Document RpBddMwBo3Or5Zk36zxzkgpva

Environmental Health Perspectives Vol. 21, pp. 99-105, 1977 Pharmacokinetics of Vinylidene Chloride in the Rat by M. J. McKenna,* P. G. Watanabe,* and P. J. Gehring* The metabolism of inhaled vinylidene chloride in rati represents a balance of biotransformation path ways leading to the formation of a reactive alkylating species which is normally detoxified by conjugation with glutathione. Detoxification of the reactive intermediate formed from inhaled YDC is dependent upon the availability of hepatic glutathione (GSH); as YDC exposure concentrations are increased, the fraction of the dose detoxified by conjugation with GSH decreases markedly, commensurate with depletion of hepatic GSH. This reactive intermediate in the absence of GSH alkylates hepatic macromolecules and causes cell death. Similarly, hepatic GSH plays a vital role in the detoxification of the reactive metabolite formed from Inhaled vinyl chloride I VC), However the dose-response relationships for the utilization of GSH and the accumulation of alkylating metabolites following inhalation exposure to either YDC or YC point to distinct differences which may explain the differing biological activities of the two materials. Finally, preliminary pharmacokinetic data for inhaled YDC in mice indicate an enhanced susceptibility to VDC by virtue of an increased ability for production of alky lating YDC metabolites over that observed in the rat. The importance of these findings in light of recent evidence for a carcinogenic effect of VDC in mice is discussed. Introduction Vinylidene chloride (1,1-dichloroethylene, VDC) is used extensively as a monomeric intermediate in the production of plastics. Studies by Jaeger et al. (/) have demonstrated that acute inhalation exposure to VDC results m a decrease in liver glutathione (GSH) concentrations of rats exposed to high VDC concentrations by inhalation. Further. VDC-induced hepatotoxicity is enhanced when hepatic GSH levels are lowered by fasting for 18 hr prior to VDC exposure. Previous studies conducted in this laboratory (2) have described the pharmacokinetics and metabolism of VDC after oral and inhalation ex posure of rats to 14C-VDC. In these studies the fate of VDC was shown to be dependent upon the mag nitude of the dose. An 18-hr fast prior to VDC ad ministration decreased the excretion of VDC uri nary metabolites associated with the GSH metabolic pathway in rats given a 50 mg/kg oral dose of VDC. The diminished ability of fasted rats Toxicology Research Laboratory, Health and Environmental Research, Dow Chemical Company. Midland, Michigan 48640. to detoxify VDC correlates with the enhancement of VDC-induced toxicity in fasted rats observed by Jaeger, et al. (/), The results of previous experiments have led to the hypothesis that the hepatotoxicity of VDC is mediated by a reactive electrophilic metabolite of VDC. At low levels of exposure this reactive in termediate is detoxified by conjugation with GSH. Exposure to toxic concentrations of V DC result' m depletion of hepatic GSH and consequently the al kylation of tissue macromolecules rathei than GSH. The toxicity of VDC may also be enhanced if GSH levels are depleted, by fasting or other means, prior to VDC exposure. Vinyl chloride (VC) is also biotransformed to an intermediate which is detoxified by conjugation with GSH. The dose-dependent pharmacokinetics have been described in studies of vinyl chloride (VC) pharmacokinetics and metabolism (J. -t). Similarities in the chemical structures of VC and VDC notwithstanding, marked differences in the biological actions of the two materials have been observed which may be accounted for by differ ences in their pharmacokinetics and metabolism in laboratory animals. Therefore the purpose of this December 1977 99 CMA. 050868 report is to summarize our recent experiments on the fate of inhaled VDC in rats and provide a com parison with similar data for VC. In addition, pre liminary data on the fate of VDC in mice are also presented. Results and Discussion Fate of MC-Vinylidene Chloride in the Rat Following Inhalation Exposure Male Sprague-Dawley rats (normally fed or fasted for 18 hr) were exposed to 10 or 200 ppm I4C-VDC for 6 hr. Immediately after exposure the animals were placed in individual glass metabolism cages and the elimination of 14C-activity was fol lowed for 72 hr. A detailed description of'the ex perimental procedure has been published previ ously (4), Maintenance of a quantitative collection system for 14C-activity and analysis of residual radioactivity at termination of the experiment (72 hr) enabled the calculation of a total ,4C-balance for each animal. The total recovery of l4C-activity from each rat was assumed to approximate the total amount of ,JC-activity in the body (body burden) at the end of the inhalation exposure. The body burden of l4C-activity, inclusive of VDC perse and metabolites formed from VDC, for rats exposed to 10 or 200 ppm 14C-VDC for 6 hr are given in Table I. Little difference between fed and fasted rats was observed in the net retention and metabolism of l4C-VDC during the 6-hr inhalation exposure to 10 ppm ,4C-VDC. However, fasted rats exposed to 200 ppm 14C-VDC had significantly lower body burdens of 14C-VDC following expo sure than did fed rats of the same exposure group. Furthermore the increase in body burdens of both fed and fasted rats at 200 vs 10 ppm was less than the proportional increase in the VDC exposure concentration. Thus, if biotransformation of ,4C-VDC was a major factor in the accumulation of 14C-activity during inhalation exposure, the data indicate that the capacity for VDC metabolism may Tabic 1. Body burdens of "C-aetlvity in rata exposed to 10 or 200 ppm l'C-VDC for ( hr. Exposure concentration, ppm Body burden, mg-eq *4C-VDC/kg" Fed ruts Fasted ms Fed ms Fasted ms 10 200 2.89 a 0.12 2.30 a 0.0J 44.33 a 3.05 35.93 a 0.30` `X - SE, r * 4. Defined as the total "C-activity (VDC plus metabolites) in the body at the end of the inhalation exposure. 'Significant fed-fasted difference, p < 0.0S. (-test. have been exceeded at the higher VDC exposure level. This dose-dependent effect was enhanced by fasting prior to VDC inhalation exposure. Fasted rats exposed to 200 ppm l4C-VDC showed definite signs of VDC-induced liver and kidney toxicity fol lowing exposure. These effects, characterized by hepatocellular degeneration and necrosis as well as proximal renal tubular epithelial degeneration, were evident only in fasted animals exposed to 200 ppm VDC. Microscopic examinations of liver and kid ney tissues from all other animals were normal. Since the total amount of VDC biotransformed by fed rats exposed to 200 ppm was greater than that observed in fasted rats, the data clearly indicate that fasting augments the process whereby reactive intermediates formed from VDC induce tissue damage. The disposition of l4C-activity by rats exposed to ,4C-VDC for 6 hr is shown in Table 2. The elimi nation of ,4C-activity via the lungs was totally ac counted for as unchanged l4C-VDC and l4CO.,,>. No additional unchanged 14C-VDC or volatile ,4C-activity was found in urine, feces, or tissues, indicating that the radioactivity in these samples represented nonvolatile metabolites of l4C-VDC. Tabic 2. Recovery of "C-activity from rate during 72 hr following "C-VDC exposure for 6 hr. Body burden. tT* 10 ppm 200 ppm Expired VDC "CO, Urine Feces Carcass Cage wash Fed ms Fasted ms 1.63 8.74 74,72 9,73 4 75 0.44 1.60 8.27 78.19 6.75 5.28 0.27 Fed ms Fasted ms 4 17' 8.22 74.66 6.39 6.18' 0.34 8.36r 7.24 70.41 2.72 10.52'' 0.76 "X of 4 rats/group Expressed as the percentage of the endexposure body burden values given in Table 1. 'Significant difference from 10 ppm group, p < 0.05. 'Significant fed-fasted difference, p < 0.05. The data in Table 2 are presented as the percen tage of the total ,4C-activity eliminated during 72 hr after exposure plus that remaining in the tissues at 72 hr following the inhalation exposure. If all of the processes involved in the disposition of ,4C-VDC in the rat could be described by first-order phar macokinetics. i.e., if the rate constants for all pro cesses were independent of the amount of VDC available, then the proportions of ,4C-activiiy eliminated or retained in the body w-ould be the same for all animals regardless of the 14C-VDC ex posure concentration. The data in Table 2 indicate clearly that the fate of 14C-VDC is dependent upon both the VDC exposure concentration and the physiological status of the rats. 100 Environmental Health Perspectives CMA 050869 Rats exposed to 200 ppm UC*VDC exhaled a greater percentage of their acquired body burden of IJC-activity as unchanged l4C-VDC than did the animals exposed to 10 ppm l4C-VDC. More impor tantly. rats exposed to the higher concentration of VDC showed a greater percentage of the body bur den remaining in the carcass at 72 hr after exposure. Retention of 14C-activity in the carcass was greater in fasted rats exposed to 200 ppm ,4C-VDC. de spite a smaller fraction of the body burden biotrans formed by fasted than fed rats (Table I). The in creased elimination of unchanged VDC with in creasing exposure concentrations indicates that the biotransformation of l4C-VDC may be a saturable process. The effect is enhanced by fasting prior to exposure to l4C-VDC. indicating that fasting prior to VDC exposure reduces the capacity for biotrans formation and detoxification of VDC in the rat. A comparison of the retention and metabolism of 14C-VDC with that for 14C-vinv| chloride (14C-VC) is shown in Table 3, Inhalation exposures of nor mally fed rats to both radio-labeled chemicals were conducted under identical conditions. Values are given in micromole equivalents of 14C-VDC or l4C-VC per kilogram body weight to facilitate com parison of the data. Rats exposed to 10 ppm l4C-VDC acquired approximately twice the body burden of those exposed to 10 ppm l4C-VC. Al though the percentage of the end-exposure body burden metabolized to nonvolatile t4C-metabolites was comparable in both the VDC- and VC-exposed animals, the greater accumulation of l4C-activity during exposure to l4C-VDC suggests a faster rate of biotransformation for VDC in the rat than for VC. The reduction in the percentage of the body burden metabolized following the higher exposure concentrations of VDC and VC indicates the dose-dependent character of the pharmacokinetics of both materials. with the hypothesis that hepatic glutathione plays a major role in the detoxification of VDC. Evidence for the conjugation of VDC or some reactive me tabolite of VDC with glutathione was obtained in these studies by analysis of the urinary metabolites of 14C-VDC in the rat. High pressure liquid chromatography of rat urine on a strong anion ex change column gave four major peaks of uC-activity, as shown in Figure 1. The chromato graphic profiles of urinary l4C-acttvity were quali tatively similar among all animals regardless of l4C-VDC exposure concentration or pretreatment (fed vs. fasted rats). Metabolites B and C were identified by gas chromatograph)-mass spec trometry as iV-acetyl-5-(2-hydroxyethyi (cysteine and thiodiglycolic acid, respectively. The mass spectra of these metabolites were identical to those of synthesized reference materials. Their identifica tion was further substantiated by quantitative coelution of the ,4C-labeled reference materials with the urinary metabolites using high pressure li quid chromatography. The presence of these mercapturic acid derivatives in rat urine following VDC exposure substantiates the proposed conjuga tion with glutathione as a major detoxification pathway for VDC. Combined, these two urinary metabolites account for 40-509c of the total urinary 14C-activity following 14C-VDC exposure. Both metabolites have also been identified-as major metabolites in rat urine following inhalation expo sure to l4C-VC. Efforts to identify the two remain ing urinary metabolites of VDC are still in progress. `iiC'4 *MISu*C UOUIQ CHft3*4tTQGluu Qg HtT u*'v( *OLl(3w**Q Cv^a*'3VM Tabic 3. Body burdens and metabolism of "C-VC and l4C-VDC in rats after inbaiatioo exposure for 6 hr." Exposure concentra tion, ppm Body burden, jimole/kg Metabolized. /imole/kg* % Metabolized VDC VC 10 200 10 1000 30.10 * 1.23 29.38 x 1.33 463.83 x 63.02 445.10 x 33.13 13.97 x 0.81 13.63 x 0.81 433.23 x 14.03 380.48 x 16.29 98 % 98 88 `X x SE. n - 4, `Calculated as the body burden minus the 14C*VDC or VC expired unchanged. Role of Glutathione in VDC Metabolism As mentioned previously, studies by Jaeger ei al. (/) and those in this laboratory (2) were consistent FlGline 1. Typical separation of rat urinary "C-activtty by high-pressure liquid chromatography, into four major frac tions of unnary MC-activity. (ri) unknown; (fll \-acetyl-5-f2 hydroxyethyllcysteine; (O thiodiglycolic acid; (DI unknown. Recovery of 14C-activity from the column was 99 3 5 1.65% (J? x SD, n = 301. December 1977 101 CMA 050870 Metabolism of VDC Associated with Toxicity In several instances, alkylation of tissue subcellular macromolecules either directly or by a reactive metabolite has been demonstrated to precede chem ically induced tissue necrosis. Both the biotrans formation of VDC to a reactive intermediate and its acute hepatotoxic action favored the possibility that evaluation of this phenomenon may prove a useful tool in assessing the biotransformation of VDC as associated with VDC induced hepatotoxicity. Ini tial experiments were conducted in the same ani mals employed in the MC-balance studies described earlier. Hepatic tissue obtained from rats 72 hr after IJC-VDC exposure was repeatedly extracted as described by Jollow et al. (5). Values for nonextractable or so-called "covalently bound" ,4C-activity in hepatic tissue of rats exposed to 10 or 200 ppm 14C-VDC are shown in Table 4. Values for total metabolism of ,4C-VDC are also presented to allow evaluation of the relationship between covalent binding and overall metabolism of ,4C-VDC. Metabolized l4C-VDC was calculated from the total and end-exposure body burden minus the l4C-activity expired as VDC. Fed rats showed a 15-fold increase in biotransformed l4C-VDC with the 20-fold increase in exposure concentration (from 10 to 200 ppm). However, the concentration of covalently bound 14C-activity in the liver in creased approximately 26-fold, appreciably greater than the observed increase in VDC metabolism. Fasted rats exposed to 200 ppm 14C-VDC metabolized less VDC than did fed rats. However, the concentration of covalently bound l4C-activity in the liver was greater in fasted than in fed rats following the 200 ppm exposure. The increase in covalent binding in the livers of fasted rats was more apparent after normalization of the data to account for differences in metabolism of VDC (B/A ratio). The data presented in Table 4 indicate that an increase in the covalent binding of VDC or some reactive metabolite to hepatic tissue is associated with VDC hepatotoxicity. The greater increase in macromolecular binding relative to that observed for metabolism of VDC with increasing exposure concentrations or after fasting may result from overwhelming the capacity to detoxify the reactive intermediate formed from VDC. Therefore as the capacity to detoxify VDC is exceeded <i.e.. as tis sue GSH stores are depleted), covalent binding to tissue constituents is enhanced. The results of the experiments described above were formulated into a working hypothesis for the biotransformation of VDC which is represented schematically in Eq. (I). Apparently, VDC me tabolism represents a balance between biotrans formation pathways leading to detoxification via GSH or to covalent binding and subsequent tissue damage. Evidence for the initial biotransformation of VDC to one or more reactive intermediates has been discussed in several papers presented in this symposium. In addition experiments conducted on liver homogenates in our own laboratory have dem onstrated that VDC conjugation with GSH re quires the presence of a microsomal enzyme s\ stem. We have been unable to demonstrate conjuga tion of VDC with GSH either directly or using only the soluble fraction of a liver homogenate (GSH alkvl transferases) in vitro. H H (Electrophile Reactive Metabolite(s) -epoxide?) /Detoxification \ Toxicity Conjugation with GSH Covalent binding to tissue nucleophiles Urinary mercapturic acid excretion Table 4. Metabolism of "C-VDC and covalent binding of "C-activity to rat hepatic tissue after inhalation exposure to "C-VDC." Exposure concn. ppm 10 200 Pretreatment Fed Fasted Fed Fasted (A) Metabolized "C-VDC mg-eq/kg 2.84 = 0.13 2.26 0.06 42 73 3.18 32.92 er 0.32" (B) `C-VDC bound. Sig-eq/liver protein 2.49 e 0.17 2.47 = 0.29 64.18 7.97 79 46 x 4.90" "All values represent the X x SE for four rats. "Covalent binding data normalized to account for difference in metabolized VDC. "Significant fed-fasted difference p ' 0 OS. /-test. (BVtAI 0 88 = 0 08" 1.10 = 0.1? 1,49 = 0 10 2,42 e 0 17' 102 Environmental Health Perspectives CMA 050871 Subsequent experiments have been directed by defining the relationship between liver GSH deple tion and covalent binding of l4C-activity following inhalation exposure to 14C-VDC. The reactive in termediate formed from vinyl chloride is also detox ified in vivo by conjugation with GSH and. in addi tion. some degree of covalent binding of MC-VC metabolites in the liver has been demonstrated (Watanabe et al., unpublished data). Therefore it was decided to conduct the experiments on l4C-VDC in a manner which would allow direct comparison of the data for both materials. Male rats were exposed to constant concentra tions of 14C-VDC ranging from 5 to 200 ppm. Ex posure duration was 6 hr. Immediately after expo sure the animals were sacrificed and hepatic non protein sulfhydryl levels and covalent binding of 14C-activity to hepatic tissue were assayed by the methods of Sedlak and Unsay (6) and Jollow et al. (5). respectively. The results of these experiments are shown in Figure 2. velopment of VDC hepatotoxicity. A comparison of dose-response relationships for hepatic glutathione depletion and covalent binding to liver protein following IJC-VDC or l4C-VC in halation exposure is shown in Figure 3. Inhalation exposure of rats to "C-VC is less effective in pro ducing hepatic NPSH depletion than observed with VDC. Furthermore covalent binding of IJC-VC metabolites to liver protein approaches saturation at VC exposure concentrations greater than about 500 ppm. Thus at VC exposure concentrations as high as 5000 ppm the accumulation of reactive VC metabolites is not sufficiently great enough to result in hepatotoxicity. The dose-response relationships shown in Figure 3 indicate that VDC is more rapidly metabolized than VC to reactive metabolites which can deplete hepatic glutathione or covalentl> bind to liver ma cromolecules. The apparent saturation of covalent binding of VC metabolites in the liver likely results from saturation of the biotransformation of VC to a reactive intermediate. Figure 2. Dose-response relationship for () hepatic NPSH de pletion and (o) covalent binding of "C-VDC metabolites to liver protein following inhalation exposure of rats to "C-VDC. Each point represents the mean for three rats. Hepatic NPSH concentrations declined with in creasing VDC exposure concentrations. The dose-dependent or saturable character of VDC de toxification is apparent, indicating an initial devia tion from first-order or linear kinetics at about 50 ppm. However, appreciable covalent binding of l4C-activity to liver protein was found only after VDC exposure which depleted hepatic glutathione by 30% or greater. Thus the metabolic events as sociated with VDC-induced hepatotoxicity con tinue to increase markedly when the VDC expo sure concentration is sufficiently high to produce enough reactive metabolite to exceed the availabil ity of glutathione for detoxification. The data are consistent with the hypothesis that covalent binding of VDC metabolites to tissue macromolecules rep resents a biochemical event which precedes the de Figure 3. Comparison of dose-response relationships for <) hepatic NPSH depletion and to) covalently bound "C-activity in the liver following MC-VDC or "C-VC expo sures. Each point represents the mean for three or five rats Fate of uC-VDC in the Mouse The results of preliminary pharmacokinetic ex periments on the fate of inhaled ,4C-VDC in mice are summarized in Table 5. In these experiments, male Hal ICR) mice were exposed to 10 ppm ,4C-VDC for 6 hr. Immediately after exposure, these animals were placed in glass metabolism cages and excreta collected for 48 hr according to the UC-balance study protocol outlined for rats (4). Table 5 shows the disposition of HC-VDC in rats and mice following exposure to 10 ppm 14C*VDC. The single 6-hr exposure to 14C-VDC resulted in a December 1977 103 CMA 050872 body burden of 5.3 mg-eq. 14C-VDC/kg in the mouse, nearly twice that obtained in the rat in an identical experiment. Total metabolism of MC-VDC was more efficient in the mouse than the rat, the former eliminated less than \% of the body burden as unchanged VDC in expired air. The higher body burden and more rapid metabolism of VDC by mice suggested that pro duction of toxic metabolites of VDC by mice may be greater than that observed in rats and thus render them more susceptible to the effects of VDC ex posure. A comparison of values obtained for cova lently bound MC-activity in liver and kidney of mice and rats immediately following 14C-VDC exposure is shown in Table 6. This data indicate clearly the enhanced production of reactive metabolites of VDC in mice as evidenced by the marked increase in covalently bound HC-activity in both liver and kidney when compared to the rat. In the context of previous findings which indicated the relationship between covalent binding and VDC-induced tissue damage in rats, the data clearly indicate that the mouse is much more susceptible than the rat to the adverse effects of VDC. Ttblt 5. Disposition of "C-activitv in rats and mice following inha lation exposure to 10 ppm "C-VDC. the response of the two mouse strains employed in these studies, both investigators have reported moderate to severe chronic tissue damage indica tive of VDC toxicity in the tumor-bearing organs. Only in Maltoni's study was a "no-effect" level for VDC-induced renal damage realized (10 ppm), and at this exposure concentration no kidney tumors were observed. Therefore, given the enhanced susceptibility of the mouse to VDC, it appears uncertain as to whether the tumorigenic responses observed are truly the direct result of VDC exposure or whether they arose subsequent to chronic insult which re sulted in appreciable tissue damage. Based upon the preliminary pharmacokinetic data obtained thus far. it is conceivable that VDC-induced toxicity may well occur long before a tumorigenic response to VDC is realized. Future studies on the fate of VDC in mice will be oriented towards hazard assessment and the rela tionship between the development of the aforemen tioned biochemical and morphological events in VDC-induced hepatic and renal toxicity. It is ex pected that these studies will provide data which will aid in interpreting the results obtained in chronic toxicity and carcinogenicity studies ofVDC in mice. Expired VDC, 9c" Expired "CO,. %" Body burden. 9c" Urine Feces Carcass Cage Wash Body burden, mg-eq "C-VDC/kg Total metabolized VDC. mg-eq "C-VDC/kg X -SE,n = 4. Mice 0.65 2 0.07 4.64 2 0.17 Rats 1.63 0.14 8.74 3.72 80.83 1.68 74.72 2.30 6.58 2 0.81 9.73 0.10 5.46 0 41 4.75 0.78 1.83 0.84 0.44 0.28 5.30 0.75 2.89 0.24 5.27 * 0.74 2.84 0.26 T>hk 6. Covalently bound "C-actlvity in rat and mouse tissue following exposure to 10 ppm "C-VDC. Mice Rats "C-VDC, nt-eq-lg protein (X SE. n - 4) Liver Kidney 22.29 3.77 5.28 2 0.14 79.55 19.11 13.14 2 1.15 Two studies reported by Maltoni and Lee at this conference have found VDC to be carcinogenic in mice. Maltoni (7) has reported kidney tumors in Swiss mice exposed chronically to 25 ppm but not 10 ppm VDC. In Lee's study (5), mice (CD-I) ex posed to 55 ppm VDC for one year showed hepatomas, angiosarcoma of the liver and pulmo nary adenomas. Despite the marked difference in Summary and Conclusions The data presented in this report indicate that the pharmacokinetics of inhaled V DC in the rat is dose dependent. Detoxification of VDC in the rat occurs primarily via conjugation of one or several VDC metabolites with glutathione and subsequent uri nary excretion of the mercapiuric acid derivatives /V-acetyl-5-(2-hydroxyethyl)cysteine and thiodiglycolic acid. The diminished ability to detoxify VDC is enhanced by fasting, and is associated with a reduction in available hepatic GSH prior to VDC inhalation. VDC-induced centrolobular hepatic necrosis ob served in fasted but not normally fed rats exposed to 200 ppm MC-VDC was associated with an in crease in covalently bound ,JC-VDC metabolites in livers of the affected animals. Subsequent experi ments have shown that significant accumulation of covalently bound VDC metabolites occurs when hepatic glutathione concentrations are depleted by greater than 309 following VDC exposure. The data are consistent with the hypothesis that VDC is rapidly metabolized to one or more alkylating metabolites which are detoxified via conjugation with GSH. When the availability of GSH is ex ceeded the reactive species may accumulate in the tissue by alkylation of subcellular macromolecules and thereby result in toxicity. 104 Environmental Health Perspectives CMA 050873 Comparison of the pharmacokinetics of inhaled VDC and VC in rats points to several marked dif ferences in the fate of the two chemicals which may account for their differing biological actions. In haled VC is less readily metabolized, and hence exposure to equivalent concentrations results in less hepatic GSH depletion and alkylation of tissue macromolecules than with VDC. Although the reactive metabolic intermediates formed from both materials are detoxified via conjugation of their re spective reactive metabolites with GSH, the data indicate that the biotransformation of VDC during inhalation exposure is quantitatively greater than that seen with similar exposure concentrations of VC. Furthermore covalent binding of VC metabolites in rat liver approaches saturation at exposure con centrations of greater than 500 ppm VC. This ob servation for VC is of particular significance since it explains the lack of a hepatotoxic effect of VC at exposures as high as 5000 ppm. In addition this non-linear or saturable character of VC phar macokinetics correlates well with previously re ported data indicating a relatively constant inci dence of hepatic angiosarcoma in rats exposed to VC concentrations ranging from 2500 to 10.000 ppm (9). Thus, although the hepatocyte is not the pri mary target site for VC tumorigenesis. the relation ship between VC metabolism and tumor incidence appears to be well supported by these experiments The preliminary data on the pharmacokinetics of inhaled VDC in the mouse deserve particular comment. In the context of our findings in experi ments on the relationship between the metabolism and toxicity of VDC in rats, the data for mice sug gest an enhanced capacity for metabolism of VDC. Furthermore, a potentially greater susceptibility of mice to VDC is evidenced by an increased produc tion of reactive VDC metabolites capable of al kylating renal and hepatic tissue constitutents over that observed in rats exposed to the same concen tration (10 ppm) of VDC. The data suggest that accumulation of VDC metabilites covalently bound to target organ macromolecules may be sufficient to produce tissue damage and necrosis appreciably sooner than the onset of neoplasia. To date this hypothesis is supported by the findings reported by both Maltoni and Lee. In neither study were tumors in mice observed in the absence of nontumor pathology attributed to VDC exposure. Further studies of the metabolism and pharmacokinetics of VDC in mice are indicated to fully explain the re lationships between metabolism, toxicity and po tential carcinogenicity of this material. Such studies are currently underway in our laboratory. This study was supported in part by the companies participat ing m the vinylidene chloride research projects administered by the Manufacturing Chemists Association, Washington. D C. REFERENCES 1. Jaeger. R. J.. Conolly. R. B.. and Murphy. S. D. Effect of IS hr fast and glutathione depletion on 1.1 -dichloroethylene-induced Hepatotoxicity and lethality in rats. Exp Mol. Pathol. 20: 187-194 (1974). 2. McKenna. M. J.. et al. The fate of "C-vinylidene chloride following inhalation exposure and oral administration in rats. Paper presented at the 16th Annual Meeting of the Society of Toxicology. Toronto. Canada. March 27-30, 1977. 3. Watanabe. P. G., McGowan. G. R., and Gehring, P. J Fate of IJC-vinyI chloride after single oral administration in rats. Toxicol. Appl. Pharmacol, 36: 339-352 (1976) 4. Watanabe. P. G.. McGowan, G. R., and Gehring. P J. Fate of IJC-vinyl chloride following inhalation exposure in rats. Toxicol. Appl. Pharmacol. 37: 49-59(1976). 5. Jollow, D. J.. et al. Acetaminophen-induced hepatic necrosis II. Role of covalent binding in vivo. J. Pharmacol. Exptl, Therap. 187: 195-202 (1973). 6. Sedlak. J.. and Lindsay. R. M. Estimation of total, proteinbound and nonprotein sulfhydryl groups in tissue with Ellman's Reagent. Anal. Biochem. 25: 192-205 (1968). 7. Maltoni, C. Recent findings on the carcinogenicity of chiorinated olefins. Environ. Health Perspect. 21: 1 (1978). 8. Lee, C. C., et al. Inhalation toxicity of vinyl chloride and vinylidene chloride. Environ. Health Perspect. 21: 25 (1978). 9. Maltoni, C., and Lefemine. G. Carcinogenicity assays of vinyl chloride: current results. Ann. N. Y. Acad. Sci. 246: 195-224 (1975). December 1977 105 CMA 050874