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INFLUENCE OF ROUTE AND PAT7ERN OF EXPOSURE

ON THE PHARMACOKINEFICS AND HEPATOTOXICITY

OF CARBON TETRACHLORIDE

J.V. Brukner, HJ. Kim, S. Muralidhara, and J.M. Galo*

Department of Pharmacology and Toxicolo,, and*Department of Phr- maceuticsý,

College of Pharmacy, The University of Georgia,Athens, GA 30602

To be published in the Proceedings of the Conference

on Route-to-Route Extrapolation, held in Hilton Head,

SC, March 19 - 21, 1990. Sponsored by ILSI & U.S.EPA.

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ntly, there are many uncertainties in risk assessment of volatileorganic compounds (VOCs), due to a paucity of lha relevant to humanexposure situations. It is unclear whether the relatively large VOC inhalationtoxicity data base can be used qualitative!y or quantitatively to forecast theconsequences of ingestion of the chemicals in food or water. It is also unclearwhether the existing VOC oral toxicity " 'a ba., which largely consists of /bolus gavage studies, is applicable to drinking water ha.:zt:-. evaluation. Theobjective of our study was to evaluate the influence of route and pattern ofexposure on the pharmacokinetics and target organ toxicity of carbontetrachloride (CCI). Male Sprague-Dawley rats of 350-400 g inhaled 100 or .4 --1000 ppjn CCI4 for 2 hr through a one-way breathing valve. -Eqbivalent oralCC14 doses of 18.9 and 186 mg/kg were giver. to other groups of rats in anaqueous emulsion by oral bolus gavage or by constant gastric infusion over 2hr. Serial blood samples were tzken from the unanesthetized animals bymeans of an indwelling arterial cannula and analyzed for CC14 by headspacegas chromatography. Blood and liver samples were taken 24 hr posi dosingfor measurement of serum and hepatic wicrosomal enzymes. Gastric infusionresulted in a lower peak blood concentration (C-MAX) and area under theblood concentration versus time curve (AUC), but a greater hepaticcytochrome P-450 loss at the high dose than did inhalation. C-MAX, AUC,and hepatotoxicity indices were substantially higher for the oral bolus than t-,corresponding gastric infusion and inhalation groups. These findingsdemonstrate that the pattern and route of exposure can significantly influencethe pharmacokinetics and acute hepatotoxicity of CC14 . (Supported by U.S.Air Force Grant AFOSR 88-0277 and U.S. EPA Cooperative Agreement CR-812267.)

INTRODUCTION

A variety of volatile organic compounds (VOCs) have been identified ascontaminants of drinking water supplies in the United States [1,21. One cassof contaminants of particular concern at present is the short-chain aliphatichalocarbon. In order to set standards which will protect against adversehealth effects from drinking such water, reliable data on the toxic potential ofeach chemical are necessary. As the majority of prior interest in healthhazards of halocarbons has centered around exposures in occupationalsettings, toxicological knowledge is largely based upon situations andexperiments involving inhalation exposure. It is unclear, however, whether theresults of inhalation studies can be used to accurately predict theconsequences of ingestion of the chemicals.

There are very few applicable pharmacokinetic or toxicologic data satsfrom which to judge the validity of route to route extrapolations. TheNational Academy d, Sciences (31 avoided the use of inhalation data in riskassessments of drinking water contaminants. They reasoned that thedisposition and ensuing bioeffects of inhaled chemicals may differ markedlyfrom that which occurs when the compounds are ingested. It wat concludedthat while inhalation studies may be of value from a qualitative standpoint,such studies may be of fimited utility quantitatively in predicting consequences

2

of ingestion of many chemicals. In contrast, the U.S. EnvironmentalProtection Agency (EPA) has applied the Stokinger-Woodward model [4] toinhalation data to derive guidelines for oral exposure to halocarbons. Thismeans of direct route to route extrapolation was used to derive acceptabledaily intake (ADI) values for a series of halocarbons, induciagtrichloroethylene, 1,1,1-trichloroethane 1,2-dichloroethane, 1,1-dichloroethylene, and t'trachlornethylene, that were published in the FederalRegister 15]. Such route to r-muie extrapolations are based on two basicassumptions: (a) pharmacokinetics of toxicants are not influenced significantlyby route of exposure; (b) target organ toxicity is independent of route ofexposure.

Another important question is the applicability of results of oral bolustoxicity studies to health effects assessments of food and drinking watercontaminants. Chemicals are usually administered as a single bolus in toxicitystudies. Actual human exposures are quite different, since people normallyconsume relatively low levels of chemicals in food and water in divided dosesover the course of a day. Therefore, another major objective of the currentinvestigation was to determine the influence of dosage regimen on thepharmacokinetics and toxicity of an ingested halocarbon.

Carbon tetrachloride (CC14) was chosen for this investigation, since CCI4is a common water pollutant, as well as a potent hepatotoxin andhepatocarcinogen [6,7]. CC14 has been widely used in the manufacture ofchlorofluorocarbons, in the fumigation of grain, in various industrial solvents,and in cleaning agents. CC14 also is produced as a by-product of themanufacture of a number of other chlorinated materials. Production of CCI4was approximately 600 million lbs. in 1983, although its use has steadilydeclined since 1974. Nevertheless, humans may still be exposed to CC14 insome occupatiotal settings and in the environment. CCI4 is often found as acontaminant of ground water and surface waters around hazardous wastedisposal sites [71.

Specific aims of the cur ent investigation were: (a) to characterize thepharmacokinetics of equivalent inhaled and ingested doses of CC14 over thesame time-frame; (b) to contrast the hepatotoxicity of equivalent oral andrespiratory doses of CC14; and (c) to assess the influence of different patternsof ingestion of Cd 4 on the relative bioavailabil-ty and hepatoxidty of thechemical.

METHODS

Chemicals. Analytical-grade (99.9% pure) CC14 was obtained from J.T. BakerChemical Company (Phillipsburg. NJ). All other chemicals and boologicalswere purchased from Sigma Chemical Company (St. Louis, MO) and AldrichChemical Company, Inc. (Milwaukee, WI).

Animals and treatment. Male Sprague-Dawley rats were purchased fromHarlan Sprague-Dawley, Inc. (Indianapolis, IN). The animals weremaintained on a 12-hr light/dark cycle, with light front 0600 to 1800 h anddarkness from 1800 to 0600 h. The animals were randomly assigned to Stoups

3

and housed in stainless-steel cages in a ventilat.d animal rack. Purina RodentChow 50010 (Ralston Purina Co., St. Louis, MO) and tap water were providedad libitum. The rats were used after a 2-week acclimation period, at whichtime their body weight ranged from 350-400 g. Chemical exposures wereperformed at approximately the same time each day (1000 to 1200 h). Atleast 24 hr before dosing, each rat was aurgically prepared v.ith an indwellingcarotid artery cannula and gastric cannula. The rats were anesthetized for thesurgical procedure by im injection of 0.8 ml/kg of a mixture consisting ofKetamine HCI (100 mg/ml): Acepromazine maleate (10 mg/ml): XylazineHCI (20 mg/ml) in a proportion of 3:2d1(V:V:V). The cannulas wereexteriorized at the bark of the neck through a harness, which allowed theanimals relative freedom of movement, but prevented them from disturbingtheir cannulas.

CC!4 inhalation and ingestion exposures. The inhalation exposure system wasprepared as described by Dallas et aL [8,9]. Each camnulated rat was placedinto a restraining tube of the type used in nose-only inhalation exposurechambers (Battelle-Geneva, Switzerland). A miniaturized one-way breathingvalve (Hans Rudolph, Inc., St. Louis, MO) was attached to the face mask sothat the valve entry port was directly adjacent to the nares of the test animal.CC14 inhalation exposures were initiated only after stable breathing patternswere established. One hundred ppm or 1000 ppm of CCI4 was inhaled for 2hr, and the respiration of each animal was continuously monitored. Theminute volumes for the 100 and 1000 ppm groups, calculated by averaging themeasurements taken at 5- to 5-rmin intervals during the 2-hr exposure, were192.2 :t 17.8 and 209.8 ± 19.1 ml/min, (mean ± SE), respectively.

The administered dose for the inhalation exposures was estimated usingthe following equation: 0.5 x minute volume x thae x inhaled concentration.The anatomic, valvular and mask deadspace tctaled approximately 50% of therat's tidal volume. It was assumed that CCI4 residing in this deadspace didnot participate in gas exchange. Thus, the factor of 0-5 represented theperccntage of inhaled CCI4 thought to reach the alveoli and be available forsystemic absorption. The administered doses for the 100 and 1000 ppmexposures were calculated to be 13.9 and 186 mg/kg bw, respectively. Thesetwo doses were given orally as an aqueous Eniulpnors emulsion, either bybolus gavage or by constant intragastric infusion, in a total volume of 5 ml/kgbw. All solutions were kept chilled, and the actual concentration of CCI4determined prior to dosing by gas chromatograph headspace analysis. Gastricinfusions were carried out using gas-tight Hamiltone glass syringes mountedon a Harvard Microinfusione pump calibrated to deliver accurate volumes ata consistent, predetcrmined rate. The infusions were carried out for 2 hrthrough the gastric cannula. Each orally-dosed animal was maintained in arestraining tube for the 2-hr period.

Blood sampling and CCI4 analysis. Blood samples were withdrawn from thearterial cannula via a 3-way stopcock into a 1-ml syringe. Serial 25-od bloodsamples were taken at time intervals of 2 to 60 min for up to 12 hr during andfollowing dosing. Each blood withdrawal was followed by a heparinized salineflush (10 U heparin/mi) of the same volume, in order to replace lost fluidvolume. Blood samples fr',m high-dose exposure groups were diluted in ice-cold saline solution, in order that they could be analyzed within the linear

4

range of the clectron capture detector of the gas chromatograph (GC). Bloodsamples were quickly transferred to dry, ice-chilled headspace vials (Perkin-Elmer, Norwalk, CT). These vials were capped immediately with PTFE-linedbutyl rubber septa and washers and tightly crimped. Each sample vial wasthen placed into the HS-6 auto-sampler unit of a SIGMA 300 GC (Perkin-Elmer, Norwalk, CT), where it was heated to 80"C and then injectedautomatically into the GC colunn for analysis.

Calculation of pharmacokinetic parame:ers. Blood concentration versus timeprofiles were evaluated by the LAGRAN [101 computer program fordetermination of relevent pharmacokinetic parameters.

The following pharmacokinctic parameters were calculated from the bloodconcentration versus time data:

S,/. 0-693K

where t% is the elimination half life of CCI4 and K is the terminal elimination.rate constanL

DoweAUC

where CLm.is the apparent oral clearance and AUC is the area under theblood CCI4 cccentration versus time curve from time zero to time infinity,as calculated by the Lagrange method.

Clinical chemistry and hepatic nicrosomal analyss. Twenty-four hr afterdosing each animal was anesthetized with ether and blood collected bycardiac puncture into evacuated serum separation tubes (SST9, BectcnDickinson,'Rutherford, NJ). The blood was kept on ice to allow it tocoagulate and then centrifuged. The serum samples wert transferred topolypropylene microcentrifuge tubes and stored in a freezer at -80"C prior toanalysis. The conversion of NADH to NAD over time was used to measuresorbitol dehydrogenase (SDH) activity [111 and glutamic-pyruvic transaminase(GMT) [11i activity in serum. The liver was perfused in situ with chillednormal saline. Liver samples were taken, and the hepatic micosomal fractionwas prepared from the 9000 g supernatant, suspended in T.IS-KCI buffercontaining 20% glycerol, 1 mM DTT and 1 mM EDTA, and stored at -80C.The protein conceelration in the hepatic micromesa was determined by themethod of Lowry et al. [131. Hepatic micro:omal cytochrome P-450 levelswere measured by the method of Omura and Sato [14.

Statistical analysis. The statistical significance of differences betweeninhalation and corresponding ingeswion groups, and oral bolus andcormespooding gastric infusion groups was evaluated by Student's t test, withp < 0.5 set as the minimum level of significance.

5

RESULTS

The arterial blood concentration versus time profiles in rats during andiollowing hrLtlation of 100 or 1000 ppm CC14 for 2 hr are presented in Figure1. CC14 w is rapidly absorbed from the lung. in that substantial levels of CC14were detected in the arterial blood at the initial sampling time (5 min), andnear steady-state was soon achieved. The curves were asymptotic, in that theycontinued to gradually rise throughout the 2-hr exposurt period. The increasein the inhaled concentration from 100 to 1000 ppra produced a proportionalincrease (Le, 10-fold) in the near steady-state blood concentrat;ons. Uponcessation of CCl4 inhalation, the blood levels initially fell very rapidly, thendlimnised at a slower rate during the 10-hr post-exposure monitoring period.The terminal elimination half-lives (t½) were comparable for the two exposurelevels (Table 1). Clearance appeared to be somewhat lower at the higherexposure level, but values for the two groups were not significantly different.The total administered doses were determined to be 18.9 and 186 mg CCI4/kgbw for the 100 and 1000 ppm groups, respectively.

Parallel blood concentration versus time profiles were manifest in animalsfollowing admininration of 18.9 and 186 mg CCI4 /kg as an oral bolus (Figure2). CC14 was r,.adily absorbed from the gastrointestinal (GI) tract. Peai.concentrations of CC14 in the blood (C-MAX) were reached within 10 minafter gavage at both dosage-levels. The blood levels decreased more sowlythan in the inhalation groups in the initial minutes post dosing. indicative ofcontinuing absorption during this period in the orally-dosed animals. The tYvalucs of 239 and 233 min for the 18.9 and 186 mg/kg groups, respectively,were quite similar to the ti values for inhalation exposure (Table 1).

The pattern of systemic uptake and elimination of CC14 is quite differentfrom inhalation when the 18.9 and 186 mg/*g doses are given by constantgastric infusion. Blood levels increase steadily over the entire 2-hr exposureperiod, and then decline at P relatively slow, constant rate post-exposure(Figure 3). The curves for the 1&9 and 186 mg/kg groups appear to beparallel, but close inspection of the pattern of uptake reveals some disparityduring the 2-hr administration period. Although blood concentrationsgenerally are directly proportional to dose in the inhalation and oral bolusgroups, there is a 35-fold increase in blood concentration with the 10-foldincrease in dose during the initial minutes of gastric infusion. The bloodlevels remain relatively low for the first 20-30 min of the infusion in the low-dose animals, then increase steadily. In contrast, the uptake curve in the high-dose infusion animals more nearly resembles that for inhalation exposure (i.e.,relatively rapid attainment of near steady-state).

The effect of pattern of ingestion of CC14 on selected pharmacokineticparameters can be seen in Table L Maximum blood levels were about 5 to6 times higher folowing oral bolus dosing than during gastric infusion. Thesedifferences are readily apparent when the blood levels are plotted on a linearscale (Figure 4). The oral bolus gro."e hae. significantly greater AUC valuesthan corresponding gastric infus.on gi'ups. Clearance, therefore, wassgnificantly increased in the rats receiving CC14 by constant infusion. Thzrate of clearance was particularly high in the low-dose infusion group.

6

The influence of route of exposure on CCI4 pharmacokinetics can also beseen in Table 1 and Figure 4. Throughout the 2-hr exposure periodconcentrations of CC14 in arterial blood were signilicaant, higher in animalsinhaling CCI4 than in animals receiving it by gastric infusion. As a result,AUC values were higher for the inhalation exposures, though intersubjectvariability precluded a statistically significant difference at the high dosage-level Clearance was significartly greater and tY, shorter in rats receiving thelow dose by gastric infusion than in the corresponding inhalation group.

The magnitude of CCI4 hepatotoxicity was more affected by pattern thanby route of CC14 exposure. Inhalation of 100 ppm CCI4 or gastric infusion ofthe equivalent administered dose (i.e., 18.9 mg/kg) had relatively little effecton SDH and GPT activities. Rats receiving 18.9 mg/kg as a single oral bolus,however, exhibited a significant elevation of each serum enzyme. The. highdosage-level (i.e. 1000 ppm/186 mg/kg) produced comparable increases inenzyme levels in the inhalation and gastric infusion groups. Three- to four-fold greater increases in SDH and GPT occurred in response toadministration of 186 mg/kg as a single oral bolus. The low dosage-levelappeared to cause modest reductions of comparable magnitude in all 3 groupsin hepatic microsomal cytochrome P-450, though the decreases were notsufficient to be statistically significanL There were significant reductions in P-450 in animals receiving the high dos, of CC14 by gastric infusion or as anoral bolus.

DISCUSSION

Delineation of the relative pharmacokinetics and toxcity of inhaled andingested VOCs requires innovative approaches. Physiologicaily-basedpharmacokinetic (PBPK) models are finding increasing use in predicting thedynamics of VOCQ in blood and selected tissues under different exposureconditions. Paustenbach et al. [161, for examplc, describe a PBPK modelwhich adequately forecasts the kinetics of CC14 and its metabolites for varyingit.hiation scenarios. Data from direct measurements of VOC levels and toxiceffects in animals are required, nevertheless, to validate and adjust PBPKmodels to improve their accuracy. Results of direct measurement studiesreported to drte, in which comparable doses of VOCs have been given orallyand by inhaltion (17211, are of limited utility in route to route extrapolation.Modt such studies have involved administration of the oral dose as a singlebolus by gavage and inhalation exposure over a period of hours. VOCs, as achemical class, are quite rapidly absorbed following oral dosing, and thenquickly eliminated from the bloodstream. Prolonged inhalation of VOCs, incontrast, serves as a constant infusion into the systemic circulation. A morelogical way to directly determine the influence of exposure route on VOCdisposition would be to give equivalent doses over the same dime-frameintragastrically and by inhalation. This is the experimental approach employedin the current investigation.

On the basis of physiological and anatomical considerations, it wouldappear that the route of exposure should significantly iafluence the quantityof chemical reaching a particular target tissue and the resulting degree of toxiceffect on the tissue. The lung is an optimal site for systemic absorption

7

because of its large surface area, intimate alveolar-capillary interfaces and highrate of blood perfusion. As VOCs are small, uncharged, lipophilic molecules,they are rapidly absorbed from the lung into the systemic circulation [221.Compounds absorbed into the pulmonary circulatio.- are transported via thearterial blood directly to tissues of the body, without first having to passthrough an eliminating organ. The GI tract is also well suited for absorptionof VOCs, though its total surface area is less than that of the lung, and itreceives only a fraction of the total cardiac output. A.lso, VOCs absorbedfrom the GI tract irto the portal blood are subject to 'first pass' eliminationby the liver and lungs. In the current study, CCI4 was rapidly absorbed fromboth the lung and GI tract, in that substantial levels of CCd4 were detected inthe arterial blood of the inhalation and oral bolus groups at the earliestsampling times. A substantial proportion of the low dose (18.9 mg/kg) didnot reach the s-yemic circulation in the gastric infusion group, as reflected bysignificantly lovwr AUC and C-MAX values in this than in the correspondinginhalation grcup. A similar pattern was seen at the high dose (186 mg/kg),though the difference between the gastric infusion and inhalation AUCs wasnot statistically significant. Thus, it appears that a significant amount of CC14absorbed from the GI t-act will be metabolized by the liver and/or exhaledbefore reaching the arterial circulation and extrahepatic organs. First-passelimination will be most efficient at low doses of CC!4, since the liver'scapacity to metabolize the chemical is limited [16]. Also, in high doses, CC14can cause liver injury and thereby inhibit its own metabolism I23]

If ingested CC 4 vedergoes extensive first-pass extraciion by the liver, itis logical to assume that the liver will accumulate a higher dose andexperience more pronourced injury than following an equivalent inhalationexposure. The aforementioned arterial CC14 concentration-time data support:his assumption, though it will be necessary to measure the concentration ofCC(4 in the liver over time (Le., the tissue dose), in order to ascertain therelative contribution of the liver and lungs to presystemic elimination. Thecylochrome P-450 data in Table 2 suggest that ingested CC14 is morehepatotoxic than inhaled CCI4 . These and the serum enzyme data are notconclusive, however, due to pronounced intersubject variability. Thus, thestudy is being repeated to clarify whether the liver is actually at greater riskof injury upon oral exposure to CC!4 . As cumulative uptaL. (Le, thesystemically absorbed dose) can be measured during inhalation exposures,both the 'absorbed* dose and the *administered* dose for inhalation will begiven to rats within the same time-frame by gastric infusion. Blood and liverCC14 levels, as well as a variety of indices of hepatotoxicity, will be monitoredover time, in order to more adequately assess the influence of route ofexposure on target organ toxicity.

Although VOCs such as CC 4 are commonly given by gaWage as a singlebolus in oral toxicity studies, human exposures to VOCs in tbh atmosphereand in drinking water typically occur on a repetitive, or continuing basis as aperson inhales 3ir or consumes water over the coursc of a day. The relativelysmall quantities of chemicals absorbed upon repetitive ingestion may bereadily metabolized and eliminated, so that toxic levels are not reached in theblood and target organs. Single high dose& are more likely to exceed a toxicthreshold and produce injury. Our study demonstrates that administration ofCC14 as an oral bolus results in C-MAX and AUC values which are

substantially greater than when the same doses are given over 2 hr by gastricinfusion or inhalation. As expected, CC!4 is significantly more hepatotoxicwhen given as a single oral bolus. Chloroform (CHCI3 ) and a number offther halocarbons have been found to produce a high incidence ofhepatocelulur carcinoma, when given to B6C3FI mice in coen oil by gavag:.Jorgcnson et al. [241, however, saw no evidence of hepatic tumorigenesis whenthese mice were given the same doses of CHCI3 in drinking water. Similarly,Klaunig et al. 125] found that CIdC3 , 1,1-dichloroethylene and 1,2-dichloroethane were not carcinogenic when given to mice in their drinkingwater, although each halocarbon has been reported to be a hepatocarcinogenwhen given daily as a single oral bolus. Thus, results of oral bolus studiesmay not accurately represent/predict risks of environmental exposure tohalocarbons and other VOCs in drinking water and air.

9

BIBLIOGRAPHY

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9. Dall"a CE., R. RamanalhAn, S. Muralidhara, J.M. Callo, and J.V.Bruckner. 1989. The uptake and elimination of 1,1,1-tric.horoethanedurL4g and following inhalation exposures in rats. T'acol. Appi.Pharmaco. 98:3&5-397.

10. Rocci, M.L. and WJ. Jusko. 1983. LAGRAN program for area tandmoments in pharmacokinetic analysis. Comput. Prog. Biomed 16:203-216.

11. Gerlach, U. and W. Wiby. 1974. Sorbitol dhydrogenase. H.O.Bergmeyer, eAd. Methods in Enzymatic Analysis, VoL 11, pp. 569-573.New York: Academic Press.

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12. Mattenheimer, H. 1971. Glutamate pyruvate transaminase (GFr')(alanine aminorzrsferase). Cinical Enzymoko, *dncipk1 andApplications, Engl. ed., pp. 149-151. Ann Arbor, MI. Ann Arbor Sci.Publ.

13. Lowry, O.H., NJ. Rosebrough, A.L. Farr, and RJ. Randall. 1951.'Protein measurement with the folin phenol reagent. J. Biol. Chem.193:265-275.

14. Omura, T. and R. Sato. 1964. The carbon monoxide-binding pigment ofliver microsomes. I. Biol. Chem. 239:2370-2378.

15. Lam, F.C., C.T. Hung, and D.G. Perrier. 1985. Estimation of variancefor harmonic mean half-lives. J. Phann. Sci. 74:229-231.

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17. Pyykko, K., H. Tahti, and H. Vapaatalo. 1977. Toluene concentrationsin various tissues of rats after inhalation and oral administration. Aich.Ta/co. 38:169-176.

18. M,:Keuma, MJ., JA. Zemple, E.O. Madrid, WJ. Brown, and PJ.G,;hring. 197 8a. Metabolism and pharmacokinet.-c profile of vinylidenechloride in rats following oral administration. TaicoL Appl PhwrmacoL45:821-835.

19. McKenna, MJ., JA. Zempel, E.O. Madrid, and PJ. Gehring, 1978b.The pharmacokinetics of [14 Cjvinylideae chloride in rats followinginhalation exposure. TaxicoL Appl. Phamwco. 45:599-610.

20. Pegg. D.G., JA. Zempel, W.H. Braun, and P.G. Watanabe. 1979.Disposition of tetrachloro(14C)etbylene following oral and inhalationexposure in rats. Taxico/. Appl. Pharmacol. 51:465-474.

21. Reitz, R.H., T.R. Fox, J.C. Ramsey, J.F. Quast, P.W. L.angvardt, and P.G.Watanabe. 1982. Pharmacokinetics and macromolecular interactions ofethylene dichloride in rats after inhalation or gavage. Tax/coJ. Appt.PhanncoL 62.169-176.

22. Astrand, I. 1975. Uptake of solvents in the blood and tissues of man.A review. Scand J. Work Environ. Health 1:199-21&

23. Reynolds, ES., RJ. Treinen, H.H. Farrish, and M.T. Moslen. 1984.Metabolism of I1 4C]carbon tetrachloride to exhaled, excreted and boundmetabolizes. Biochen. Phamcol. 21:3363-3374.

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24. Jorgenson, TA., E.F. Meicrhezuy, CJ. Rushbrook, RJ. BULL and M.Robinson. 1985. Caucinogenicity of chloroform in drinking water to maleOsborn-Mendel rats and female B6C3FI mice. Fund AppI. Taco.it5:760-769.

25. Klaunig, J.E., RJ. R'ch, and M.A. Pereira. 1986. Carcinogenicity ofchlorinated methane and ethane compounds administered in drinkingwater to mice. Envu'oa. Health P.rspec. 69:89-95.

12

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(INFUSION) TIME (MIN)

Flom & looe OC1 4 cowalrat;o ,WAM tune prdriks ma ramsdwlqn &ad fodow* Cong weaseliri Wfusice o( 1&9 or 4S6 ml;(Th4/k kw 2 hr. Arteria Ample wm take st 2- to W0-uaaWguyeb. valme Are sof 2 SE for 54 rfn.

17

0 - ORAL BOLUS 15.9 m,*go . GASTRFC WFUSXO 189 nmgt.j0 2000

0

z

00

0 60 120 1SO 240 300 360 420 400 540 600 $60 720t I

EXPOSURE TIME (MIN)

Fnpre 4. Effed of route and pattern o( exposure on blood CC4coocentratios-time pro,"La. Rats inhaled 100 pp of cW4 for 2 hr.An equivalent oral dose of 18.9 mgg was em m an aqueowemulsion cither by pvage a a oral bolus and by constant astric

fusion for 2 hr. Arterial CC4 coeantrsttons wer me sued at 2.to 60-min intervals. Values mae OnA t SE for 54 ras.

18