Document mmmMdgj43XQO58vJRwEbRn37Q

r &S 107163 BIO-MEDICAL RESEARCH DOCUMENT DESCRIPTION FORM * 63 68 69 76 Duplicate in all cards:--> year as-1961- ; :Q#$0107 F:Jle number {Right justify [Numeric only] Author(s), as Last Name FS (No Punctuation) and coden for journal as JAMA preceeded -by. one blank space r'ft f 5* e. J-M 20 -21 1W. -- 40 41 \ 77 78 Vc^ 1 Sub-Index Code 60 61 62 11 12 13 Title of Report: end with space-hyphen-hyphen-space. Follow with Index Terms, seoarated from each other with -comma-soace. Avoid other mmf'tnnMcm- * do not abbreviate. 12 61 '62 :_________________________________ 21 ~t~^h<Krr^G. k i'n rcS *'O'T //c _ 4-' ^v hy / 22 ^ /' _______________ 23 24 Source (Journal, Vol., Number, Pages, Date) 12 ftrdi O n i C a /- ........... ... yi. 04,. '>rt~ rbC. ~f iCf-fc, 61 62. 31 32 Brief Summary- 61 62 61 62 63 64 Arch. Toxicol. 42, 123-136 (1979) TOXICOLOGY Springer-Verlag 1979 0000107 Pharmacokinetics of Halogenated Ethylenes in Rats J. G. Filser and H. M. Bolt Institut fur Toxikologie der Universitat Tubingen, Lothar Meyer-Bau, Wilhelmstrasse 56, D-7400 Tubingen 1, Federal Republic of Germany Abstract. Inhalation pharmacokinetics of the halogenated ethylenes vinyl fluo ride (VF), vinylidene fluoride (VF^), vinyl chloride (VC1), vinylidene chloride (VClj), cis- and trans-dichloroethylene (cis-DCE and trans-DCE), trichloro ethylene (Tri), perchloroethylene (Per), and vinyl bromide (VBr) have been com paratively studied in the rat Rats were exposed in a closed inhalation system to various initial atmospheric levels of halogenated ethylenes, and the decline of atmospheric concentration was followed using gas chromatographic analysis. From pharmacokinetic analysis of the experimental curves the following general patterns of the halogenated ethylenes were derived. Distribution of the compounds in the organism and in the gas phase is determined by physical factors. For practical purposes, a relation of the equilib rium constants with the volatilities of the compounds, expressed by the boiling points, may be used: compounds with a low boiling point are enriched in tissues much less than those of a higher boiling point, and vice versa. Compounds with high accumulation in tissues (Tri, Per) need much more time for completion of the equilibration process than more volatile compounds. Metabolic elimination of halogenated ethylenes is a saturable, dose-dependent process. If animals are exposed to atmospheric concentrations of a haloge nated ethylene which exceed the "point of saturation (Sp)", elimination is deter mined by a zero-order law, i.e., its rate is independent of the concentration of the compound. In contrast, below saturation normal first-order kinetics apply. If the rate of metabolic elimination is related to the concentrations of the compounds in the tissue compartment, very similar rates for first-order elimina tion of the different halogenated ethylenes are found. This suggests a common rate limiting factor applicable for the lower concentration range. The maximal velocities (Kmai) of metabolic elimination of halogenated ethyl enes which are reached above the "saturation points" depend on the chemical structures of the individual compounds. In general, with the exception of Tri, Send offprint requests to: H. M. Bolt at the above address 0340-5761/79/0042/0123/S 02.80 3J 124 J. G. Filser and H. M. Bolt further halogen substitution inhibits metabolic conversion. Of the halogenated ethylenes, VFj and Per are extremely slowly metabolized. The present report also provides the data necessary for calculation of the rates of metabolism of halogenated ethylenes in rats at a given concentration of atmospheric exposure. Key words: Halogenated ethylenes -- Vinyl fluoride -- Vinylidene fluoride -- Vinyl chloride -- Vinylidene chloride -- Cis-dichloroethylene -- Trans-dichloroethylene -- Trichloroethylene -- Perchloroethylene -- Vinyl bromide -- Pharma cokinetics -- Metabolism. Introduction Since the cancerogenic effects of vinyl chloride have been detected many experimen tal studies are concerned with the action of halogenated ethylenes on the living organism. It is now generally recognized that the cancerogenic and hepatotoxic effects of vinyl chloride, and also hepatotoxic effects of compounds related to vinyl chloride, arc not directly elicited by these substances themselves, but by reactive metabolites (cf, Bonse and Henschler, 1976). Gehring and coworkers (1977, 1978) very recently pointed out for vinyl chloride that toxicological dose-response considerations have to take into account the rates by which the compound is metabolically transformed. They also showed that metab olism of vinyl chloride is a saturable process, and that increase in atmospheric vinyl chloride concentrations does not similarly enhance the hepatic tumor rate. The gen eral concept has been inferred that the toxicologically effective dose of a chemical which requires metabolic activation is not identical to the total dose acting upon the organism, but must be calculated from appropriate kinetic and metabolic data, ft For the congeners of vinyl chloride such data are still lacking. The present report describes a pharmacokinetic approach which allows calculation of the rate of metab olism of vinyl fluoride (VF), vinylidene fluoride (VF2), vinyl chloride (VC1), vinyl idene chloride (VClj), cis- and trans-dichloroethylene (cis-DCE and trans-DCE), trichloroethylene (Tri), perchloroethylene (Per), and vinyl bromide (VBr), at a given atmospheric concentration. The considerations extend a previous assessment of the pharmacokinetics of vinyl chloride in the rat (Bolt et al., 1977). Materials and Methods Materials. VC1, VF and VF, were donated by Dynamit-Nobel AG, Troisdorf, Germany. VBr, VClj, trans-DCE and Per were products of Aldrich Europe, Brussels, Belgium. Cis-DCE was manufactured by Ferak, Berlin, and Tri by Merck, Darmstadt. The purity of all the compounds was checked by GLC. If necessary, the compounds were redistilled to achieve a purity of at least 99%. Animals. Male Wistar rats (250 g) of Ivanovas, Kisslegg. Germany, were used for the experiments. In some experiments metabolism of the halogenated ethylenes was inhibited by injecting (i.p.) 150 mg/kg 6-nitro-I,2,3-benzc)thiadiazole (Bolt et a!,, 1976. 1977) or 100 mg/kg dithiocarb (diethyldithiocarbamate, Merck, Darmstadt), 30 min prior to the exposure. Pharmacokinetics of Halogenated Ethylenes in Rats 125 Exposure ofAnimals. Animals were exposed to the volatile halogenated hydrocarbons in a closed all glass system as previously described (Bolt et al., 1976, 1977). The animals were placed into a desicca tor which contained soda lime for CO, absorption and which was connected via a water trap to an oxygen supply. Air samples could be drawn via a teflon-coated rubber septum. After injection of the calculated amount of compound into the gas phase its concentration in the system's atmosphere was determined by GLC in short intervals. Gas-Liquid-Chromatography (GLC). Air samples, drawn from the exposure system, were injected into a 5-ml loop, connected by a 6-port valve with a Varian 1440 gas chromatograph. The column (3 m, 1/8" stainless steel) was filled with Porapak Q (50-80 mesh). Column temperatures varied according to the individual compounds which were to be analyzed. Detector (FID) temperature was set 500 above column temperature. Gas flow rates were: nitrogen. 60 ml/min; hydrogen, 30 ml/min; air, 300 ml/min. Calculations. In general, the computations followed the system previously imployed for assessment of pharmacokinetics of vinyl chloride (Bolt et al., 1977) with the modifications described below in the results section. For the computations a programmed Texas Instruments TI59 calculator, equipped with a PC 100 A printer, was used. Results Decline of Halogenated Hydrocarbon Concentration In the Gas Phase Figures 1--3 show representative examples of declines in our exposure system of atmospheric concentrations of VBr, cis-DCE and trans-DCE. The air volume of the system was 10.3 1; it was occupied by three rats, each of 200--250 g. For analysis. 30 CD O-4 o> a> Fig. 1. Decline of concentration of vinyl bromide (VBr) in the gas phase of a closed exposure system (volume 10.3 I), occupied by three male Wistar rats, each of 200--250 g. Top: logarithmic plot (linear section = first order decline); bottom: linear plot (linear section = zero order decline) 126 J. G. Filser and H. M. Bolt the curves obtained can be divided into three sections. The first phase in which the compound equilibrates between gas phase and the organism takes V, h for VBr (Fig. 1), about 2h for cis-DCE (Fig. 2) and about 1.5 h for trans-DCE (Fig. 3). After the initial equilibration period, a further decline in concentration of the com pounds in the system represents the velocity by which metabolic conversions take place. At higher atmospheric concentrations of the compounds the equilibration phase is followed by a phase of apparent zero-order decline, as visualized in the linear graphs (Figs. 1 and 2). As time proceeds, a third phase is apparent (see Figs. 1 and 2) in which the kinetics now strictly follow a first-order law, as visualized in the logarithmic graphs. Trans-DCE (Fig. 3) is an example for a compound with an only slow metab olism. However, Figure 3 shows that the pharmacokinetic behaviour also of this compound is dose-dependent; it shows different metabolic rates at high and at low concentrations. Also for this compound the experimental values can be described by assuming zero-order kinetics at the higher, and first-order kinetics at the lower con centration range. The same type of analysis as shown for the three compounds in Figure 1--3 has also been performed with VF2, VF, VC1, VC12, Tri and Per. Individual curves for these compounds are not shown in separate figures; they exhibited the same features as those in Figures 1--3. Experiments with the same compound were always re- ppm os-DCE 1000' w- \ 2 4 & 10 12 h ppm eu-DCE 500* 400- j \300- 100- i s 12 Fig. 2. Decline of concentration of cis-dichloroethylene (cis-DCE) in the gas phase of a closed exposure system (volume 10.3 1), occupied by three male Wistar rats, each of 200--250 g. Top: logarithmic plot; bottom: linear plot Pharmacokinetics of Halogenated Ethylene* in Rat* 127 R8tS 107168 Fig. 3. Decline of concentration of transdichloroethylene (trans-DCE) in the gas phase of a closed exposure system (volume 10.3 1), occupied by three male Wistar rats, each of 200--250 g. Top: logarithmic plot; bottom; linear plot of the same experimental values. Two sets of experiments at a higher and a lower concentration range are shown, demonstrating dose-dependent pharmacokinetics 1000- 900- 500 h peated, and the pharmacokinetic parameters derived from different sets of analogous experiments showed always variations of less than 10%. For all compounds the `points of saturation" and the velocities of metabolism (zero and first order kinetics) were established (see below). Equilibration of Halogenated Ethylenes with the Organism The process of equilibration of the halogenated ethylenes between the gas phase and the animal organism was analyzed in rats in which the oxidative metabolic enzymes had been inhibited by previous administration of 6-nitro-l,2,3-benzothiadiazole, as described earlier (Bolt et a!., 1976, 1977). During the course of the present investiga tion it was found that a nearly complete inhibition of metabolism, lasting a few hours, could also be achieved by a single i.p. dose of 100 mg/kg dithiocarb (in H20). Under these conditions the first model of Figure 4 was applied. In contrast to the previous pharmacokinetic approach (Bolt et a!., 1977) the present calculations were done in a more generalizing manner, also taking into account different possible volumes of the gas phase and the tissue compartment. Concentrations of the halo genated compounds, in atmosphere as in organism, were considered both as concen trations by volume. 128 Cptl) k,, Cp|2] J. G. Filscr and H. M. Bolt (atmosphere) .equilibration ; (animal) metabolism inhibited Cpll) , ------ 1 k|2 Vi L----- -- -----' k21 (atmosphere) Cp(2) V2 (animal) b.equilibration and metabolism me a o ism FTg. 4. Pharmacokinetic models for (a) equilibration of atmospheric halogenated ethylenes with the organism in a closed exposure system. Conditions of negligible metabolic turnover or inhibited metabolism; (b) distribution, as in (a), and metabolism. Metabolic elimination may follow zeroorder or first-order kinetics On influx of a volatile compound from the closed gas phase into the animal compartment fCp{l) and Cp(2) of Fig. 41 the concentration in gas phase fol lows: dy _ ku yi dt (1) For the opposite process of efflux from the body into the gas phase the concentra tion in tissues follows = - kn yi dt (2) y, and y2 are the concentrations of the volatile compound in question in the gas phase and in the tissues, respectively. " If we do not consider metabolic processes (conditions of low metabolic turnover or inhibited metabolic enzymes; Fig. 4a) the solution of (1) and (2) is k V, y2(<) = yno) k)j Vi + k2i V! [1 (3) y,(0) = initial concentration in gas phase K, = gas phase volume in the individual experiment V2 = volume of tissue compartment (animal volume) in the individual experi ment. For Vx Vv and accordingly k2, k12, Eq. (3) approximates to ku V, yTM33 yno) --*21 v2 l1 (4) For conditions of equilibrium (t -* ), we obtain kn F/ M-) *= yn) ---rVi ~ yi(o)' Kcq (5) Keq = equilibrium constant. Pharmacokinetics of Halogenated Ethylenes in Rats Table 1. Equilibration of atmospheric halogenated ethylenes with the rat organism, according to model fo Figure 4 a. = equilibrium constant; k|j Ei and kj, V2 = clearance values for the partial processes, see Eqs, (3-7) Compound vf2 VF VC1 VBr VClj trans-DCE cis-DCE Tri Per K* 0.23 0.91 5.3 11.3 7.8 11.5 20 52 105 kn(ml/h) 5 000 4 200 34 700 61 500 11 600 11 300 14 500 13 000 7 200 k,, ki (ml/h) 21 200 4 600 6 600 5 400 1 500 985 730 250 68 129 R&S 107170 In this calculation V2 is the "animal volume" in the particular experiment. For any other "animal volume" (F^) to be considered the concentration y*(t) in the animal compartment, according to (4) and (5), is: , |^1 - e k,` y-'j. y2(i) ~ Keq yi(0) (6) Table 1 shows, for the different compounds examined, the "clearance values" for the partial processes, kt2F, and k21F2, and the "constant of equilibrium" between gas phase and animal compartment, i.e.. Keq ~* k, y, ku Vi (7) The values of Table 1 are derived from experimental determinations of K(q and from the observed first order rate constants k whereby k equals k12 + k21 (see Eq. 3). Metabolic Elimination of Halogenated Ethylenes The linear (zero-order) and logarithmic (first-order) sections of all experimental curves obtained were subjected to regression analyses (lin or log, respectively). The constant velocity for each compound during the linear phase was called "Fm,," and calculated as ^mol/h per kg body weight (Table 2). For the logarithmic section, y0 and Id of the process y = y e~kt (8) were calculated. In every individual regression analysis done r amounted to > 0.99. From k' and the apparent volume of distribution the clearance values for firstorder elimination of all compounds were calculated, expressed as 1/h per kg body 130 J, G. Filser and H. M. Bolt Table 2. First-order and zero-order metabolic elimination of halogenated ethylenes from a closed exposure system: saturation points (Sp) and metabolic rates, expressed as and clearance, based on I kg body weight Compound vf2 VF VC1 VBr VC12 trans-DCE cis-DCE Tri Per Sp (ppm in air) (100) (75) 250 55 150 (15) 20 65 First order clearance liter h - kg body wt 0.29 2.5 U.O 18 17 11 30 77 Zero order pmol h kg body wt 1.1 7 110 40 100 7 25 210 <7 * Because of the very low metabolic elimination of Per, the appropriate values cannot be given Sp-values given in brackets are not observed experimentally, but are obtained by extrapolation (intersection of observed zero-order and first-order declines) weight (Table 2). For most of the compounds (VC1, VBr, VC12, cis-DCE, Tri) the atmospheric concentration point where elimination shifted from zero-order into firstorder kinetics ("saturation point") could directly be taken from the experimental concentration curves (e.g.. Fig. 1 or Fig. 2). However, for those compounds which were slowly eliminated a direct assessment was not possible because of the very prolonged experimental observation periods required. Therefore, with VF, VF2 and trans-DCE different experiments were carried out at higher and at lower concentra tions (e.g., see Fig. 3). From these figures the kinetic parameters for the zero-order and first-order sections were derived. The theoretical "saturation point" (Sp) then was calculated as that point at which dl~-ygk'e~k' dt equals Fmax. It is obtained by (9) Sp = ~r U) The appropriate values for Sp (expressed as ppm of compound in air at which "saturation" occurs) are included into Table 2. However, Per is so extremely slowly metabolized in rats that we were not able to experimentally differentiate between zero and first order kinetics. Hence, the equiva lent data cannot be given for Per (Table 2). Pharmacokinetics of Halogenated Ethylenes in Rats K IbfthAj pM| 131 Fig. 5. Connection between distribution behaviour of halogenated hydrocarbons (equilibration con stant Kt, on the abscissa) and volatilities (boiling points on the ordinate). Results of a log regression analysis are given. The calculated curve is drawn Further Analysis Using the Kinetic Data The behaviour of the volatile compounds during the initial phase of equilibration with the organism is largely determined by Ktq (Eq. 7); the latter depends on the physical properties of the compound in question. Figure 5 shows the connection between volatilities, expressed as the boiling points of the compounds, and Keq,' The curve also includes data for methyl chloroform (1,1,1-trichloroethane) and for CC14 which, in terms of distribution, behave in a manner similar to the halogenated ethyl enes. In order to give an idea how fast equilibrium is achieved, theoretical curves of concentration of the volatile xenobiotics in the tissues were drawn, assuming a con stant concentration of xenobiotic in the atmospheric environment. Metabolic pro cesses were not taken into account. Figures 6 and 7 show the calculated courses of concentrations of the halogenated ethylenes in tissues of rats exposed to a constant concentration of the individual compounds in the air of 100 ppm. VF, VF2, VC1 and VBr reach their equilibrium very rapidly, within 30 min after beginning of exposure. VC12, cis-DCE and trans-DCE take a middle position, and Tri and Per equilibrate so slowly that real steady-state conditions are not reached within an exposure period of 8 h. * The metabolic parameters (Table 2) can be used to plot the metabolic rates against the level of atmospheric exposure. This is shown in Figure 8. It must be stressed that this calculation presupposes adjustment of equilibrium between the compound in the gas phase and in the organism. All the compounds (Fig. 8) show different and apparently unrelated characteristics as far as first and zero order veloc- R&S 107172 132 cone, tn iwu* J. G. Filser and H. M. Bolt 33 e iv i v Fig. 6. Calculated concentrations of VC1. VF, and VFj in the tissue compartinent on exposure to a constant atmospheric level of 100 ppm of the compounds, according to the data of Table 1 Cnc m tissi* Fig. 7. Calculated concentrations of Tri, Per. cis-DCE, trans-DCE, VBr, and VC12 in the tissue com partment on exposure to a constant level of 100 ppm of the compounds, according to the data of Table 1 | 4 Pharmacokinetics of Haiogenated Ethylene* in Rats 133 R&S 107174 Fig. 8. Calculated velocities of metabolic elimination of haiogenated ethylenes by rats at different levels of atmospheric exposure. (O) "Saturation points (Sp)"; transition from first-order to zero-order kinet ics IlllU* vq| Fig. 9. Calculated velocities of metabolic elimination of haiogenated ethylenes by rats at different concentrations of the compounds in the tissue compartment. (O) Transition points (Sp) from first-order to zero order kinetics 134 J. G. Fiber and H. M, Bolt ities of metabolic elimination and the transition points (Sp) between both kinetics are ^concerned. However, the picture completely changes when the (calculated) concen trations of the compounds in the tissue compartment are taken into account (Fig. 9). Based on similar concentrations in tissues, the velocities of first order elimination of all the compounds are similar. The coefficient of correlation for the line of Figure 9 connecting the points of tissue concentration where metabolic elimination shifts from zero to first order law is 0.987. The rates of zero order elimination (very dissimilar among the individual compounds, even if the concentration of the compounds in tissues is consid ered. Discussion Pharmacokinetic Approach Several recent publications have shown that the pharmacokinetic behaviour of halogenated ethylenes is dose-dependent; this phenomenon is due to saturability of the metabolic capacity of the organism (Gehring et al., 1977, 1978; Bolt, 1978; Reichert and Henschler, 1978; McKenna et al., 1978). Therefore we have here separately treated the distribution patterns of the compounds and the velocities of metabolic breakdown. The present system may be used to calculate the amount of an halogenated ethylene which is metabolized during exposure of rats in a toxicological experi ment. From Figure 8a or from the data of Table 2 the metabolized amount of the compound can be taken, according to the underlying first-order or zero-order kinet ics, below or above the point of exposure (Sp) where saturation occurs. However, this calculation presumes an immediate adjustment of an equilibrium between the volatile compound in the air and in the animal compartment. Figures 6 ad 7 show that the error introduced by this assumption is negligible for rapidly quilibrating compounds (VBr, VC1, VF, VF:). It may be tolerable for VCljV cisDCE and trans-DCE. However, as Tri and Per equilibrate with the organism only very slowly, this simple approach cannot be applied to these two particular com pounds. In their recent analysis of the pharmacokinetic behaviour of VC1 in rats Gehring et al. (1978) used a Michaelis-Menten approach to account for the dose-dependent metabolism of VCI. Our calculation differs from theirs in that we assumed strict zero-order and first-order behaviour, respectively. We decided to do so from rea sons of practicability, and because we could in fact describe our experimental curves (e.g., Fig, 1--3) with excellent accuracy by dividing them into linear and logarith mic sections. The r values for the individual lin or log regression analyses amounted always to > 0.99. Both approaches, that of Gehring et al. (1978) and ours, are describing essentially the same patterns, i.e,, dose-dependence and "saturability" of metabolism in vivo of the compounds in question. Biochemical Implications Differences in pharmacokinetics and disposition of the halogenated ethylenes are seen in their distribution behaviour, in the atmospheric concentrations (Sp) of com- R&S 107175 Pharmacokinetics of Halogenated Ethytenes in Rats 135 pounds necessary to achieve saturation of metabolism, and in the individual veloci ties by which metabolism occurs. The first feature, distribution, is apparently dependent on the physical properties of the compounds (Fig. 5) whereas the metabolic parameters are determined by interaction of the compounds with the metabolizing enzymes. An important factor is the different enrichment of halogenated ethylenes in the organism; the same atmo spheric exposure levels of different compounds lead to very dissimilar concentrations at the metabolic site, i.e., to dissimilar "substrate concentrations" in enzymological terms. When comparison of metabolic elimination of the different compounds is based on their concentrations in tissues (Fig. 9) the rates of first order elimination of all the halogenated ethylenes are very similar. This suggests that, at lower concentra tions of the compounds in tissues, a process must limit the rate of metabolic elimina tion which obeys the first order law and is apparently independent of the chemical reactivities (or the reactivities towards the monooxygenase enzyme system) of the compounds. Such a partial process may be transport to the enzymic site. On the other hand, under Vmn conditions (Fig. 9) the enzymic turnover must be ratelimiting; hence, the different substrate qualities of the individual compounds towards the metabolizing enzyme system determine the comparative rates of metabolic elimination. On the basis of studies using the isolated perfused rat liver preparation and of theoretical considerations Bonse et al. (1975) have established a tentative rule for the metabolic behaviour of chlorinated ethylenes. They showed that in general an in verse relation exists between the number of chlorine substituents and the rate of conversion. Our present experiments in vivo confirm this as the Vmax values (Table 2, Fig. 8) decrease in the order VC1 > VC12 > cis- and trans-DCE > Per. However, according to our data, this rule does not apply for Tri which has the highest Vmtx at all. An explanation for this unique behaviour of Tri is difficult to give, but studies in vitro using the rat liver microsomal system point to special properties of Tri as a substrate of the hepatic monooxygenase system. Under condi tions of substrate saturation about 1 nmol of Tri metabolites was covalently bound within 60 min to 1 mg microsomal protein (Bolt et al., 1977a) whereas the corre sponding value for VC1 was only 0.44 nmol (Kappus et al., 1976) and that for VBr (Wistar rats) was about 0.2 nmol (Bolt et al., 1978). The present pharmacokinetic data are valid only for Wistar rats. Other strains may show some differences as to the particular rates of metabolism of the haloge nated ethylenes. Thus, it could be demonstrated that liver microsomes from Sprague-Dawley rats metabolize VBr about 50% faster that those obtained from Wistar rats (Bolt et al., 1978). Also, other species metabolize halogenated ethylenes at rates which differ from those calculated from rat experiments. For instance, mice and gerbils metabolize VC1 faster than rats whereas in rabbits and in man metabolism of VC1 is much slower than in the rat species (Buchter et al.. 1978). Because toxic actions of VC1 and its congeners are attributed to activated me tabolites, species variations in metabolism of these compounds must well be consid ered in evaluation of toxicological effects. Acknowledgement. The Financial assistance of the "Deutsche Forschungsgemeinschaft" (grant No. Bo 491/2) is gratefully acknowledged. R&S 107176 136 J. G. Filser and H. M. Bolt References 1 Bolt, H. M.: Pharmacokinetics of vinyl chloride. Gen. Pharmacol. 9, 91--95 (1978) Bolt, H. M., Kappus, H., Buchter, A., Bolt, W.: Disposition of l,2-'4C-vinyl chloride in the rat Arch. Toxicol. (Berl.) 35, 153-162 (1976) Bolt, H. M., Laib, R. J,, Kappus, H., Buchter. A.: Pharmacokinetics of vinyl chloride in the rat Toxicology 7, 179-188 (1977) Bolt, H. M., Buchter, A., Wolowski, L., Gil, D, L., Bolt, W.: Incubation of ''C-trichloroethylene vapor with rat liver microsomes: uptake of radioactivity and covalent protein binding of metabolites. Int Arch. Occup. Environ. Health 39, 103--111 (1977a) Bolt, H. M., Filser, J. G., Hinderer, R. K.: Rat liver microsomal uptake and irreversible protein binding of l,2-l4C-vinyl bromide. Toxicol. Appl. Pharmacol. 44, 481--489 (1978) Bonse, G-, Henschler, D.: Chemical reactivity, biotransformation, and toxicity of polychlorinated alj. phatic compounds. CRC Crit Rev, Toxicol. 3, 395--409 (1976) Bonse, G., Urban, Th., Reichert, D., Henschler, D.: Chemical reactivity, metabolic oxirane formation and biological reactivity of chlorinated ethylenes in the isolated perfused rat liver preparation. Biochem. Pharmacol. 24, 1829-1934 (1975) Buchter, A., Bolt, H. M., Filser, J. G., Goergens, H. W., Laib, R. J., Bolt, W.: Pharmakokinetik und Karzinogenese von Vinylchlorid; Arbeitsmedizinische Risikobeurteilung. Verh. Dtsch. Ges. Arbeitsmedizin (A. W. Gentner Verlag, Stuttgart) 18, 111--124 (1978) Gehring, P. J., Watanabe, P. G., Young, J, D.: The relevance of dose-dependent pharmacokinetics in the assessment of carcinogenic hazard of chemicals. In: Origins of Human Cancer (H. H. Hiatt, J. D. Watson, J. A. Winsten, eds.), pp, 187--203. Cold Spring Harbor Conferences on Cell Proliferation, Vol. 4. Cold Spring Harbor Laboratory 1977 Gehring, P. J., Watanabe, P. G., Park, C. N.: Resolution of dose-response toxicity data for chemicals requiring metabolic activation: example vinyl chloride. Toxicol. Appl. Pharmacol. 44, 581--591 (1978) Kappus, H., Bolt, H. M., Buchter, A., Bolt, W.: Liver microsomal uptake and transformation to protein alkylating metabolites in vitro. Toxicol. Appl. Pharmacol. 37, 461--471 (1976) McKenna, M. J., Zempel, J. A., Madrid, E. O., Braun, W. H., Gehring, P. J.: Metabolism and pharma cokinetic profile of vinylidene chloride in rats following oral administration. Toxicol. Appl. Pharma col. 45, 821-835 (1978) eichert, D., Henschler, D.: Uptake and hepatotoxicity of 1,1,-dichloroethylene by the isolated blood- r perfused rat liver. Int. Arch. Occup. Environ. Health 41, 169--178 (1978) Received December 6, 1978