Document K6o2x9RRD15vGyEG1oB2JZ7gK

TOXICOLOGY AND APPLIED PHARMACOLOGY 95, 230-240 (1988) Physiologically Based Pharmacokinetic Model for Vinylidene Chloride R. W. D'Souza and M. E. Andersen* The Procter & Gamble Company. Miami Valley Laboratories. Box 398707. Cincinnati, Ohio 45239-8707. and *Armstrong Aerospace Medical Research Laboratories. Biochemical Toxicology Branch, Wright Patterson AFB. Ohio 45433 ReceivedJanuary 15.1988; accepted May 22,1988 Physiologically Based Pharmacokinetic Model for Vinylidene Chloride. D'Souza, R. W., and Andersen, M. E. (1988). Toxicol. Appl. Pharmacol. 95, 230-240. Vinylidene chloride (VDC), a potent hepatotoxin and suspected carcinogen, is metabolized by mixed-function oxi dases into a reactive metabolite(s) which is responsible for its toxicity. The metabolite is detoxi fied by glutathione (GSH), and liver GSH status is an important factor in the expression of VDC toxicity. A physiologically based pharmacokinetic (PB-PK) model has been developed for VDC in the rat based on oxidative metabolism ofVDC and subsequent GSH detoxification ofmetabo lite. The model ofTers insight into the complex interrelationship between the processes ofabsorp tion, metabolism, and GSH conjugation, and simulates the manner in which these factors oper ate in regulating VDC toxicity. The PB-PK model successfully predicts blood, tissue, and ex haled air concentrations of VDC, and liver GSH levels as a function of dose and route of administration. The model also explains the complex dose-response mortality curves seen with VDC. Because of the low blood:air partition coefficient of VDC and its saturable metabolism, the amount of VDC dose that is metabolized is sensitive to the rate of absorption. After an intravenous bolus dose, most ofthe administered VDC is exhaled unchanged within a few min utes. Blood VDC half-life is not representative of metabolism rates but to reequilibration of VDC from fat. Rats with greater fat content, therefore, display longer VDC blood half-lives. Simulations are shown to demonstrate the strength of PB-PK modeling techniques in under standing the kinetic behavior of VDC in the rat under a variety of experimental conditions. 1988 Academic Press, loc. Mathematical modeling of physiologic sys tems to determine the kinetic behavior of compounds, commonly known as physiolog ically based pharmacokinetic (PB-PK.) mod eling, has recently been introduced in the area of cancer risk assessment. The utility of this type of modeling has been demonstrated in explaining differences in cancer bioassay data after oral and inhalation exposure to methylene chloride (Andersen et al., 1987) and in improving the process of estimating cancer risk for low level exposure to methyl ene chloride (Andersen et al,, 1987) and eth ylene dichloride (D'Souza et al., 1987). This modeling can also be used to explain or pre dict potential for toxicities other than cancer. To illustrate this utility a PB-PK model has been developed for vinylidene chloride (VDC; 1,1-dichloroethylene) in the rat. The model demonstrates the manner in which the interaction of various, often nonlinear pro cesses, can affect both the pharmacokinetics and toxic potential of VDC. VDC is a large volume chemical that is an air and drinking-water contaminant (U.S. EPA, 1982, 1986). VDC has been shown to be a potent hepatotoxin (Jenkins et al, 1972; Reynolds et al., 1975; Andersen et al., 1979) and is also a suspected carcinogen (Maltoni et al, 1977). The metabolism of VDC has been well studied (Henschler, 1977; Leibman and Ortiz, 1977). VDC is metabolized by mixed- 004I-008X/88 $3.00 Copyright <> 1988 by Academic Press, Inc. All rights of reproduction in any form reserved. SL 40454 functioi ide is ui reactive tyl chlo therdet macron toadih expir dose is; reactivi ent VC hepatot 1977, 1 lism, a affectec level, ri dosing VDC sponse plex, in ppm ir ing viri for a 4al,, 19' kinetic been o dose a McKei Kenna intrave peritoT of exp (1977, of cov VDC-' ter ora crease inhala are dei tive to theref (Jaege 1977) tratioi ery ve atoto> dosin' with ' ation ofchemical reaci. Eng. Symp. Ser. No. I. J., Gargas, M. L., H., (1987) Physiologind the risk assessment . Toxicol. Appl. Phar- and Ramsey, J. c. aetics: Evaluating systabolism, and the time inhaled styrene in rats r concentration ratios. 76-187. -ION, A. B., AND ARMweights of the Rat. I. ssection and chemical led. 91,122-126. . W. (1981). Structure ideal model ofthe disinhaled anaesthetics. ral pharmacokinetics. dSME, September, P. G. (1983). Tetrassue distribution in inking-water adminol. 69,66-72. Mathematical model'ombust. Toxicol. 3, M. E,, (1988) A gas 'Jie kinetic constants M. Toxicologist, 8, ; potency of various "dative to their narToxicol. Appl. Phar- I., (1979). A review ly based pharmaconet. Biopharm. 7, Nation Protection >n Reference Man, ' et al., Eds). Perga- ews, H. B., Eung, 77). A preliminary chlorinated biphe' S, 386-396. * CCoNNELL, E. E,, sions in mice after hloroethane. Lab METHYLCHLOROFORM PHARMACOKINETICS 199 National Academy of Sciences (1986). Dose route ex trapolations: Usingftnhalation toxicity data to set drinking water limits. In Drinking Water and Health Vol. 6, Chap. 6, pp. 185-236. National Academy Press, Washington, DC. National Academy ofSciences (1987). Proceedings ofthe Pharmacokinetics in Risk Assessment Workshop, Washington DC, October 7-9,1986. National Acad emy Press, Washington, DC. \ Nolan, R. J., Freshour, N. L., Rick>Q. L., Mc Carty, L. P., and Saunders, J. H. (1984)^Kinetics and metabolism of inhaled methylchloroform (C~t trichloroethane) in pale volunteers. Fundam. Appl ' Toxicol. 4,654-662.! Quast, J. F., Calhoun, L. L., and McKenna, M.> (1984). Chloroethena VG. A chronic inhalation toxi cologic and oncogenic study in rats and mice. Rut I. Results of findings in Alice. Toxicologist, 5,14./ Rampy, L. W,, Quast,U. F,, Leong, B. L/j., and Gehring, P. J., (1978l Results oflong-teym inhala tion studies on rats of 1.1.1 -trichloroethylene and perchloroethylene formulation. In Proceedings ofthe 1st International Congress on Toxicology(G. L, Plaa and W. A. M. Duncan, Eds.). Academic Press, New York. Ramsey, J. R,, and Andersen, M.&(1984). A physio logically based description of the Inhalation pharma cokinetics of styrene in rats and humans. Toxicol. Appl. Pharmacol. 73, 159--tyS. Sato, A., and Nakahma, Tl (1979) Partition coeffi cients of some aromatic hydrocarbons and ketones in water, blood, arid oil. Brit. J. Ind. Med. 36,231-234. Schumann, a/M., Fox, T. R., and Watanabe, P. G. (1982a). (/*C]Methylchloroform (1,1,1-trichloroeth ane): Pharmacokinetics in rats and mice following in- halatiojr exposure. Toxicol. Appl. Pharmacol. 62,390- 401. / I Schumann, A. M., Fox, T. R., and Watanabe, P. G. (1^82b). A comparison of the fate of inhaled methylilorofonn (1,1,1 -trichloroethane) following single or peated exposure in rats and mice. Fundam. Appl. Toxicol. 2,27-32. Teorell, T. (1937) Kinetics distribution of substances ministered to: the body. Arch. Im. Pharmacodyn. Ther^l, 205-240. TorkelsOn^T. R-i and Rowe, V. K. (1981). Halogenated alipnhtic hydrocarbons containing chlorine, bromine, and iodine. In Patty's Industrial Hygiene and Toxicology (Q. D^Payton and E. Clayton, Eds.), 3rd cd,, pp. 3502-1510. Wil^y, New York. U.S. Environmental! ProtectioiN^gency (1983). Draft HealthAssessmentDocumentfor 1,1,1-Trichloroethyl ene (Methylchlorofyrm). Section 3, p. 29. Office of Health and Enviroi ttal Assessment, Research Tri- angle Park, NC 277 1. Withey, J. R,, Coi B. T., and Collins, P, G., (1982) Effect of vel le on the pharmacokinetics and uptake of four hali mated hydrocarbons from the gastrointestinal tract >f the rat / Appl. Toxicol. 3, 313-332. SL PB-PK MODEL FOR VINYLIDINE CHLORIDE 231 function oxidases into an epoxide. This epox ide is unstable and can decompose to highly reactive intermediates, primarily chloroacetyl chloride (CAC). The intermediates are ei ther detoxified by glutathione (GSH), bind to macromolecules, or are further metabolized to adihydrodiol, resulting in C02 elimination in expired air. A portion of unchanged VDC dose is also eliminated in exhaled air. It is the reactive intermediates like CAC, and not par ent VDC, that is responsible for the acute hepatotoxic effects of VDC (Andersen et ai, 1977, 1978; Henschler, 1977). The metabo lism, and hence the toxicity, of VDC is affected by several factors. These include dose level, route ofexposure, GSH status, and oral dosing vehicle employed. VDC metabolism is saturable and dose-re sponse curves for mortality in rats are com plex, increasing sharply between 100 and 200 ppm inhalation concentration, but remain ing virtually flat between 200 and 1000 ppm for a 4-hr inhalation exposures (Andersen et al., 1979). Large differences in the pharmaco kinetics and metabolic fate of VDC have been observed with an increase in exposure dose after oral (Jones and Hathway, 1978; McKenna et al., 1978a), inhalation (Mc Kenna et ai, 1978b; Andersen et al., 1979), intravenous (Putcha et al., 1986), and intraperitoneal (Jones and Hathway, 1978) routes of exposure. Furthermore, McKenna et al. (1977, 1978a,b) have shown that the amount of covalent binding, VDC exhalation, and VDC-related l4C02 exhalation is different af ter oral and inhalation exposures, and an in crease in dose has different effects for oral and inhalation exposures. Since VDC metabolites are detoxified by GSH, VDC toxicity is sensi tive to liver GSH concentration. Toxicity is, therefore, affected by such factors as fasting (Jaeger et al., 1974; Andersen and Jenkins, 1977) and diurnal variation in GSH concen trations (Jaeger et ai, 1973). Effects of deliv ery vehicle on the pharmacokinetics and hepatotoxicity have also been observed after oral dosing. Cheico et al. (1981) dosed rats orally with VDC in com oil, mineral oil, and an TABLEi Parameters Used in the VDC PB-PK Model Partition coefficients Liverblood Richly perfused:blood Slowly perfused:blood Fat.'blood Blood:air 1.1 1.1 0.6 18.4 5.0 Kinetic constants ^TM(mghr-') Km (mg liter*1) K^a OiM-1 hr-1) k.(hr1) a:,TM (hr1) Acoj(m_1 hr ') HjO(m) 2.6 0.25 0.33 50 9000 1.82 x 10 55 aqueous Tween vehicle and observed that hepatotoxicity was greater with vehicles like com oil and mineral oil from which absorp tion is expected to be slow compared with Tween from which absorption ofVDC would be more rapid. They also observed that the half-life of exhaled VDC was more prolonged after com oil and mineral oil, compared to Tween vehicle. Our purpose was to develop a physiologi cally based pharmacokinetic model for VDC in the rat that could be used to explain the changes in VDC behavior seen with the above factors and could also predict the phar macokinetic behavior and toxic potential of VDC in untested conditions. METHODS PB-PKModel Development The basic structure of the PB-PK model is similar to that developed by Ramsey and Andersen (1984) for sty rene, utilizing a lumped-tissue group approach. Com partments are the "richly perfused" group comprising such tissues as the lung, kidney, and spleen; the "slowly perfused" group, comprising muscle and skin; and a fat compartment. Metabolism was assumed to take place solely in the liver. The various parameters that were used in developing the PB-PK model are shown below. Partition coefficients. Partition coefficients for blood; air and tissue:blood (Table l) were determined by the SL 040456 232 D'SOUZA AND ANDERSEN method of Sato and Nakajima (1979). Briefly, tissues were homogenized with normal saline using a Polytron homogenizer. Homogenates were spiked with low levels ofVDC and allowed to equilibrate in sealed reaction ves sels at 37*C in a water bath for I -3 hr. A sample of the headspace vapor above the homogenate was then ana lyzed for VDC content. By appropriate mathematical corrections, the tissue:air partition coefficient was com puted. Tissuerblood partition coefficient was obtained by dividing the tissue:air partition coefficient by the blood: air partition coefficient. Experience with partition co efficient measurements of over 30 chemicals in one of our (M.E.A.) laboratories has indicated that reaction of chemical with tissue homogenate does not typically take place. This may be due to the fact that cofactors, oxygen, etc., are not supplied, and also because the tissues are not handled in a particularly gentle manner. As a rule, two to three measurements are made a few hours apart. Ifthe results are identical, this indicates to us that metabolism is not occurring. Physiologic parameters. Values for organ volume and blood flow were taken from Gargas etai.il986). Modeling VDCmetabolism. The pathway ofVDC me tabolism (Scheme I) contained a single activation step. Metabolism rate constants for conversion ofVDC to the epoxide (F,,, and Km, Table 1) were determined by gas uptake studies (Gargas et al., 1986). Metabolism was found to be saturable, as previously reported in the litera ture (Andersen et al., 1979). It was assumed that VDC epoxide convened almost instantaneously to CAC or re acted with water to form C02 as the end product. The reaction ofepoxide with water resulting in COj was mod eled similar to that reported by Dekant et al. (1984) for trichloroethylene oxide. Based on the data of McKenna et al. (1978), where 10% of radiolabel was recovered as CO2 in expired air after a 1 mg/kg oral dose ofradiolabel VDC, these two reactions were split in a 9:1 ratio (epox ide to CAC or CO2, respectively). In order to simulate virtually instantaneous reactions while maintaining the 9:1 ratio, the first-order rate constant for conversion of epoxide to CAC (A|,,,) was arbitrarily set to 9000 and the second-order reaction rate constant of epoxide with water (Aco, 55 m water in liver) was therefore 1000, re sulting in a value of 1.82 10-5 for Kco, (Table 1). Cl,C=CH, V-*,i^=. jo,c--bnj [ochJL] H> \ MAT GCM Precursors ;\ GSH Synthetase ' Precursors Breakdown ,, Products - GSH Breakdown Products [oCH^Cl] / Detoxified Products Scheme II. GSH synthesis and depletion by chloroacetyl chloride. For simplicity, it was assumed that the reaction with GSH was only with CAC and not with VDC epoxide or other intermediates. This assumption is justified based on the data of Liebler et al. (1985) where CAC reacted with model thiols (in vitro) I03 times faster than VDC epoxide and 103 times faster than minor metabolites formed from the epoxide. The relative contribution of the GSH pathway in detoxifying CAC, compared to the ability ofthis intermediate to elicit toxicity by binding to macromolecules (metabolite available for toxicity, MAT), was modeled as two first-order processes: reaction ofCAC with GSH (rate constant A,,) and reaction with "everythingelse" (rateconstant A*.). Values for and (Table 1) were obtained by using the PB-PK model for VDC developed until this level and varying (trial and error) until GSH depletion model predictions were a visual fit to GSH depletion data reported by McKenna et al. (1977), for inhalation exposures to increasing VDC concentrations. Starting values for K^, and A** were ap proximated based on previous experience with modeling GSH conjugation with reactive metabolites (Andersen et al., 1986; D'Souza etal., 1987,1988). Modeling GSHturnover. The model for GSH turnover and reaction with CAC (Scheme II) is similar to that re ported for GSH depletion by ethylene dichloride (D'Souza et al., 1988). It is somewhat simpler in that, unlike ethylene dichloride, parent VDC does not conju gate with GSH. Except for K^, and A**, the other rate constants are similar to those utilized by D'Souza et al. (1988). Mathematical construction of model. Mass-balance differential equations were written for the various com partments in the model (Appendix A). Briefly, for non metabolizing compartments differential equations were written to describe the influx, binding, and efflux of VDC. The lung compartment was described similarly, but included a description of gas exchange between the lung and the ambient air. The equations for the liver compartment also accounted for VDC metabolism and GSH turnover. These equations were solved simulta neously by numerical integration, using Gear's algo rithm for stiff systems. The ACSL1 computer program CO, Scheme I. Model for VDC metabolism pathways. 1 Advanced Continuous Simulations Language, Mitchell and Gauthier, Concord, MA. o 10' 10" Fig. VDC c< mg a sir centrati a single "richly (Data & was en these e< Anir were pi and to edat lm t abolis 1986) ments tionsr sourer taken the da 040A57 SL uwkdown loxiftod aducts l depletion by chloro- that the reaction with with VDC epoxide or >tion is justified based >) where CAC reacted mes faster than VDC in minor metabolites lative contribution of AC, compared to the toxicity by binding to ailable for toxicity, der processes; reaction and reaction with ,,). Values for and ing the PB-PK model and varying (trial odel predictions were reported by McKenna res to increasing VDC and were apsrience with modeling lites (Andersen et del for GSH turnover I) is similar to that re ethylene dichloride what simpler in that, VDC does not conjuid Kt,*, the other rate zed by D'Souza et al. model. Mass-balance for the various com\ A). Briefly, for nonential equations were inding, and efflux of is described similarly, exchange between the quations for the liver ^DC metabolism and were solved sirpultai. using Gear's algocomputer program mlations Language, 1A. PB-PK MODEL FOR VINYLIDINE CHLORIDE 233 Fig. 1. Model predicted (--) and observed (symbols) VDC concentrations in different tissues ofthe rat follow ing a single SO mg/kg iv bolus dose. Predicted VDC con centrations for blood, liver, lung, and brain are shown as a single curve, as tis$ue:blood partition coefficient for the "richly perfused" tissue group was approximately one. (Data source: D'Souza, 1984). was employed on an IBM PC-AT computer to solve these equations. Animal studies. Although some experimental studies were performed to validate various portions ofthe model and to demonstrate predictability ofthe model, much of the data utilized to confirm model predictions were taken from the literature. Because the literature for VDC me tabolism and acute toxicity is quite extensive (U.S. EPA, 1986) it was unnecessary to repeat many of the experi ments. In all the figures, curves are PB-PK model predic tions and symbols are experimental data. When the data source is acknowledged and referenced, the data were taken from the literature. When no reference is made, the data were collected by the authors for this study. difference in VDC concentrations was ex pected between this group of tissues and blood. Therefore, model prediction is shown as a single curve for the richly perfused tis sues. Simulated and experimentally deter mined fat concentrations are also shown. As predicted, VDC concentrations in fat are considerably higher than concentrations in other tissues. The PB-PK model appears to predict VDC biodistribution in these iv dos ing studies. Dose level and route-of-exposure effects. The model was used to gain insight into the disposition ofVDC as a function ofdose level and route ofexposure (Fig. 2). Because ofsat uration of VDC metabolism, there is a de crease in the percentage of dose that is con verted to the epoxide as dose is increased. The percentage ofVDC dose that reacts with GSH (glutathione-conjugated metabolite, GCM), or is eventually exhaled as CO2, decreases with increasing dose (Fig. 2). This decrease begins at about a 50 mg/kg dose of VDC, in dicating the dose where saturation of the oxi dative pathway occurs. Although a decrease is seen in the parameters noted above, be cause ofthe complex interaction between the processes of absorption, metabolism and GSH conjugation, the relative decreases in these parameters do not run parallel. At the RESULTS Once the PB-PK model was constructed, simulations could be generated to predict the disposition of VDC, its metabolic products, or liver GSH. In order to validate the model, predictions were generated for blood and tis sue VDC concentration-time profiles after an iv bolus dose. These predictions were con firmed with experimental data (Fig. 1). Since the tissue:blood partition coefficient for the richly perfused group of tissues was 1.1, no Oral Dose (mg/kg) Fig. 2. Model simulations (--) and observed data for the effect of oral dose level on percentage of VDC dose that is exhaled unchanged (O), exhaled as CO? (), or GCM (glutathione-conjugated metabolite (no observed data)). K. of 1.0 hr'1 used in simulations. (Data source: 1 and SO mg/kg doses, McKenna et al.. 1978a; 350 mg/ kg dose, Jones and Hathway, 1978.) SL 040458 234 D'SQUZA AND ANDERSEN Inhalation Concentration (ppm) Fig. 3. Model simulations (--) for the effect of inhala tion dose level (6-hr exposures) on body burden () and amount of dose exhaled as C02 (O). (Data source: Mc Kenna etai., 1978b.) point where metabolism is saturated, an in creasing percentage of absorbed VDC dose is also exhaled in the breath (Fig. 2). Experi mental data for exhaled VDC and 0O2 are also shown to confirm model predictions. Once again, model predictions agree quite well with reported data. A series of simulations were performed to study the effects of inhalation exposures (6 hr) on the disposition of VDC (Fig. 3). After inhalation exposures an apparent linear rela tionship exists between exposure concentra tion and the parameters shown, up to about 200 ppm concentration. At concentrations exceeding 200 ppm, VDC metabolism is sat urated and the model predicts a less than pro portional increase in the parameters shown with an increase in exposure concentration. Data from the literature (McKenna et al., 1978) for body burden and C02 exhalation (Fig. 3) are shown along with the simulations. Saturation of VDC metabolism at about 200 ppm concentration has also been confirmed by Dallas et al. (1983). GSH depletion effects. Figure 4 illustrates the effects of inhalation (Figs. 4a and 4b) and oral (Fig. 4c) VDC exposures on liver GSH levels. Experimentally measured liver GSH concentrations for inhalation (McKenna et al., 1977; Reynolds et al., 1980) and oral ex posures agree quite well with model predicted values. As GSH levels are inversely related to toxicity, prediction ofthe time course ofGSH depletion indirectly allows for the estimation of toxic potential. Absorption rate effects. While simulating various aspects of the behavior of VDC, sev eral observations were made that would not be readily noticed without the PB-PK model. For instance, because of the low bloodrair partition coefficient and saturable metabo lism of VDC, its disposition is sensitive to the rate of entry into the body. Figure 5 depicts the effects of increasing absorption rates on the percentage of VDC dose that will be ex haled unchanged at different oral dose levels. These simulations were performed for doses ranging from I to 200 mg/kg. Experience in studying the oral absorption rates of halogenated hydrocarbons (data not shown) sug gests that the absorption rate constant for these compounds from aqueous vehicles, like Tween/water mixtures, is typically 5.0 hr-1, while the rate constant from com oil vehicle is typically 1.0 hr-1. As absorption rate in creases, particularly at higher doses, a greater percentage of VDC dose is exhaled un changed. The toxicological implications of this observation are obvious. That is, greater toxicity will be seen if com oil is used as the oral delivery vehicle compared to aqueous vehicles. Data collected by Cheico et al. (1981), where greater hepatotoxicity was seen with com oil and mineral oil vehicles com pared to a Tween vehicle, are consistent with this finding. Pharmacokinetic data in the lit erature also confirm this observation. Mc Kenna et al. (1978a) noted that from a com oil vehicle approximately 3 and 20% of the oral doses of 1 and 50 mg/kg, respectively, were exhaled. Our model predicts similar re sults ifan absorption rate constant of0.5-1.0 hr"1 is used. Similarly, the model predicts data reported by Jones and Hathway (1978) where 0.6 and 62% were exhaled after oral and 11 and 90% were exhaled after intraperitoneal doses of 0.5 and 350 mg/kg, respec tively, (ka of 10 hr-1 was used to simulate rapid absorption for the intraperitoneal doses; simulations not shown). SL 040459 PB-PK MODEL FOR VINYLIDINE CHLORIDE 235 Fig. 4. Predicted (--) and observed () liver GSH concentrations following inhalation (a and b) and oral (c)exposure to VDC. (Datasource: (a) McKennaet at., 1977; (b) Reynoldsetal., 1980.) Intravenous dosingstudies. The model also predicts that after a bolus intravenous dose of VDC, because ofthe rapid input ofVDC into the body, most ofthe dose will be exhaled un changed within the first few minutes (simula tions not shown). This prediction can be con firmed by studying the data reported by Jones and Hathway (1978), where 80% of an intra venous dose was exhaled after a low 0.5 mg/ kg intravenous dose. Furthermore, if VDC metabolism was partially blocked or induced Flo. 5- Simulated effect of oral absorption rate con stant on the percentage ofVDC dose exhaled unchanged at various dose levels. It can be observed that the effect is negligible at low doses, very dramatic at the intermediate doses, and minor at doses of 200 mg/kg, where virtually all of the dose is exhaled, regardless of the absorption rate. This prediction suggests that VDC toxicity at say 50 mg/kg dose will be much greater with com oil vehicle (absorption rate constant 0.5-2 hr"1) compared to an aqueous vehicle (absorption rate constant 3-6 hr-1). (Fig. 6a) no change in blood VDC half-life would be seen after an iv bolus dose. A sim ilar simulation was performed for oral doses with absorption rate constants of 1 and 5 hr-1 to illustrate the effect of absorption rate on blood VDC kinetics. As absorption rate decreases, metabolism differences due to changes in V-- become more apparent. These observations suggest that unlike stud ies with nonvolatile compounds, intravenous bolus dosing has limited utility when study ing the pharmacokinetic behavior or metabo lism of volatile materials for which exposure from the environment is not intravenous. Body-fat effects. PB-PK model simulations suggested that VDC blood half-life was due to reequilibration of VDC from fat depots back into the blood. Therefore, an increase in body fat would be expected to cause an increase in blood VDC half-life. Simulations were per formed to determine blood VDC half-life in rats ofthree body-weight levels, 180,300, and 600 g, and the simulations compared to liter ature data (D'Souza, 1984) to confirm predic tions (Fig. 7). The only parameter that was changed when performing these simulations was percentage of body fat. For the three body-weight groups, body fat was set at 7% for the 180-g group, 12% for the 300-g group, and 22% for the 600-g group (Zucker and Zucker, 1963). Effect ofdose level on mortality. Andersen et al. (1979) showed an interesting dose-re sponse relationship between inhaled VDC dose level and mortality in rats. They noted a very sharp increase in mortality, from 0 to SL OA0460 236 D'SOUZA AND ANDERSEN e Time (hour) Time (hour) Fig. 6. Simulated blood VDC concentrations following intravenous bolus (a), and oral (b and c) doses. These simulations demonstrate that after intravenous bolus doses effects ofenzyme inhibition or induction will not be observed from studying blood VDC levels. These effects will manifest themselves in blood VDC levels after oral dosing. The slower the absorption rate constant, the more pronounced the effect. 50%, when inhalation exposure concentra tion for a 4-hr exposure was increased from 100 to 200 ppm. However, from 200 to 1000 ppm there was virtually no increase in per centage mortality. The PB-PK model was used to study the relationship between inha lation exposure concentration and MAT. This relationship was then plotted on the dose-response plot of Andersen et al. (1979). The result is shown in Fig. 8. It is clear from this plot that the dose-response relationship for VDC is similar to the model predicted dose-MAT relationship. It can be seen that 50% mortality is achieved when about 12 pmol MAT is produced. Using this kind of information, mortality can be predicted for, say, other routes of intake. That is, regardless of the VDC dose and exposure route, 50% mortality is predicted when 12 pmol MAT is formed. Preliminary extrapolation to humans. Al though none ofthe parameters for construct ing a PB-PK model were obtained in hu mans, a preliminary attempt was made to ex trapolate the model to humans. The PB-PK model for the rat was scaled to the human by correcting for known physiologic differences between the rat and human, and by estimat ing VDC metabolism rates in humans based on allometric scaling principles (Dcdrick et al.. 1973). In extrapolation to the human. Time (hour) Fig. 7. Predicted (--) and observed (symbols) effects ofbody weight (body fat) on VDC blood concentrations in rats of different body-weight levels following a single 50 mg/kg iv dose. (Data source: D'Souza 1984). Concentration (ppm) Fig. 8. Predicted (--) metabolite available for toxicity (MAT, chloroacetyl chloride uot detoxified by glutathi one (see text for more detailed explanation) and mortal ity dose response for 4-hr VDC inhalation exposures. (Data source: Andersen et al., 1979.) SL 040461 * On* ka 1.0 2.67 4.00 to (hour) al (b and c) doses, iition or induction i ves in blood VDC ie effect Using this kind of i be predicted for, That is, regardless xwure route, 50% i 12 fitnol MAT is >n to humans. Al ters for constructobtained in hu rt was made to exnans. The PB-PK d to the human by ^ftpgic differences ^Fand by estimatin humans based :iples (Dedrick et n to the human, 25 20 3 O 15 I 10 5 5 0 750 1000 ">> available for toxicity etoxified by glutathianation) and mortalnhalation exposures. ) PB-PK MODEL FOR VINYLIDINE CHLORIDE 237 cardiac output and pulmonary ventilation was scaled as a function of body weight (BW) to the 0.7 power, l/miUl as BW to the 0.74 power, and body fat was changed from 7% for a 200 g rat to 20% for a 70-kg human. This type of scaling has been successfully used in extrapolating animal data to the human for halogenated hydrocarbons using PB-PK models (Gargas et al., 1986; Andersen et al., 1987; D'Souza et al., 1987). Figure 9 displays the relationship between VDC exposure dose level and the amount of epoxide formed for the rat and human. This simulation was con ducted for both oral (Fig. 9a) and inhalation (Fig. 9b) exposures. Although in previous simulations MAT was used as the "internal dose," in this case, because humans are un likely to be exposed to VDC concentrations that would decrease liver GSH, the shape of the curves for MAT and epoxide (versus VDC dose) are expected to be similar, and therefore epoxide is being used. Additionally, the use of epoxide serves to minimize the number of rate constants to be scaled from rat to human. It can be seen that saturation effects in VDC metabolism begin at about 10 mg/kg for oral, and 200 ppm for inhalation exposures for both the rat and human. Also, the amount ofepoxide formed is predicted to be lower in the human compared to the rat at equivalent mg/kg oral or ppm inhalation concentrations. DISCUSSION The PB-PK model developed for VDC demonstrates the complex interplay of the many factors that contribute to the kinetic behavior and subsequently the toxicity of the compound. In terms of metabolism and pharmacokinetics at least, data from the liter ature appear to be different from one study to the other and at times appear to contradict each other when only minor differences in ex perimental design exist. The PB-PK model, however, demonstrates that given the physi cal, chemical, and biochemical properties of Fig. 9. Simulated amount of VDC epoxide produced at different (a) oral and (b) inhalation dose levels in the rat and human. After oral exposure saturation effects in VDC metabolism are expected in both rat and human at doses of 10 mg/kg, while after inhalation exposure satu ration is predicted after about 200 ppm (6-hr) exposures. VDC, such differences are expected and can be predicted. For example, VDC metabolism profile, in terms of percentage VDC exhaled, percentage metabolized and percentage con jugated with glutathione is different for different routes of exposure and dose levels as reported by McKenna et al. (1978a,b) and Jones and Hathway (1978). It appears that slightly different experimental conditions can have large effects on the kinetic behavior of VDC. For example, Putcha et al. (1986) have shown that although ether anesthetic used in their experiments may have had potential to inhibit VDC metabolism, no differences were seen in the kinetic behavior of VDC when ether-treated and untreated rats were com pared after intravenous bolus injections of 10 mg/kg. The PB-PK model was used to study such a situation and simulations indicate that with intravenous bolus dosing no differences will be seen in VDC blood kinetics even if metabolism is blocked. Also, Putcha et al. (1986) noted a slight increase in the blood clearance ofVDC with an increase in intrave nous dose, while most of the other studies suggest a decrease in clearance because of sat uration of metabolism. Putcha et al. (1986) calculated clearance from the area under the blood VDC time plot from the first time point (2-3 min postdose) to infinity. The PB-PK model demonstrates that for an intravenous dose most ofthe injected dose will be exhaled within the first few minutes, and, therefore. SL 040462 238 D'SOUZA AND ANDERSEN under these experimental conditions VDC blood clearance will not show a decrease in clearance with increasing dose, but may actu ally show an increase, depending on when the first sample was taken (simulations not shown). Similarly, the effect of com oil vehi cle on hepatotoxicity (Cheico el al., 1981) can also be readily explained on the basis of different rates of absorption of VDC from various dosing vehicles. The purpose of obtaining animal kinetic data is to help in predicting the risk ofchemi cals to humans under realistic exposure situa tions. Because of the extreme sensitivity of VDC kinetics to these various factors, any model that is used to predict risk of VDC ex posure to humans must be sensitive to these factors in order to meaningfully extrapolate the animal data to humans. A model that is restricted to extrapolation within a narrow data base cannot offer insight in untested conditions and, therefore, would have lim ited utility in extrapolation of animal data to the human. Scaling ofthe PB-PK model from the rat to the human presented here serves mainly the purpose ofillustrating the manner in which these models can be used to extrapo late dose and toxicity information from ani mal to human for risk assessment. The PBPK model for the human has parameters (V__and Km, for example) that were not ob tained or verified in the human, nor have the final blood VDC concentrations in the hu man been validated by experimental data. In summary, the metabolism and pharma cokinetics of VDC is complex and is affected by many factors. A composite model, physio logically realistic in design, has been devel oped to explain the kinetic behavior of VDC, predict untested situations, and help assess human risk to VDC exposure. APPENDIX A Mass Balance Equationsfor VDC Model Mass balance differential equations for lung uptake of compound and for influx and efflux in noneliminating compartments were] similar to those for methylene chloride (An-1 dersen et al,, 1987), while equations for] glutathione synthesis and depletion can be] found in D'Souza et al. (1988). Differen tial equations describing VDC metabolism,! which are unique to this model, are shown < below. = (PTM*CVL)/Km + CVL) ^ammM = dAM/dt -- dACoJd, -- d^cAc/d, dACoJd, = Kcq^CMMiHiOaVL dACAc/d, - Kiam * AMM ^acacmM " dKCac ~~ ^amatM -- docu/d, ^amatM = Xfce*ACACM doaJd, = A^,,, * GSH * CCACM * VL APPENDIX B Nomenclature ACAC Amount of chloroacetyl chloride formed. ACACM Amount of chloroacetyl chloride present at time, t. AC02 Amount ofCO2 formed. AM Amount ofepoxide formed. AMAT Amount of metabolite available for toxicity. AMM Amount of epoxide present at time, t. AGCM Amount of glutathione-conju gated metabolite. CCACM Concentration of chloroacetyl chloride at time, t. CMM Concentration ofepoxide at time, t. d/d, Differential. GCM Glutathione-conjugated metabo lite. GSH Glutathione concentration. H20 Water concentration. Acoj Rate constant for reaction of ep oxide with water. SL 040463 MAT Vtamx ANDERSEN, M ANDCONOL kinetic mod by inhaled 1 148_ stISKf- ` cally based process for macol. 87,' ANDERSEN, t Jenkins, L acute toxic Pharmacol Andersen, I toxicity of sex, age a 157-167. Andersen, 1 (1978). Th dichloroet tion and i on mortal: Cheico, P-, (1981). Ef chloroeth 146-155. Dallas, C. andBru tion of l, exposure impairments were lene chloride (Anhile equations for i depletion can be (1988). DiiferenVDC metabolism, model, are shown ] )/Km + CVL) oo~ dACAc/dt i*H20*VL atId, -- VI CCACM * VL :B ire >roacetyl chloride i 5acetyl chloride 2,/. formed, ide formed, abolite available >xide present at itathione-conjute. of chloroacetyl '1 t epoxide at time, ugated metffbo- entration. ion. reaction of ep- r. PB-PK MODEL FOR VINYUDINE CHLORIDE 239 Xf K\am Km Kq Kty Ki K\' MAT Knu* First-order rate constant for for mation of MAT. First-order rate constant for for mation ofGOM. First-order rate constant for chlo roacetyl chloride formation. Michaelis constant for oxidative pathway. Zero-order glutathione synthesis, time and GSH dependent. Glutathione synthetase forma tion. First-order rate constant for gluta thione breakdown. First-order rate constant for gluta thione synthetase breakdown. Metabolite available for toxicity. Maximum velocity of oxidative pathway. REFERENCES ANDERSEN, M. E., Clewell, H. J., Ill, Gargas, M. L., and Conolly, R. B. (1986). 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Nutr. 80,6-19. 2 iXICJH-O tier 1*6^ 0* sv Huma (DBE) fruit ft knock be mu et al,, tal ani metab ceeds tomi, chroir 'Thi.' Occupa 2To'