 83, 101, 141
Fig. 2. A typical linear response of analytes by GC/EI/SIM-IS-MS: (A) DIBC (0.1–120 mg/mL); (B) DIBK (0.002–120.0 mg/mL).
match those of the DIBC alcohol metabolite (Fig. 1) found in probe experiment plasma from Group 1 animals. Based on the GC/MS spectral analysis, the DIBC alcohol metabolite was identified as 2,6-dimethylheptane-1,4-diol, arising from hydroxylation at the s position of DIBC (Scheme 3). In order to identify any potential conjugated metabolites of DIBC, DIBK, the collected estimated probe experiment plasma samples from both DIBC and DIBK were also analyzed by LC/MSMS with neutral loss scan (for glucuronide conjugate characterization) (Beaudry et al., 1999; Fabregat et al., 2013; Poon et al., 1999; Qu et al., 2001). However, no detectable conjugates or other novel metabolites of either test material were found (data not shown).
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After DIBC, DIBK, DIBC-alcohol and DIBK-alcohol were identified from plasma or blood samples, a more sensitive SIM-GC/EI-MS method based on the abundant ions of major biomarkers (Table 1) was developed for the definitive quantitation of these biomarkers in Group 3 and 4 whole blood samples. Ions of stable-isotope labeled internal standards of DIBK, DIBC (DIBC-IS, DIBK-IS, Scheme 2, Table 1) were also incorporated into the quantitation method. A typical standard curve is shown in Fig. 2. The final quantitation results are shown in Table 2. Overall, DIBK, DIBK alcohol and DIBC alcohol were quantifiable in most blood samples from rats administered DIBK (Group 3). In a similar manner, DIBC, DIBK alcohol, and DIBC alcohol were also quantifiable in blood samples from rats administered DIBC (Group 4). Based on the metabolite identification conducted with probe experiment plasma and selected Group 4 whole blood samples (described above) and the final quantitation results (Table 2), the
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Table 2 Final biomarker concentrations in blood samples from oral administration of DIBC and DIBK. Concentration of biomarker (mg/g blood) from DIBC oral administration Biomarker name
DIBC
Time point (h)
Average
STDEV
Average
STDEV
Average
STDEV
0.08 0.17 0.25 0.50 1.0 2.0 3.0 6.0 12.0 24.0 48.0 72.0 96.0 120
0.548 1.21 1.51 2.33 1.43 3.54 5.04 2.01 0.418 0.414 0.404 0.432 0.454 0.288
0.192 0.618 0.727 1.19 1.36 1.78 2.18 1.90 0.00423 0.0318 0.00458 0.0427 0.0741 0.0360
0.189 0.182 0.198 0.179 0.202 0.269 0.365 0.311 0.196 0.194 0.189 0.202 0.213 0.135
0.0423 0.0140 0.0104 0.0201 0.0196 0.0607 0.119 0.140 0.00198 0.0149 0.00215 0.0200 0.0347 0.0168
0.189 0.182 0.198 0.179 0.192 0.209 0.340 0.590 1.03 0.534 0.189 0.202 0.213 0.135
0.0423 0.0140 0.0104 0.0205 0.0146 0.0309 0.0509 0.263 0.965 0.618 0.00215 0.0200 0.0347 0.0168
BIBK alcohol
DIBC alcohol
Concentration of biomarker (mg/g blood) from DIBC oral administration
278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304
Biomarker name
DIBK
Time point (h)
Average
STDEV
Average
STDEV
Average
STDEV
0.08 0.17 0.25 0.50 1.0 2.0 3.0 6.0 12.0 24.0 48.0 72.0 96.0 120
0.566 2.05 3.18 2.21 2.49 4.01 4.27 1.27 0.181 0.173 0.159 0.183 0.169 0.173
0.415 0.960 1.79 1.45 0.543 2.61 2.46 1.21 0.0132 0.0107 0.00353 0.00441 0.0134 0.0283
1.17 4.40 7.20 9.19 10.7 15.0 16.0 9.22 0.533 0.201 0.184 0.212 0.196 0.201
0.915 1.71 2.96 4.24 2.43 7.49 7.93 6.22 0.371 0.0124 0.00408 0.00510 0.0155 0.0327
0.302 1.05 1.69 4.02 8.11 16.0 22.4 33.0 24.6 2.77 0.205 0.212 0.196 0.201
0.169 0.363 0.401 0.993 1.76 2.49 5.20 12.7 19.1 1.21 0.0277 0.00510 0.0155 0.0327
BIBK alcohol
metabolic pathways of DIBK and DIBC in rats can be proposed as described in Scheme 3. DIBC and DIBK are initially interconverted by alcohol dehydrogenase and ketone reductase. DIBK and DIBC are further oxidized to their corresponding metabolites (DIBK alcohol and DIBC alcohol, respectively) by CYP 450-based hydroxylation at the s-1 or s positions, respectively. Note that with these unique sites of hydroxylation for DIBK and DIBC, there is no possibility for initial hydroxylation of either test material, followed by redox interconversion. 3.3. Time-course concentration and pharmacokinetics of DIBC, DIBK and metabolites in blood The time-course blood concentrations of the four quantifiable major biomarkers in Group 3 (DIBK) and Group 4 (DIBC) animals are shown in Fig. 3. Overall, the Cmax for the highest concentration of DIBC, DIBK, and DIBK-alcohol was about 3 h, while the Cmax for DIBC-alcohol was about 10 h (Fig. 3). These results showed that DIBC and DIBK are both readily absorbed after oral gavage. The pattern of an initial, lower Cmax, at about 30 min, was also observed for both DIBC (from DIBC administration) and for DIBK (from DIBK administration). This dual-peak kinetic pattern possibly reflects enterohepatic recirculation of DIBC and DIBK, possibly through biliary elimination of yet-unidentified Phase II conjugates of the test materials and/or their metabolites. Due to this apparent enterohepatic recirculation, some of the pharmacokinetic parameters (such as elimination half-life) were not calculated. Therefore only AUC (area under the curve) values were calculated and summarized in Table 3. The average AUC
DIBC alcohol
values for both major test material (DIBK and DIBC) were similar (26.8 and 35.3 ug/g h, respectively), indicating that DIBK and DIBC have a similar bioavailability after excluding the contribution from their potential metabolites. The total AUC of all biomarkers from administration of DIBK is about 10 times of the total AUC from administration of DIBC (Table 3). There are potentially two factors contributing to this difference in systemic bioavailability. One factor may be a higher rate of phase II conjugation DIBC (Fig. 3) and its metabolites than that of DIBK or DIBK metabolites. Another factor may be a lower oral absorption of DIBC than DIBK. Regardless of these quantitative differences, these toxicokinetics analysis results show a high degree of metabolic similarity between DIBC and DIBK, with only lower bioavailability seen for the DIBC compound. Similar systemic metabolic bioequivalence arguments have been previously made to predict certain aspects of systemic toxicity of alcohol isomers based on the available toxicity data of the corresponding ketone isomer (such as methyl ethyl ketone and sec-butanol (OECD, 2000); MIBK and MIBC (Gingell et al., 2003)). MIBC and MIBK had similar total AUC (similar systemic metabolic bioequivalence) (Gingell et al., 2003). The 6-week inhalation toxicity study in rats with MIBC showed that LOAEL (low observed adverse effect level) was 900 ppm (Gingell et al., 2003); in the similar study conditions, the LOAEL for MIBK is 1000 ppm. These inhalation toxicity results for MIBC and MIBK are correlated very well with the similar bioequivalence of MIBC and MIBK. Therefore, it can be predicted that the systemic toxicity of DIBC may be lower than that from DIBK based on the current study results.
Please cite this article in press as: Zhang, F., et al., Comparative metabolism and pharmacokinetics of diisobutyl ketone and diisobutyl carbinol in male SD rats. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.10.027
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A)
B)
Fig. 3. Kinetics of various biomarkers in blood from rat orally dosed with DIBK and DIBC: (A) DIBC; (B) DIBK.
Table 3 Area under the curve (AUC) of DIBK, DIBC, DIBK alcohol and DIBC alcohol. Test material
DIBK
Major quantifiable biomarkers in blood AUC (mg/g h) Total AUC (mg/g h)
DIBK 22.5 599
DIBC DIBK alcohol 106
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4. Conclusions
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The current study showed that DIBC and DIBK are well interconverted metabolically in rats. Both DIBC and DIBK have similar metabolic pathways to form the major common metabolites (DIBC-alcohol and DIBK-alcohol). Both DIBC and DIBK are rapidly absorbed following oral administration, with substantial evidence of enterohepatic recirculation. Quantitatively, DIBK was shown to have higher bioavailability (AUC) than DIBC after oral administration. This higher bioavailability may be due to lower absorption of DIBC or a higher rate of Phase II conjugation of DIBC and metabolites, followed by biliary elimination. The lower total systemic bioavailability of DIBC than that of DIBK, and the common major metabolite formation from DIBC and DIBK, imply that DIBC may have lower toxicity than DIBK. The current study results support that toxicological data on DIBK could be used to characterize the hazards of DIBC.
336 337 338 339 340 341 342 343 344 345 346 347 348 349
DIBC alcohol 493
DIBC 24.5 48.5
DIBK alcohol 2.8
DIBC alcohol 21.9
Conflict of interest The authors declare no conflict of interest.
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Transparency document
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The Transparency document associated with this article can be found in the online version.
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