development of a physiologically based pharmacokinetic model for chlorobenzene in f-344 rats

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Development Of A PhysiologicallyBased Pharmacokinetic Model ForChlorobenzene In F-344 RatsKarla D. Thrall a , Angela D. Woodstock a & Melissa R. Kania ba Biological Sciences Division, Pacific Northwest NationalLaboratory , Richland, Washington, USAb U.S. Department of Energy Pre-Service Teacher Program,University of Washington , Seattle, Washington, USAPublished online: 12 Aug 2010.

To cite this article: Karla D. Thrall , Angela D. Woodstock & Melissa R. Kania (2004) Development OfA Physiologically Based Pharmacokinetic Model For Chlorobenzene In F-344 Rats, Journal of Toxicologyand Environmental Health, Part A: Current Issues, 67:7, 525-536, DOI: 10.1080/15287390490425731

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Journal of Toxicology and Environmental Health, Part A, 67:525–536, 2004Copyright© Taylor & Francis Inc.ISSN: 1528–7394 print / 1087–2620 onlineDOI: 10.1080/15287390490425731

525

DEVELOPMENT OF A PHYSIOLOGICALLY BASED PHARMACOKINETIC MODEL FOR CHLOROBENZENE IN F-344 RATS

Karla D. Thrall,1 Angela D. Woodstock,1 Melissa R. Kania2 1Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA 2U.S. Department of Energy Pre-Service Teacher Program, University of Washington, Seattle, Washington, USA

A physiologically based pharmacokinetic (PBPK) model to describe the absorption, distribution,metabolism, and elimination of chlorobenzene in rats was developed. Partition coefficientswere experimentally determined in rat tissues and blood samples using an in vitro vial equili-bration technique. These solubility ratios were in agreement with previous reports. The in vivometabolism of chlorobenzene was evaluated using groups of three F344 male rats exposed toinitial chlorobenzene concentrations ranging from 82 to 6750 ppm in a closed, recirculatinggas uptake system. An optimal fit of the family of uptake curves was obtained by adjustingMichaelis–Menten metabolic constants, Km (affinity) and Vmax (capacity), using the PBPK model.At the highest chamber concentration, the uptake curve could not be modeled without theaddition of a first-order ( Kfo ) metabolic pathway. Pretreatment with pyrazole, an inhibitor ofoxidative microsomal metabolism, had no impact on the slope of the uptake curve. The com-pleted PBPK model was evaluated against real-time exhaled breath data collected from ratsreceiving either an intraperitoneal (ip) injection or oral gavage dose of chlorobenzene in cornoil. Exhaled breath profiles were evaluated and absorption rates were determined. Develop-ment of the chlorobenzene PBPK model in rats is the first step toward future extrapolations toapply to humans.

Chlorobenzene (monochlorobenzene) is an industrial solvent used in themanufacture of pesticides, resins, inks, and paints. Human exposure to chloro-benzene can occur by ingestion of water contaminated by chlorobenzene, orby dermal and inhalation routes during manufacture. Chlorobenzene ismetabolized into 4-chlorocatechol, ortho-, meta-, and para-chlorophenol, and4-chlorophenylmercapturic acid (Kumagai & Matsunaga, 1995). In humans,urinary concentrations of the primary metabolite, 4-chlorocatechol, has beenshown to be a reliable biological indicator of occupational exposure to chloro-benzene (Ogata & Shimada, 1983; Ogata et al., 1991; Yoshida et al., 1986;Kamugai & Matsunaga, 1994). Studies indicate that chlorobenzene is toxic tothe liver and kidney of animals exposed by inhalation exposures (Dilley & Lewis,

Accepted 24 October 2003. This work was conducted under U.S. Department of Energy (DOE) contract DE-AC06-76RLO 1830. Address correspondence to Karla D. Thrall, Biological Sciences Division, Pacific Northwest National

Laboratory, 902 Battelle Blvd., Mail Stop P7-59, Richland, WA 99352, USA. E-mail: [email protected]

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526 K. D. THRALL ET AL.

1978). However, it is not clear whether the toxicity of chlorobenzene is due tothe parent chemical itself, or can be attributed to one or more of its metabolites(Kumagai & Matsunaga, 1995).

A physiologically based pharmacokinetic (PBPK) model has been devel-oped to describe the dose-dependent kinetics of urinary 4-chlorocatechol andblood chlorobenzene concentrations following inhalation exposure to chloro-benzene in man (Kumagai & Matsunaga, 1995). However, the human-specificparameters of this model and the lack of time-course experimental data makeit difficult to extrapolate beyond the limited occupational data and to under-stand species-dependent differences in parent and metabolite kinetics. In aneffort to better understand the dose-dependent and route-specific kinetics ofchlorobenzene, the current objectives focused on development and evalua-tion of a chlorobenzene PBPK model for the rat through the use of controlledin vitro and in vivo animal studies. Development of an accurate representationof the rodent absorption, distribution, metabolism, and elimination kinetics willultimately enable extrapolation of the PBPK model to describe human kineticrelationships.

MATERIALS AND METHODS

Animals Adult male F344 rats (200–275g body weight) were obtained from Charles

River Breeding Laboratories (Raleigh, NC). The animals were housed in solid-bottom cages with hardwood chips and were acclimated in a humidity- andtemperature-controlled room with a 12-h light/dark cycle for at least 5d priorto use. Certified Purina rodent chow (Ralston Purina Co., St. Louis, MO) andwater were provided ad libitum throughout the acclimation period.

Test Material Chlorobenzene (99+% purity) was obtained from Aldrich Chemical Com-

pany (Milwaukee, WI). Pyrazole (1,2-diazole) of >98% purity was obtainedfrom Fluka (Ronkonkoma, NY). Clinical grade saline (0.9%) was obtained fromAbbott Labs (Chicago) and contained 0.9% benzyl alcohol to prohibit bacterialgrowth. All other chemicals were reagent grade or better.

Partition Coefficients Substrate-to-air partition coefficients were determined for rat blood, epididy-

mal fat, thigh muscle, and liver using a vial equilibration method as describedby Sato and Nakajima (1979) and Gargas et al. (1989). Headspace concentra-tions were analyzed by gas chromatography (GC) using a Hewlett-Packardmodel 6890 system (Avondale, PA) with a hydrogen flame ionization detector(FID). The column was DB-Wax, 30m×0.53mm ID, 1µm film thickness (J&WScientific, Folsom, CA). The detector was operated at 250 °C, the inlet at210 °C, and the final oven temperature was 230 °C. Under these conditions,

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PBPK MODEL FOR CHLOROBENZENE 527

chlorobenzene had a retention time of approximately 1.7 min. Tissue-to-blood partition coefficients were calculated by dividing tissue-to-air partitioncoefficients by blood-to-air partition coefficients.

Gas Uptake Studies Gas uptake studies were conducted using a closed-atmosphere exposure

system as described by Gargas et al. (1986) with modifications as described byThrall et al. (2000). In brief, the system consisted of a 9-L desiccator jar with gasinlet and outlet fittings fashioned into a 1/4-in-thick stainless-steel lid. A siliconerubber gasket was fitted between the glass rim of the desiccator and the stainless-steel lid, and the assembly was clamped in place using thumbscrew bracketsplaced around the perimeter. Preliminary studies conducted with an emptychamber found the nonspecific loss of chlorobenzene to be independent of con-centration, and less than 7%/h. Although this loss rate is slightly higher than ratesreported by others using similar types of systems, no additional loss was observedwhen dead animals were placed in the chamber. The chamber atmosphere wasrecirculated using a Bellows (model MB-41, Metal Bellows Corp., Los Angeles, CA)stainless-steel metal pump at 2 L/min. Carbon dioxide was removed with Soda-Sorb (W. R. Grace & Co., Atlanta, GA). Separate experiments to evaluate nonspe-cific chamber loss without the use of SodaSorb were not conducted. Relativehumidity was maintained by placing the glass chamber directly in ice, as describedby Gargas et al. (1986). Oxygen concentration in the chamber was maintained at19–21% by slowly adding ultra-high-purity (UHP) O2 when an audible O2 alarm(Cole-Parmer, Vernon Hills, IL) signaled concentrations dropped below 20%. Thepressure in the chamber was continually monitored using a Cole-Parmer (VernonHills, IL) digital pressure gauge and stayed constant throughout the experiments.

Each experiment utilized three rats per exposure concentration. Animalswere acclimated to the closed system prior to exposure. Chlorobenzene wasadded as a liquid through a heated septum fitting 12 in upstream of the chamberin a volume sufficient to achieve the desired initial chamber concentrations.Chamber atmosphere was monitored prior to addition of chlorobenzene, andup to 6h thereafter.

Atmospheric concentrations of chlorobenzene in the chamber were deter-mined every 5min by GC using a Hewlett-Packard model 5890 series II systemin place of the model 6890 system utilized in the partition coefficient studies.The 5890 series II GC used a hydrogen FID and the same column and similartemperature settings as described previously. Specifically, the detector wasoperated at 250 °C, the inlet valve at 150 °C, and the final oven temperaturewas 210 °C. Under these conditions, chlorobenzene had a retention time ofapproximately 1.3min.

To evaluate the impact of inhibition of oxidative microsomal metabolismon the shape of the gas uptake curve, animals of a separate group (n=2) werepretreated with a single intraperitoneal (ip) injection of aqueous pyrazole at320mg pyrazole/kg body weight, 1/2 h prior to gas uptake exposure, as describedpreviously by Gargas et al. (1986).

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In Vivo Studies Separate groups of naive animals received a single ip injection (n=3;

131mg/kg; 1ml/kg dose volume) or oral gavage (n =3; 127mg/kg; 2 ml/kg dosevolume) dose of chlorobenzene in corn oil. Immediately following dosing, ratswere placed individually in small off-gassing chambers and exhaled breathcontinually monitored for up to 5h postexposure, as described by Thrall et al.(2002). In brief, certified pure breathing air was supplied to each rat throughthe lid of the off-gassing chamber at a calibrated rate of 200ml/min. A TeledyneDiscovery II ion-trap mass spectrometer (MS/MS) equipped with an atmosphericsampling glow discharge ionization (ASGDI) source sampled directly from theoff-gassing chamber approximately every 1.6 s. The intensity data from the MS/MS was converted to concentration (ppb) through the use of external standardsprepared in Tedlar bags and a calibration curve. A new calibration curve wassimilarly generated for each day of experimentation.

PBPK Model The structure of the rat PBPK model was similar to that used to describe

styrene kinetics in rats (Ramsey & Andersen, 1984; Cohen et al., 2002). Forchlorobenzene, the PBPK model consisted of four compartments (fat, liver,rapidly perfused tissues, and slowly perfused tissues), plus the exchange ofchlorobenzene between lung blood and alveolar air (Figure 1). In this model,

Alveolar Air

Lung Blood

Liver

Slowly Perfused Tissues

Rapidly Perfused Tissues

Inhalation Exposure Exhaled

Fat

Metabolites

Oral Exposure

IV

IP Exposure

FIGURE 1. PBPK model for chlorobenzene.

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metabolism of chlorobenzene was assumed to occur in the liver. Gas uptakeinhalation exposures were modeled according to Gargas et al. (1986). Absorp-tion of chlorobenzene following ip injection or oral gavage was described as afirst-order process with a rate constant (h−1) of ka or kas, for ip or oral exposures,respectively. For in vivo kinetic studies using the off-gassing chambers, thechanging concentration of chlorobenzene in the off-gassing chamber (CCH) wasdescribed in terms of input from the exhaled breath and removal from thechamber, either by rebreathing or to the MS/MS system. In the PBPK modelthis is written as:

where ACH is the amount of chlorobenzene in the chamber (µmol), QP is thealveolar ventilation rate (L/h), CEX is the concentration of chlorobenzeneexhaled from the animal (µmol/L), CCH is the concentration of chlorobenzenein the chamber (µmol/L), and FCH is the air flow through the chamber (L/h).

To develop and validate the rat PBPK model, partition coefficients weredeveloped as already described. Values for breathing rate, organ volumes, andblood flow rates specific for the rodent (Table 1) were taken from the literature(Thrall et al., 2000). Metabolic parameters for chlorobenzene were obtainedby computer optimization of the gas uptake data, as described previously(Gargas et al., 1986). In brief, a PBPK model containing all parameters exceptrate of chemical removal due to metabolism was used to simultaneously predictthe family of gas uptake data. A maximum likelihood search algorithm in SimuSolv(version 3.0; Dow Chemical Co., Midland, MI) was used to vary values of theMichaelis–Menten constants Km and Vmax until an optimal fit was achieved thatdescribed all the time-course data with a single set of constants.

dACH

dt------------- QP CEX QP CCH×–×( ) FCH CCH×( )–=

TABLE 1. Physiological Parameters for the Rat PBPK Model

Parameter Rat

Body weight (kg) 0.25Cardiac output (L/h) 5.4 Alveolar ventilation (L/h) 5.4 Blood flow (% cardiac output)

Liver 25 Fat 4 Rapidly perfused 51 Slowly perfused 20

Tissue volume (% body weight) Liver 4 Fat 8 Rapidly perfused 5 Slowly perfused 74

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Statistical Analysis Partition coefficient values are given as the mean± standard deviation of

n=4 to 14 samples. The variability of sample size was due to additional testing ofsome tissues in order to minimize the statistical error. All sets of gas uptakecurves were simultaneously optimized; no statistical evaluation of variability inthe optimized values was possible. Estimates of metabolic and absorption modelparameters were based on the ability to describe data from the gas uptake andin vivo studies using the software optimization routines supplied with the com-mercial software package SimuSolv (Dow Chemical Company, Midland, MI).The percent variability explained for the optimized values was always ≥80%.The use of these routines has been described previously (Agin & Blau, 1982).

RESULTS

Partition Coefficients Substrate-to-air partition coefficients for chlorobenzene were measured in

saline, and rat blood, liver, epidyimal fat, and thigh muscle (Table 2). Musclewas used to represent the slowly perfused tissue compartment, and the liverwas considered representative of the rapidly perfused tissue compartment inthe PBPK model. Since an integral component to development of a PBPKmodel is estimation of blood-to-air and tissue-to-blood partition coefficients,reassessment of chlorobenzene partition coefficient values on a limited scale

TABLE 2. Substrate-to-Air Partition Coefficients for Chlorobenzene (Mean ± SD)

a Current study. b Gargas et al. (1989), studies in male F344 rats. c Kumagai and Matsunaga (1995), human values calculated as described by Fiserova-

Bergerova and Hughes (1983). d Béliveau and Krishnan (2000a). e Béliveau and Krishnan (2000b).

Substrate to air Rat Human Other

Blood 52.1 ± 8.1a 30.0 ± 0.3b 59.4 ± 1.0b 30.8c 61.8 ± 2.8d 57 ± 2.5 – 59 ± 3.2e Liver 98.9 ± 13.9a 110.9c 86.1 ± 3.0b Fat 1268 ± 136a 2649c 1277 ± 43b Muscle 44.6 ± 4.9a 55.4c 34.0 ± 3.9b Saline 3.1 ± 0.1a 2.8 ± 0.07b 4.1c Oil 465 ± 5b 3763c

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was considered to be a worthy effort. In vitro techniques to determine partitioncoefficients, such as described by Gargas et al. (1989), can be conducted in ashort period of time, using just a small number of animals.

A comparison of the partition coefficient values determined in this studywith literature values is provided in Table 2. The blood-to-air partition coeffi-cient reported here (52.1±8.1) compared well with the rodent values of59.4±1.0 reported by Gargas et al. (1989) and 61.8±2.8 reported by Béliveauand Krishnan (2000a). In a separate concentration dependency study, Béliveauand Krishnan (2000b) reported chlorobenzene blood-to-air partition coefficientsranging from 57±2.5 to 59±3.2 for concentrations of 0.11 to 0.90µmol, whichencompasses the 0.25µmol concentration used in the current studies.

Gas Uptake Studies Closed, recirculating chamber exposures of naive rats were conducted with

initial exposure concentrations ranging from approximately 100 to 6800ppm(Figure 2). A comparison of the shape of the series of uptake curves reveals thatthe slopes of the curves decreased progressively with increasing chamber con-centration. This behavior is representative of a saturable metabolic pathway, andis in agreement with observations of metabolic saturation reported by Sullivanet al. (1983) for chlorobenzene inhalation exposure in the rat. An optimal fit ofthe family of uptake curves was obtained by adjusting Michaelis–Menten meta-bolic constants, Km (affinity) and Vmax (capacity), using the PBPK model. At thehighest chamber concentration, the uptake curve could not be modeled with-out the addition of a first-order (Kfo ) metabolic pathway. Model simulations

1

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1000

10000

0.0 1.0 2.0 3.0 4.0 5.0 6.0Time, hr

Chl

orob

enze

ne C

ham

ber

Con

c., p

pm

FIGURE 2. Uptake of chlorobenzene from a closed, recirculating atmosphere by three naive F344 malerats per exposure. The initial chamber concentrations were 82, 471, 1250, or 6750 ppm. The smoothcurves were generated by the PBPK model using the constants given in Tables 1, 2, and 3.

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incorporating two saturable pathways (high and low capacity, high and lowaffinity) to describe chlorobenzene metabolism did not improve the model pre-diction, and were not pursued further (data not shown). The best fit of the familyof uptake curves was achieved with a Km (affinity) of 0.04mg/L, Vmax (capacity) of5.97mg/h/kg, and Kfo (first-order) of 13.5h−1 (Table 3). Pretreatment of animalswith pyrazole had no impact on the slope of the gas uptake curve.

In Vivo Studies To evaluate the PBPK model, a series of in vivo studies was conducted

with animals exposed to chlorobenzene in a corn oil matrix by ip injection ororal gavage and exhaled breath concentrations monitored for chlorobenzenelevels (Figures 3 and 4 data points). Oral and ip absorption of chlorobenzene

TABLE 3. In Vivo Metabolic and Absorption Rate Constants

Parameter Value

Metabolic rate constants Vmax (mg/h/kg body weight) 5.97 Km (mg/L) 0.04 Kfo (h

−1) 13.5 Absorption rate constants

Kas (h−1), oral absorption 0.56

Ka(h−1), ip absorption 0.49

0

5000

10000

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20000

25000

30000

35000

0.0 0.5 1.0 1.5 2.0 2.5 3.0Time, hr

Chl

orob

enze

ne C

onc.

, ppb

FIGURE 3. PBPK model prediction of the chamber concentration of chlorobenzene exhaled (ppb) from arat receiving a single ip injection of 131mg/kg chlorobenzene in corn oil (solid line) compared to the measureddata from this study (data points).

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was assumed to occur in a first-order fashion, with uptake modeled as directintroduction into the liver. The PBPK model, incorporating partition coefficientand metabolic parameters determined as described earlier, was used to simulatethe ip or oral exposures and estimate the absorption rate constants in order tofit exhaled breath levels. Exhaled breath data from one animal per exposureroute were used to estimate the oral or ip absorption rate constants, and theseabsorption rates were used to simulate the remaining data sets. In this manner,one data set could be used for estimating the absorption rate and the otherdata sets could be used to test the validity of the estimated rate constant.

For ip exposures, exhaled breath concentrations of chlorobenzene increasedrapidly to peak within approximately 1h of administration of the dose (Figure 3).The IP absorption rate constant (Ka) was estimated by the PBPK model to be0.49h−1 (Table 3), and the optimized absorption coefficient fit all ip-exposuredata sets from n=3 animals. The PBPK model simulations predict that approxi-mately 77% of the injected amount of chlorobenzene was absorbed systemically,of which roughly 25% was exhaled during the 3h the animals were monitored.

Systemic absorption of chlorobenzene was somewhat slower following oralgavage dosing compared to that observed following ip administration. Peakexhaled breath chlorobenzene concentrations following oral exposure wereachieved within approximately 2h (Figure 4). The absorption rate constant (Kas)for oral exposure was estimated by the PBPK model to be 0.56h−1 (Table 3) andprovided a good fit to all n=3 animal oral-exposure exhaled breath profiles.The PBPK model simulations of these oral exposures predict that approximately

0

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20000

25000

30000

0.0 1.0 2.0 3.0 4.0 5.0Time, hr

Chl

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enze

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ham

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Con

c., p

pb

FIGURE 4. PBPK model prediction of the chamber concentration of chlorobenzene exhaled (ppb) from arat receiving a single oral gavage dose of 127 mg/kg chlorobenzene in corn oil (solid line) compared to themeasured data from this study (data points).

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96% of the oral dose of chlorobenzene was absorbed systemically, of whichroughly 36% was exhaled during the 5h the animals were monitored.

Because chamber concentration is analyzed by the ion-trap mass spec-trometer every 1.6 s, the resulting data have a tendency to appear variable.However, this variability likely reflects temporary variations in breathing ratesand any movement by the animal. For model simulations, the breath data areaveraged every 10 data points, and the ion intensity for chlorobenzene is nor-malized to the intensity of the air peak (m/z ratio of 31) to minimize variabilityand accommodate any system drift.

DISCUSSION

A PBPK model can be used to describe the biokinetics of a chemical in experi-mental animals, and can be used to predict human tissue levels of a compoundfollowing occupational or environmental exposure. Thus, the objective of thepresent work was to systematically develop a PBPK model to describe the kineticsof chlorobenzene in rats following different routes of administration.

Metabolic rate constants determined from gas uptake studies reported hereindicate chlorobenzene is metabolized in a saturable manner. This is consistentwith prior reports of saturable chlorobenzene metabolism in the Sprague-Dawleyrat (Sullivan et al., 1983). Based on tissue burden and excretion information, thedata by Sullivan et al. (1983) suggest that metabolic clearance of chloro-benzene from the blood becomes saturated at exposure concentrations as lowas 400ppm for 8 h. In the studies conducted here, animals were exposed tochlorobenzene by inhalation at initial concentrations ranging from 82 to6750 ppm, with the slopes of the resulting uptake curves decreasing for expo-sures above the lowest exposure at 82 ppm.

Previous investigators have established that chlorobenzene is oxidized mainlyto 4-chlorophenol and to a lesser degree to 2-chlorophenol and 3-chlorophenol(Nedelcheva et al., 1998). In the studies reported here, however, pretreatmentof animals with pyrazole, an inhibitor of oxidative microsomal metabolism,was found to have no impact on the slope of the gas uptake curve. While thisin no way implies that the metabolic process is different from that previouslydescribed, it does suggest that for chlorobenzene the technique of gas uptakeis not appropriately sensitive to detect metabolic inhibition under these exper-imental conditions.

The real-time exhaled breath data demonstrate that chlorobenzene is wellabsorbed following oral administration, with peak exhaled breath concentrationsoccurring approximately 2h following exposure. In contrast to more sophisticatedgastrointestinal tract absorption models, the single one-compartment first-orderdescription of absorption used here was found to adequately simulate theexhaled breath profiles following oral dosing. In addition, the Kas of 0.56h−1

estimated in the current study is within the range of rate constants reported fora similar compound, benzene, of 0.25 to 0.6h−1 (Medinsky et al., 1989; Traviset al., 1990). Although environmental exposures to chlorobenzene are more

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likely to occur from contaminated water supplies, the limited aqueous solubilityof chlorobenzene (0.05%) precluded oral absorption studies in a water vehicleat a concentration sufficient to achieve meaningful exhaled breath concentrations.

Similarly, the real-time exhaled breath data indicates that absorption ofchlorobenzene following ip administration was rapid, with an estimated rateconstant of 0.49h−1. Data in the literature suggest that this ip absorption rateconstant is consistent with similar low-molecular-weight halogenated com-pounds administered in oil (D’Souza & Andersen, 1988; Reitz et al., 1988).

In summary, the studies described here provide kinetic information onchlorobenzene following oral and ip injection exposures. Expansion of the currentPBPK model to include a kinetic description of chlorobenzene metabolites,4-chlorocatechol, ortho-, meta-, and para-chlorophenol, and 4-chlorophenyl-mercapturic acid will greatly enhance our understanding of the role of thesemetabolites in the toxicity of chlorobenzene. Interpretation of the current databy a validated PBPK model is the first step toward understanding human kineticsand, ultimately, health risks associated with chlorobenzene exposures.

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development of a physiologically based pharmacokinetic model for chlorobenzene in f-344 rats

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