Document 4JX0d2gOJD711Zb1VRXpqgy4R

% /tjun^iT-s OuTy /7t /?7/ RESOLUTION OF DOSE-RESPONSE TOXICITY DATA FOR CHEMICALS REQUIRING METABOLIC ACTIVATION: EXAMPLE - VINYL CHLORIDE BY: P. J. Gehring, P. G. Watanabe and C. N, Park May 28, 1977 COKFISEtTIAIr' I1 Subject to Protective Order In Ross V. Conoco, Ire., No. 90-4837. "Q.4tli 'Judicial Court Ju *Calcasieu Toxicology Research Laboratory Health and Environmental Research Dow Chemical U.S.A. Midland, Michigan 48640 Thii itady won funded by the companies iu.ppoA.ttng the vinyl ehloxide pA.ojeeti being adminiiteA.ed by the Manu^aetaA.ing Chemiiti Aiiociation, Olaihington, V.C. CMA 001222 ABSTRACT The toxicity of many chemicals results from biotransformation products formed from the chemical rather than to the chemical per se. In such cases, the incremental response may become diminishingly smaller with increasing dose or exposure because activation of the chemical to the toxic form follows apparent Michaelis-Menten rather than apparent first-order kinetics. To illustrate this concept, rats were exposed to concentrations ranging from 1.4 to 4600 ppm vinyl chloride for 6 hours and the total amount metabolized determined. The amount metabolized followed apparent Michaelis-Menten kinetics. Subsequently, it was found that the tumorigenic response to vinyl chloride was linear with respect to the amount of vinyl chloride metabolized rather than the concentration of vinyl chloride to which rats were exposed. Extrapolation of the data analyzed in this manner indicated that an incidence of 0.01% hepatic angiosar coma may be expected from an exposure to 4.6 ppm vinyl chloride. The concepts presented herein are important in designing and interpreting experiments to determine the dose-response t relationship for chemicals requiring metabolic activation to a toxic form. r V BuBJeet to Gr^r ^ ,, Boss v- Csrcoo. me.. Ho. 9 - JU'iicl&i District Court ;Calcasiru farish, Louisiana CMA 001223 -1- INTRODUCTION There exists a great deal of uncertainty in predicting the potential response of exposure to chemicals at concentra tions below those producing an experimentally discernible response. This is particularly true when the response to the chemical in question is tumorigenesis. It has been suggested that a logarithm probability plot (probit plot) of the incidence of the response versus dose may be projected to doses smaller than those producing a discernible response. For conservatism, Mantel and Bryan (1961) promoted using a slope of one. These procedures are strictly a descriptive exercise for the most part which are not applicable in all instances. The use of high doses to reveal the chronic toxicity incurred with exposure to a chemical is a common, scienti fically defensible practice if judgment and scientific rationale is used in designing the experiments and in assessing the resulting data. Such doses frequently overwhelm the enzymatic processes needed for activation of the chemical to the toxic form and the subsequent deactiva tion to an innocuous form. In this paper, it is demonstrated how the dose-dependent activation of vinyl chloride to a Ross v nr "Jr? I A! Louisiana CMA 001224 2- - tumorigenic product must be considered in resolving the dose-response to vinyl chloride exposure in rats. This concept leads to a much more meaningful interpretation of the dose-response data and permits more meaningful extrapo lations of that data. *+ \<^ . \M tf,f 'VVJ-'C* - ' 4 V*" V CMA 001225 -3- Material. METHODS ^ -: .; - . : , 7 ,.0-'^ .'7 DliTrirt Court C.2lo2.3isu Parish, Louisiana Vinyl chloride (^4C-labeled) was synthesized from (1,2-^C) 1,2-dichloroethane (New England Nuclear, Lot #819-221 and 819-292, 5.0 and 4.8 mCi/iranole, respectively) directly prior to use (Wagner, et crl. , 1975). Non-labeled VC (Matheson Gas Products) of 99.9% purity was mixed with the 14 C-material to obtain the desired specific activity. Animals. Male, Sprague-Dawley rats (Spartan Research Laboratory) weighing 200-250 g were used throughout the study. Food and water were provided ad libitum except during the exposure. Exposures were conducted between 9:00 a.m. and 3:00 p.m. (EST). Groups of 3-6 rats were exposed to various concentrations of "'^C-VC for 6 hours. Exposure and Procedure. The rats were exposed by inhalation under dynamic conditions in a 30 l glass inhalation chamber. The mean analytical concentrations of VC measured by gas chromatography were 1.40.3 (SD), 9.3 + 0.2, 24.71.4, 51 + 2, 109 + 23, 2502, 51111, 102013, and 4600+311 ppm. Details of this exposure and the method of analytical determinations have been reported previously (Watanabe, t al., (1976a). Immediately following CMA 001226 -4- 14 the 6-hour exposure to various concentrations of C-VC (1.4-4600 ppm) the rats were killed by a blow to the head, and the carcass was analyzed for total radioactivity (Watanabe, et al., 1976b). Since radioactivity found in the carcass was non-volatile, this radioactivity represented the total amount of VC which was metabolized. ^ e, co ^ 1 rsie-a C3.1C- itv 493- - + --rft -5- RESULTS Consistent with the results of previous studies (Watanabe, et a_l. , 1976a and 1976b), the metabolism of VC does not increase proportionately with increasing concentrations of VC being inhaled (Table 1). The nonlinearity of the amount of VC metabolized during 6 hours of exposure to various concentrations of VC appeared to be in accordance with Michaelis-Menten kinetics as described by the equation: In this equation, v and V^ , are the velocity and maximum velocity respectively for the biotransformation of VC expressed as yg equivalents VC metabolized per 6 hours. S and K are the concentration of VC being inhaled and the m Michaelis constant expressed as yg VC/Z air, respectively. To ascertain whether Michaelis-Menten kinetics were applicable, the data in Table 1 were analyzed in accordance with the linear Woolf-Augustinson-Hofstee transformation of the Michaelis-Menten equation (Segel, 1976), v = -Km =S + Vm (2) U' -,0 < y.c: . 1- ,, yr\ ;`J\.O0- 9' 0-.yj*, Vl1 CMA. 001228 -6- It can be seen from the plot (Figure 1) that the data appear to lie along a straight line thus verifying, at least visually, the Michaelis-Menten model. V and K can be estimated by the ordinate intercept and the slope of the line or they can be estimated directly by fitting the nonlinear Michaelis-Menten model. Both procedures yield similar parameter estimates. The estimates derived by fitting the model directly are 8558+1147 (SD) yg VC metabolized and 860159 (SD) yg VC/ air for and Km respectively. Once a means is obtained to calculate the amount of VC metabolized as a function of exposure, it is then possible to relate the untoward effects associated with VC exposure to the amount biotransformed rather than the exposure concen tration. Hence if the untoward effects of exposure are related to the formation of toxic metabolites of VC rather than VC per se, more meaningful predictions can be made. Maltoni and Lefemine (1975) reported the incidence of hepatic angiosarcoma in rats exposed to different concentrations of VC, 4 hours/day, 5 days/week for 12 months and subsequently held for observation until death (Table 2). Before attempting to relate these data to the amount of VC biotransformed in accordance with the Michaelis-Menten equation using the previously determined values of Vm and K , the value for V^ must be adjusted for the shorter exposure duration used by CMA 001229 -7- Maltoni and Lefemine, 4 hours versus 6 hours. This adjust ment is accomplished by multiplying by 4/6. Thus, the amount of VC biotransformed daily by rats exposed to the various concentrations used in the experiment of Maltoni and Lefemine can be calculated from the equation : 5706 v S(f) 860 (^ + S(^) (3) The resulting values for v are given in Table 2. Figure 2 depicts a logarithm probability plot (probit plot) of the incidence of hepatic angiosarcoma versus the amount of VC biotransformed for 4 hours of exposure, v, or the exposure concentration, S. The incidence of hepatic angiosarcoma was linear with respect to log v but not log S. The line drawn for log v versus tumor incidence (Figure 2) was determined by using a probit regression analysis program, and the equation relating the incidence of hepatic angio sarcoma to log v was: probit response = -1.625 + 1.543 log v (4) CMA 001230 8- - Using the foregoing equation, the anticipated exposure concentration required to cause 1 angiosarcoma for 10,000 rats can be calculated. The probit percent representing an incidence of 0.01% is 1.28. Substitution of this value into the equation (4) yields: Log v = 1.8827 Antilog v = 76.33 ug VC metabolized/4 hours Using equation 3, the concentration of exposure to VC needed to give this value for v is 11.66 vg/l or 4.6 ppm (95% confidence limits are from .08-22.2 ppm). Hence, exposure of rats to 4.6 ppm VC for 4 hours daily, 5 days/ week for 1 year can be expected to produce one angiosarcoma per 10,000 rats if the dose-response curve remains valid at exposures less than those producing a discernible experimental response. -- t \ ij ,-~\T -J* ' " -----" t -to yoSgusi>3veC Vcoty.O^Oj l&Xti V3xr -Vi , ,,1 nasiv a CMA 001231 -9- DISCUSSION For many chemicals, toxicity may not be a function of exposure to the chemical per se, but rather to a biotrans formation product of the chemical. Frequently, production of the toxic metabolite is dependent upon enzymatically mediated reactions which are classically described by Michaelis-Menten kinetics. Since enzymatically mediated reactions are concentration-dependent and saturable, toxicity resulting from exposures to chemicals requiring activation to a toxic form cannot be related directly to the magnitude of exposure or dose. In such a case, it is necessary to determine the amount of the chemical undergoing biotransforma tion as a function of dose or exposure before a meaningful dose-response relationship can be established. There is considerable evidence that vinyl chloride requires bioactivation to produce tumors. Metabolic activation is required to induce mutations in bacteria exposed to VC (Bartsch, et aA. , 1975; Malavielle, et al.. , 1975; Rannug, et al^. , 1974) . Covalent binding of 14 C to hepatic macro molecules in rats (Watanabe, et_ al. , 1977) or in liver homogenates (Kappus, et_ al^. , 1976) exposed to 14 C-VC also requires bioactivation. Covalent binding of electrophiles to DNA has been associated with tumorigenesis. COlIFIDSl-IiT'IAL Subject to Protective Order In Ross v. Conoco, Inc. , No. 90-483*/ 14th Judicial Pi strict Court Calcasieu Parish, Ionicir-a CMA 001232 -10- For vinyl chloride-induced hepatic angiosarcoma in rats, a logarithmic probability plot (probit plot) of the incidence versus the amount of vinyl chloride metabolized, v, over a range of exposures from 50 to 10,000 ppm VC gives a classical straight line, Figure 2. The dose-response relationship is not a straight line when plotted as a function of the exposure concentration, S. These results support further the conclusion that VC requires biotransformation to an active metabolite for tumorigenesis. Furthermore, a more reasonable evaluation of the dose-response data for vinyl chloride induced tumorigenesis requires knowledge of the amount of VC activated as a function of exposure. Extrapolating the tumorigenicity data of Maltoni and Lefemine (1975), in the same manner as Schneiderman, et al. , (1975) predicts that a concentration of 1 ppm VC will produce an incidence of 0.01% hepatic angiosarcoma compared to our prediction of 4.6 ppm VC for the same incidence. However, the Schneiderman, et al. prediction required arbitrary exclusion of data acquired from rats exposed to concentra tions of VC exceeding 500 ppm. If all of the data had been used, a dose-response curve with an unrealistically shallow slope would have resulted and the predicted level causing 0.01% hepatic angiosarcoma would have been even smaller. CMA 001233 -11- The concepts developed herein allow use of all of the data presented by Maltoni and Lefemine (1975) to construct a doseresponse curve on a scientifically defensible basis. Assuming that the resulting dose-response curve can be projected beyond the range of the experimentally discernible responses, the exposure concentration required to produce an incidence of 0.01% hepatic angiosarcoma is 4.6 ppm. Whether or not this statistical extrapolation below the range of discernible, responses is reliable remains to be established. In this regard, there is evidence that detoxification of reactive electrophilic metabolites of VC may occur more efficiently in rats exposed to concentrations of VC below 50 ppm (Watanabe, et a3^. , 1976c) in which case the actual concen tration of VC required to produce an incidence of 0.01% hepatic angiosarcoma may be higher. The concepts presented herein are exceedingly important in evaluating toxicity data obtained for chemicals requiring activation to a toxic form. Equally important are the inherent implications when designing an experiment to assess the toxicity, including carcinogenicity, of chemicals requiring bioactivation. For these chemicals, increasing the concentration above the apparent will produce diminishingly smaller increments in the response; in i:o 90-4337 Boss v. J_---^trloLot Court Louisiana Calcasaeu CMA 001234 -12- no increase in the response is to be expected when the exposure concentration is 2 or 3 times K . Since exposure is a function of time as well as concentration, it is impor tant to determine the effect of exposure time on the response. As shown in Equation 3, the only parameter influenced by exposure time is V , which is increased linearly with time. Therefore, after the concentration to which the animals are exposed becomes 2 to 3 times K^, the amount metabolized, v, will increase linearly with increasing exposure time. For this reason, the gradation of incidence of angiosarcoma in individuals exposed to high concentrations of VC will become a function of exposure time rather than concentration. This reasoning makes determination of exposure time as well as exposure concentration important in conducting epidemiological studies of people exposed to high concentrations of VC in the work environment. Unless the dose-dependent, Michaelis-Menten type pharmaco kinetic parameters are resolved prior to designing the experiment, the results may be useless for assessing the hazard incurred via exposure to the agent. Thus, the current approach using the maximum tolerated dose and fractions thereof may be scientifically tenuous if the objective is to assess the hazard of exposure to much lower doses or exposures. CMA 001235 -13For some chemicals detoxification of reactive metabolites may also be dose-dependent and saturable leading to a build-up of toxic materials. In such cases, the incremental responses to increasing doses or exposures will become disproportionately larger rather than smaller (see Gehring and Blau, 1977) . In conclusion, rational design of experiments to obtain doseresponse data for chemicals requiring metabolic activation or deactivation and the subsequent rational interpretation of the results requires resolution of the pharmacokinetic parameters for the chemical. CMA 001236 -14- REFERENCES Bartsch, H., Malavielle, C., and Montesano, R. (1975). Human rat, and mouse liver mediated mutagenicity of vinyl chloride in Salmonella typhimurium strains. Int. J. Cancer, 15, 429-437. Gehring, P. J. and Blau, G. (1977). Mechanisms of carcino genesis: dose-response. Toxicology Laboratory, The Dow Chemical Company, in manuscript. Kappus, H., Bolt, H. M., Buchter, A., and Bolt, W. (1976). Liver microsomal uptake of ( 14 C) vinyl chloride and transformation to protein alkylating metabolites in vitro. Toxicol. Appl. Pharmacol., 37, 461-471. Malavielle, C., Bartsch, H., Barbin, A., Camus, A. M., and Montesano, R. (1975). Mutagenicity of vinyl chloride, chloroethyleneoxide, chloroacetaldehyde and chloroethanol. Biochem. Biophys. Res. Comm., 63, 363-370. Maltoni, C. and Lefemine, G. (1975). Carcinogenicity assays of vinyl chloride: Current Results. Ann. N. Y. Acad. Sci., 246, 195-224. Mantel, N. and Bryan, W. R. (1961) . "Safety" testing of carcinogenic agents. J. Nat. Cancer Inst., 27, 455-470. Rannug, U., Johansson, A., Ramel, C. and Wachtmeister, C. A. (1974) . The mutagenicity of vinyl chloride after metabolic activation, Ambio, 3, 194-197. CQflglDESTIAL Subject to Profs ofire Order in Ross v. Conoco, Ino:_,_Ko. SO-4837 14th Judicial District Court Calcasieu Parish, Louisiana CMA 001237 -15- Schneiderman, M. A., Mantel, N. and Brown, C. C. (1975) . From mouse to man - or how to get from the laboratory to Park Avenue and 59th Street. Ann. N.Y. Acad. Sci., 246, 237-248. Segel, I. H. (1976). Biochemical Calculations, 2nd Ed., pp. 236-237, John Wiley and Sons, Inc., New York. e; 9' C ! Wagner, E. R., Muelder, W. W., Watanabe, P. G., Hefner, R. E., Jr., Braun, W. H., and Gehring, P. J. (1975). Gas chromatographic method for the preparation of 14 C-labeled vinyl chloride, J, Labelled Compounds, 11, 535-542. O* f=- r--j ^ i o. VnI ao-,l Watanabe, P. G., McGowan, G. R., Madrid, E. 0., and Gehring, 14 P. J., (1976a). Fate of C-vinyl chloride following inhalation exposure in rats. Toxicol. Appl. Pharmacol., 37, 49-59. Watanabe, P. G., McGowan, G. R., and Gehring, P. J., (1976b). Fate of 14 C-vinyl chloride after single oral administra tion in rats, Toxicol. Appl. Pharmacol., 36_, 339-352. Watanabe, P. G., Hefner, R. E., Jr., and Gehring, P. J. (1976c). Vinyl chloride induced depression of hepatic nonprotein sulfhydryl content and effects on bromosulphthalein (BSP) clearance in rats. Toxicology, 6^, 1-8. Watanabe, P. G., Zempel, J. H., Pegg, D. G., and Gehring, P. J. (1977). Hepatic macromolecular binding following exposure to vinyl chloride. Toxicology Laboratory, The Dow Chemical Company, in manuscript. CMA 001238 -16- TABLE 1 Parameters for Describing the Metabolism of Inhaled Vinyl Chloride (VC) Using Michaelis-Menten Kinetics Exposure Concentration S (ppm VC) 1.4 9 25 51 109 250 511 1020 4600 5 (pg VC/A air)a 3.6 23.0 64.0 130.6 279.0 640.0 1308.2 2611.2 11776.0 k pg VC metabolized0 ^________6 hr 303C 242+26 557142 1181193 24061173 38261345 6263+355 42571765 9255+1467 v/S 8.33 10.52 8.70 9.04 8.62 5.98 4.79 1.63 0.79 a 1 ppm VC = 2.56 pg VC/2, air Determined from the total radioactivity in the carcass c Mean + standard deviation 14th CalcaJ pal' ;- , 90 ' CMA 001239 TABLE 2 Correlation Between Exposure Concentration of Vinyl Chloride, Metabolism and Induction of Hepatic Angiosarcoma in Rats Exposure Concentration S (ppm VC) 10,000 6,000 2,500 500 250 50 S (pg VC/ air)a 25,600 15,360 6,400 1,280 640 128 Viq VC metabolized*5 v 4 hr 5,521 5,403 5,030 3,413 2,435 739 log v 3.742 3.733 3.702 3.533 3.386 2.869 Percent Incidence of Hepatic Angiosarcoma 15 22 22 12 7 2 a 1 ppm VC = 2.56{^a-Y|) ,Viq VC metabolized. . /f- Vm corrected for 4 hour exposure, 8558 1 6 hr * * q/b qfug VC metabolized. 4 hr ' 57Q6(4ghr'C) * v = --------------------------------------------860 (pg/) + S(pg/) C0N7ij^:'f.r, Subject to Prots.o L j. vo e -lor in Boss y. Conoco, ].v'1, Do. 90-4337 ^TO O c From Maltoni and Lefemine (1975). 14tll Judicial I.lucjlet t\jurt Xalcaolcu Parish, Louisiana o -18- LEGENDS Figure 1. Metabolism of vinyl chloride analyzed in accor dance with the Woolf-Augustinson-Hofstee linearized form of the Michaelis-Menten equation. Values of v and v/S were taken from Table 1. The line was fit by linear regression analysis. The correlation coefficient, R, was 0.88. t Figure 2. (a) Metabolism of vinyl chloride expressed as log v (,y2-g2---V--C----m4~ehtra--b--o---l-i-z--e---d--.) versus . percent . ., incidence , of hepatic angiosarcoma (probability scale)* (b) Exposure concentration expressed as log S (ppm) versus the percent incidence of hepatic angio sarcoma. The probit equivalents of the percent incidence are shown on the right hand ordinant. cMA 001241 CONFIDENTIAL_ ii1;j1;o t co * -'i-1.'. _ i V/ o.i'dor in BSJiLL v . ifo. 50-4837 14hh Judicial District Court Calcasieu Parish, Louisiana 10,000 8,000 6,000 4,000 2,000 n o o Hto ifc> tsJ FIGURE 1 v = "Km v/S + Vm Slope = -Km CO:' LL fiubjiiOt i-:; \iz Ornor .In Eons v. .Couo;.o, Jan. , ho. 30-4837 14 th JuO.l'"rlr.l .OJ strict Court Caloasieu Parish, Louisiana FIGURE 2 Probit n o o t-1 to it* Log v Log S w TOXICOLOGY AND APPLIED PHARMACOLOGY 44, 581-591 (1978) Resolution of Dose-Response Toxicity Data for Chemicals Requiring Metabolic Activation: Example--Vinyl Chloride1 P, J. Gehring,2 P. G. Watanabe,2 and C. N. Park3 Toxicology Research Laboratory, Health and Environmental Research, and Physical Research Laboratory. Math Applications, Dow Chemical U.SA.. Midland. Michigan 48640 Received August 30.1977; accepted November 4,1977 Resolution of Dose-Response Toxicity Data for Chemicals Requiring Metabolic Activation: Example--Vinyl Chloride. Gehring, P. J,, Watanabe. P. G,, and Park, C. N. (1978). Toxicol. Appl. Pharmacol. 44, 581-591. The toxicity of many chemicals results from biotransformation products formed from the chemical rather than from the chemical per se. In such cases, the incremental response may become diminishingly smaller with increasing dose or exposure because activation of the chemical to the toxic form follows apparent MichaelisMenten rather than apparent first-order kinetics. To illustrate this concept, rats were exposed to concentrations ranging from 1,4 to 4600 ppm of vinyl chloride for 6 hr, and the total amount metabolized was determined. The amount metabolized followed apparent Michaelis-Menten kinetics. For rats, the logarithmic probability incidence of angiosarcoma versus the amount of vinyl chloride metabolized rather than the exposure concentration of vinyl chloride is linear. Assuming no threshold in spite of evidence to the contrary, extrapolation of the data below the range of doses causing experimentally observable responses predicted an incidence of 0.01% hepatic angiosarcoma in rats exposed to 4.6 ppm of vinyl chloride. Theoretical extension of the extrapolation to humans after adjusting for metabolic and body mass differences was under taken. The theoretical extrapolation for man exposed daily for 8 hr to 1 ppm suggests an incidence of 1.5 per 100,000.000. This theoretical incidence, although a likely overestimate because of a less than predicted incidence in men exposed to 200 ppm and greater, as well as evidence for a threshold in rats, is less than that expected to occur spontaneously. The concepts evolved from this analysis reveals why pharmacokinetics must be considered in designing toxicology experiments as well as in interpretation of the resulting data. There exists a great deal of uncertainty in predicting the potential response of exposure to chemicals at concentrations below those producing an experimentally discernible response. This is particularly true when the response to the chemical in question is oncogenesis. Statistical projections recommended for assessing the risk of exposure to doses of oncogenic chemicals lower than those producing an observable response include those based on logarithm probability curves (probit curves), logistic curves, or linear -curves (one-hit curves) (FDA Advisory Committee on Protocols for Safety Evaluation, 1971). One of the most commonly used statistical projections for risk assessment has been that promoted by Mantel and Bryan (1961) in which a logarithm probability pro- 1 This study was funded by the companies supporting the vinyl chloride projects being administered by the Manufacturing Chemists Association, Washington, D.C. 2 Health and Environmental Research, Dow Chemical U.S.A.. Midland. Michigan 48640, manuscript No. B 600-176-77. 1 Physical Research Laboratory. Dow Chemical U.S.A.. Midland, Michigan 48640. 58 1 0041-008 X/78 '0443-0581S02.00/0 Copyright C 1978 by Academic Press, Inc. AH rights of reproduction m any form reserved. Printed in Great Britain CMA 001244 582 GEHRING, WATANABE, AND PARK jection with a slope of 1 is utilized. A flaw innate to all of these methods is that the doseresponse information used to make the projection is based on the dose of chemical administered to the animal rather than the quantity of the administered dose giving rise to the response: the latter may either increase or decrease disproportionately as the administered dose is increased. The use of high doses to reveal the chronic toxicity incurred with exposure to a chemical is a common, scientifically defensible practice if judgment and scientific rationale are used in designing the experiments and in assessing the resulting data. However, such doses frequently overwhelm the enzymatic processes for activation of the chemical to the toxic form or for deactivation of the toxic form to an innocuous form. In this paper, it is demonstrated how the dose-dependent activation of vinyl chloride to an oncogenic product must be considered in resolving the dose-response of rats exposed via inhalation to vinyl chloride. METHODS Material. Vinyl chloride (14C-labeled) was synthesized from l,2-dichloro[ 1.214C]ethane (New England Nuclear, Lots, No. 819-221 and 819-292, 5.0 and 4.8 mCi/mmol, respectively) directly prior to use (Wagner et al., 1975). Nonlabeled VC (Matheson Gas Products) of 99.9% purity was mixed with the 14C-labeled material to obtain the desired specific activity. Animals. Male Sprague-Dawley rats (Spartan Research Laboratory) weighing 200250 g were used throughout the study. Food and water were provided ad libitum except during the exposure. Exposures were conducted between 9:00 am and 3 :00 pm (EST). Groups of three to six rats were exposed to various concentrations of 14C-VC for 6 hr. Exposure and procedure. The rats were exposed by inhalation under dynamic conditions in a 30-liter glass inhalation chamber. The mean analytical concentrations of VC measured by gas chromatography were 1.4 0.3 (SD), 9.3 0.2. 24.7 + 1.4, 51 2, 109 23, 250 2, 511 11, 1020 13, and 4600 311 ppm. Details of this exposure and the method of analytical determinations have been reported previously (Watanabe et al.. 1976a). Immediately following the 6-hr exposure to various concentrations of 14C-VC (1.4-4600 ppm), the rats were killed by a blow to the head, and the carcass was analyzed for total radioactivity (Watanabe et al.. 1976b). Previous studies have shown that only a small percentage of radioactivity (< 12%) is excreted as metabolites other than 14C-VC per se during 72 hr following a 6-hr inhalation exposure (Watanabe et al., 1976a). A large proportion of the 12% is comprised of l4CO, excretion 72 hr after exposure. Furthermore, very little urine is excreted during the exposure period. Thus, the nonvolatile radioactivity determined immediately after exposure in the tissue and carcass is a good estimate of the total amount of metabolized VC RESULTS Consistent with the results of previous studies (Watanabe et al., 1976a,b), the metabolism of VC by rats does not increase proportionately with increasing concentrations of VC being inhaled (Table 1). The nonlinearity of the amount of VC 001245 CMA- dose-response: vinyl chloride 583 metabolized during 6 hr of exposure to various concentrations of VC appeared to be in accordance with Michaelis-Vlenten kinetics as described by the equation: In this equation, r and Vm. are the velocity and maximum velocity, respectively, for the biotransformation of VC expressed as microgram equivalents of VC metabolized per 6 hr. 5 and Km are the concentration of VC being inhaled and the Michaelis constant expressed as micrograms of VC per liter of air, respectively. TABLE 1 Parameters for Describing the Metabolism of Inhaled Vinyl Chloride (VC) Using Michaelis-Menten Kinetics Exposure concentration 5 (ppm of VC) S(pg of VC/liter of air)" v(fig of VC metabolized4^ hr) 1.4 9 25 51 109 250 511 1020 4600 3.6 23.0 64.0 130.6 279.0 640.0 1308.2 2611.2 1 1776.0 30 3C 242 26 557 42 1181 93 2406 173 3826 345 6263 355 4257 + 765 9255 z 1467 " 1 ppm of VC = 2.56 fig of VC/liter of air. 4 Determined from the total radioactivity in the carcass. r Mean t SD. v/S 8.33 10.52 8.70 9.04 8.62 5.98 4.79 1.63 0.79 To ascertain whether Michaelis-Menten kinetics were applicable, the data in Table 1 were analyzed in accordance with the linear Woolf-Augustinson-Hofstee trans formation of the Michaelis-Menten equation (Segel. 1976), ' - J + Km- (2) It can be seen from the plot (Fig. 1) that the data appear to lie along a straight line thus verifying, at least visually, the Michaelis-Menten model. Vm and Km can be estimated by the ordinate intercept and the slope of the line or they can be estimated directly by fitting the nonlinear Michaelis-Menten model. Both procedures yield similar parameter estimates. The estimates derived by fitting the model directly are 8558 1147 (SD) fig of VC metabolized and 860 159 (SD) fig of VC/liter of air for Vm and Km respectively. Once a means is obtained to calculate the amount of VC metabolized by rats as a function of exposure, it is then possible to relate the untoward effects associated with VC exposure to the amount biotransformed rather than the exposure concentration of VC per se incurred by rats exposed to VC. CMA 001246 584 GEHR1NG. WATANABE. AND PARK Fig. 1. Metabolism of vinyl chloride analyzed in accordance with the Woolf-Augustinson-Hofstee linearized form of the Michaelis-Menten equation. Values of v and t'/S were taken from Table 1. The line was fit bv linear regression analysis. The correlation coefficient. R, was 0.88. Maltoni and Lefemine (1975) reported the incidence of hepatic angiosarcoma in rats exposed to different concentrations of VC. 4 hr/day. 5 days/week for 12 months and subsequently held for observation until death (Table 2). Before attempting to relate these data to the amount of VC biotransformed in accordance with the MichaelisMenten equation using the previously determined values of Vm and Km, the value for Vm TABLE 2 Correlation between Exposure Concentration of Vinyl Chloride, Metabolism and Induction of Hepatic Angiosarcoma in Rats Exposure concentration 5(ppm of VC) S(,ug of VC/ liter of air)a v{p% of VC metabolized4/4 hr) log V Percentage incidence of hepatic angiosarcoma1' 10.000 6.000 2.500 500 250 50 25.600 15.360 6,400 1,280 640 128 5.521 5,403 5,030 3,413 2.435 739 3.742 3.733 3.702 3.533 3.386 2.869 15 22 22 12 7 2 .!.('*0,vcl u 1 ppm of VC I- \liter of air) . (ui of VC metabolized\ 4 corrected for 4-hr exposure. 8558 1 ----------- --------------- 1 4/6 = 5706 - (Ml of VC metabolized^ IS / : [lMl aof VC\ 5706 ) SOug/Iiter) hr / e From Maltoni and Lefemine (1975), [860Uig/liter) + SOig/liter)l. --90-433'. iKft ^ SC ct Co'J^ Cal'-145 CMA 001247 DOST response: vinyl chloride 585 must be adjusted for the shorter exposure duration used by Maltoni and Lefemine. 4 versus 6 hr. This adjustment is accomplished by multiplying Vm by 4/6. Thus, the amount of VC biotransformed daily by rats exposed to the various concentrations used in the experiment of Maltoni and Lefemine can be calculated from the equation: i706 c= 860 -S 4 hr / \liter MS\+sl^\ liter! liter O) The resulting values for c are given in Table 2. Figure 2A, depicts a logarithm probability plot (probit plot) of the incidence of hepatic angiosarcoma observed in rats by Maltoni and Lefemine (1975) versus the amount of VC biotransformed for 4 hr of exposure, i\ or the exposure concentration. 5. The incidence of hepatic angiosarcoma in rats is linear with respect to log v but not log S. The line drawn for log v versus tumor incidence (Fig. 2) was determined by using a probit regression analysis program, and the equation relating the incidence of hepatic angiosarcoma to log v was: probit response = --1.625 + 1.543 log v (4) Using the foregoing equation, a projection below the levels of exposure producing an experimentally discernible response has been made (dashed line). Assuming no Of Tumon Pro6*t 5.0Q Pirctm IncMint Of Tumor* Profati $.00 Fio. 2. (A) Metabolism of vinyl chloride expressed as log v (Micrograms of VC metabolized/4 hr) versus percentage incidence of hepatic angiosarcoma (probability scale). (B) Exposure concentration expressed as log 5 (parts per million) versus the percentage incidence of hepatic angiosarcoma. The probit equivalents of the percentage incidence are shown on the right-hand ordinate. The solid line is the best lit for experimentally observed responses while the dashed line represents extrapolation below those doses producing an observable response assuming no threshold. '"TV ,* L log. TPyioia-1 vs- CMA 001248 586 GEHRING, WATANABE, AND PARK threshold for the induction of angiosarcoma in rats exposed to VC, the exposure concentration producing one angiosarcoma in 10,000 rats can be calculated. The probit percentage representing an incidence of 0.01% is 1.28. Substitution of this value into the Eq. t-1) yields: Log v = 1.8827, c = 76.33 fig of VC metabolized/4 hr Using Eq. (3). the concentration of exposure to VC needed to give this value for v is 11.66 ^g/liter or 4.6 ppm [approximate 95% confidence limits obtained by substituting the upper and lower 95% confidence limits for v in Eq. (3) and solving for 5 are 0.038.7 ppm]. Hence, exposure of rats to 4.6 ppm of VC for 4 hr daily, 5 days/week for 1 year can be expected to produce one angiosarcoma per 10,000 rats if the dose-response curve remains valid at exposures, less than those producing a discernible experimental response. DISCUSSION For many chemicals, toxicity may not be a function of exposure to the chemical per se, but rather to a biotransformation product of the chemical. Frequently, production of a toxic metabolite is dependent upon enyzmatically mediated reactions which are classically described by Michaelis-Menten kinetics. Since enzymatically mediated reactions are concentration dependent and saturable, toxicity resulting from exposures to chemicals requiring activation to a toxic form cannot be related directly to the magnitude of exposure or dose. In such a case, it is necessary to determine the amount of the chemical undergoing biotransformation as a function of dose or exposure before a meaningful dose-response relationship can be established. There is considerable evidence that vinyl chloride requires bioactivation to produce tumors. Metabolic activation is required to induce mutations in bacteria exposed to VC (Bartsch et al.. 1975; Malavielle et at., 1975; Rannug et at.. 1974). Covalent binding of ,4C to hepatic macromolecules in rats (Watanabe et at., 1978) exposed to UC-VC also requires bioactivation. Covalent binding of electrophiles to DNA has been associated with tumorigenesis. For vinyl chloride-induced hepatic angiosarcoma in rats, a logarithmic probability plot (probit plot) of the incidence versus the amount of vinyl chloride metabolized, v, over a range of exposures from 50 to 10,000 ppm of VC gives a classical straight line (Fig. 2A). The dose-response relationship is not a straight line when plotted as a function of the exposure concentration, S (Fig. 2B), These results support further the conclusion that VC requires biotransformation to an active metabolite for tumori genesis. Furthermore, a more reasonable evaluation of the dose-response data for vinyl chloride-induced tumorigenesis requires knowledge of the amount of VC activated as a function of exposure. After arbitrarily excluding data acquired from rats exposed to concentrations of VC exceeding 500 ppm in the experiment of Maltoni and Lefemine (1975), Schneidcrman et al. (1975) extrapolated the remaining data to predict an incidence of 0.01% hepatic angiosarcoma in rats exposed to 1 ppm of VC. This number is reasonably dose to our prediction of 4.6 ppm of VC for the same incidence. If Schneiderman et al. (1975) had used all of the data, a dose-response curve with an CMA 001249 dose-response: vinyl chloride 587 unrealistically shallow slope would have resulted and the predicted level causing 0.01% hepatic angiosarcoma would have been much smaller, on the order of 0.00001 ppm. The concepts developed herein allow use of all of the data presented by Maltoni and Lefemine (1975) to construct a dose-response curve on a scientifically defensible basis. Assuming that the resulting dose-response curve can be projected beyond the range of the experimentally discernible responses, the exposure concentration required to produce an incidence of 0.01% hepatic angiosarcoma in rats is 4.6 ppm. Aside from interpreting toxicity data for chemicals requiring activation to a toxic form, there are some practical implications of the concepts presented herein for designing experiments to assess the toxocity, including carcinogenicity, of such chemicals. For these chemicals, increasing the concentration above the apparent Km will produce diminishingly smaller increments in the response; no increase in the response is to be expected when the exposure concentration is two or three times Km. Since total dose is a function of exposure time as well as concentration, it is important to determine the effect of exposure time on the response. As shown in Eq. (3), the only parameter influenced by exposure time is Vm, which is increased linearly with time. Therefore, after the concentration to which the animals are exposed becomes two to three times Km, the amount metabolized, v, will increase linearly with increasing exposure time. For this reason, the gradation of incidence of angiosarcoma in rats exposed to high concentrations of VC will become a function of exposure time rather than concentration. This reasoning makes it imperative that the duration of exposure as well as exposure concentration be considered in evaluating the results of epidemio logical studies of people exposed to high concentrations of VC in the work environment. Unless the dose-dependent, Michaclis-Menten type pharmacokinetic parameters are resolved prior to designing the experiment, the results may be robbed of much of their value for characterizing the dose-response function for the untoward effects observed. Thus, the current approach using the maximum tolerated dose as defined presently and fractions thereof may be scientifically unsound if the objective is to assess the potential toxicity of exposure to much lower doses or exposures. For some chemicals detoxifica tion of the chemical per se or reactive metabolites formed from the chemical may also be dose-dependent and saturable leading to a build-up of toxic materials. In such cases, the incremental responses to increasing doses or exposures will become disproportionately larger rather than smaller (Gehring and Blau, 1977). In the foregoing analysis of the dose-response data for the induction of angio sarcoma in the rat, no threshold for the response was assumed. As indicated, extra polation below the range of doses resulting in an observable response may overestimate the response in rats because there is evidence that detoxification of reactive metabolites of VC may occur more efficiently in rats exposed to concentrations of VC below 50 ppm (Watanabe et ai, 1976c). Indeed, further analysis of the data reported by Maltoni and Lefemine (1975) also provides an indication of a practical threshold. In a subse quent presentation of these data, it was revealed that the latencies for the development of hepatic angiosarcoma were, respectively, 64, 70, 78, 81, 79, and 135 weeks for rats exposed to 10,000, 6000, 2500, 500, 250, and 50 ppm of VC (Maltoni, 1975). These results indicate that at some low levels of exposure the time required for induction may exceed considerably the life expectancy for rats. This is consistent with the work of others suggesting that multiples of a lifetime may be required for expression of cancer in CMA 001250 588 GEHR1NG, WATANABE, AND PARK response to low doses of a carcinogen (Druckrey, 1967; Albert and Altshuler, 1973). Thus, extrapolation of the data obtained for rats below the range of exposures causing a discernible response may be expected to overestimate the projected incidence. The ultimate objective of a toxicological study is to develop data which can be used to assess the potential risk for man. It is worthwhile to utilize the concepts presented herein to achieve this objective realizing fully that such extrapolation is fraught with uncertainties. The basic assumptions made are: (i) Induction of angiosarcoma is related to the amount of reactive metabolite of VC per unit of mass. (ii) Exposure of rats for 12 months approximates exposure of workers for their working life, (iii) There is no threshold for the induction of angiosarcoma in either rats or man which likely overestimates the assumption of risk as discussed above. (iv) The efficiency of the metabolic processes involved in the conversion of VC to the reactive form is proportional to the body surface area. Since data for the bio transformation of VC by man are not available, the most logical basis for translation of the animal data to man would seem to be on the basis of body surface area. The last assumption deserves comment. There are considerable data in the literature showing that metabolism in general and other physiological parameters as well are relatable directly to the surface area of the body (Schmidt-Nielsen, 1970; Pinkel, 1958). For this reason, administration of biologically active chemicals to various species frequently gives an equivalent response when the dose is administered in proportion to the surface area of the body, that is dose per square meter of body surface (Pinkel, 1958). This relationship is gaining recognition in estimating the risk incurred by man from exposure to chemicals in the environment (Committee on Safe Drinking Water. National Research Council, 1977). In utilizing this relationship it must, however, be recognized that the original relationship was developed for biologically active agents. Since metabolism and other physiological processes involved in detoxification are more active in smaller animals, the dose of a biologically active chemical per unit of mass required to produce a given effect increases as the body decreases, while the dose per unit surface area remains relatively constant. However, for a chemical requiring activation to the biologically active toxic form, the total amount transformed will be roughly proportional to the body surface area. Since toxicity is a function of the concentration of the active form in tissue, this total amount transformed must then be normalized for mass to estimate an equivalent response. Using aforementioned rationale, the maximum velocity, Vm, for a 70 kg man can be estimated by calculation using the Vm value obtained for a 0.250-kg rat. The Vm of man - for VC will be; 1.85 m2 0.045 m or CMA 001251 dose-response: vinyl chloride 589 where the values 1.85 and 0.045 m* are the body surface areas of a 70-kg man and a 0.250-kg rat. respectively (Pinkel. 1958). For an 8-hr exposure, the value is 469.105 jtg/8 hr. In order to use this number to theoretically estimate the response in man using data collected in rats, the Vm for man must be adjusted to a mass equivalent to that of rats since toxicity is a function of concentration in tissue. To do this the number is divided by 70 kg/0.25 kg or 280. The resulting Vm for man on a mass equivalent basis to that of rats is 1675 ,ug/8 hr. Using this value, the amount of VC transformed to a reactive form by man on a mass equivalent basis to rat is given by the equation: 1675Jug/8hr-50tg/liter) t'0tg/8 hr) = 860 /xg/liter + 5"(ug/liter) (5) Using this equation, the amount of VC transformed by man on a mass equivalent basis to rats was calculated as a function of exposure concentration and the expected incidence of angiosarcoma estimated from Eq. (4) (Table 3). For men exposed to greater than 200 ppm of VC, the incidence of angiosarcoma, has been reported to be 0.02% (Fox and Collier. 1977). Hence, the theoretical calculated incidence using data from rats exceeds that currently detected by approximately 50-fold. This may indicate that people are less sensitive than rats to the induction of angiosarcoma, or it may indicate that a practical threshold for the induction of angiosarcoma had been attained. Consistent with this latter possibility is that v on a mass equivalent basis to rats for men exposed to 200 ppm is 625. This number lies below that for rats exposed to 50 ppm. As indicated previously, angio sarcoma observed in rats exposed to 50 ppm occurred only in a rat that lived 135 weeks (Maltoni, 1975). Further, this rat did not die as a result of the angiosarcoma but was killed. Thus, as indicated previously, projection of the data collected in rats below the range of exposures producing an observable response may overestimate the incidence. TABLE 3 Theoretical Amounts of Reactive Product Formed from VC by a 70-kg Man Exposed Continuously for 8 hr and the Corresponding Expected Incidence of Angiosarcoma as Predicted from Data Collected in Rats Assuming no Threshold Exposure concentration v(pg of VC meiabolized('/8 hr) log V Probit response1' Theoretical percentage incidence of angiosarcoma1' (ppm) 200 .50 5 1 (jtg/liter)" 512 128 12.8 2.56 625 217 24.6 4.97 2.79 2.34 1.39 0.70 2.68 1.98 0.52 -0.54 1.02 0.11 3.74 x 10-J 1.5 x 10'1' " I ppm = 2.56 >ig/liter. 4 r has been calculated using the Michaelis-Menten equation after calculating the for man from the Va determined for rats. This calculation was made by assuming Vm is proportional to body surface area and subsequently adjusting it to a mass equivalent to that of rats. See teat. ` The expected probit response was calculated from probit Eq. (3) (see text). Subsequently the theoretical percentage incidence of angiosarcoma was determined from the respective probit. CL _ T T-G * r-iVOtV T.oss,v' x mi - " CMA 001252 590 GEHRING, WATANABE. AND PARK In spite of the likelihood of overestimating the incidence of angiosarcoma in rats or man, the incidence predicted for people exposed to 1 ppm, the current OSHA (Occupational Safety and Health Act) standard, is very small (1.5 per 100 million). This value which is likely an overestimate is below the expected incidence of spontaneous angiosarcoma reported to be 20 to 25 cases in the U.S. annually (Makk et al.. 1976). In summary, it has been demonstrated that the incidence of VC induced angio sarcoma in rats is related not to the concentration of exposure but rather to the amount of VC biotransformed. Biotransformation of VC by rats is a dose-dependent process characterized by Michaelis-Menten type kinetics. The concepts evolved from this analysis reveal why pharmacokinetics must be considered in designing toxicology experiments as well as in interpretation of the resulting data. Having characterized the dose-response for induction of angiosarcoma in rats as a function of the amount of VC biotransformed, a theoretical estimate of the incidence expected to occur in exposed men was undertaken using the data collected in rats. For daily 8-hr exposures to 1 ppm. the predicted incidence is 1.5 in 100,000,000 which is less than that expected to occur spontaneously. This predicted incidence is likely an onverestimate of that which will occur as a result of exposure to 1 ppm because there is some evidence for at least a practical threshold in both rats and man. There are no illusions that this estimate by extrapolation of data outside the range of doses causing experimentally observable responses and subsequently to man is without flaws. However, the rationale used represents a new approach which utilizes more logic than methods employed currently for such extrapolation. REFERENCES Albert. R. E., and Altschuler. B. (1973). 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