Document ZnZBJ0K3QEpQK1gXzwKbknyaL

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 Toxicology Research Laboratory Health and Environmental Research Dow Chemical U.S.A. Midland, Michigan 48640 This study was funded by the, companies 4u.ppoA.ttng the vinyl chloride projects being administered by the Manufacturing Chemists Association, Washington, V.C. ASI 000019469 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 t 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 relationship for chemicals requiring metabolic activation to a toxic form. ASI 00OOJ947O -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 mo'st 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 ASI 00001947] -2tumorigenic product must be considered in resolving the dose-response to vinyl chloride exposure in rats. This concept leads to a much more meaningful interpretation ol: the dose-response data and permits more meaningful extrapo lations of that data. AS I 00001 9472 -3- METHODS Material. Vinyl chloride ("^C-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/mmole, respectively) directly prior to use (Wagner, et al. , 1975). Non-labeled VC (Matheson Gas Products) of 99.9% purity was mixed with the 14C-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 Z glass inhalation chamber. The mean analytical concentrations of VC measured by gas chromatography were 1.4 + 0.3 (SD) , 9.30.2, 24.7 + 1.4 , 51 + 2, 109 + 23, 2502, 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 AS I 00001 9473 -4the 6-hour exposure to various concentrations of 14C-VC (X. 4-4600 ppm) the rats were killed by a blow to the head, and the carcass was analyzed for total radioactivity (Watanabe, et: a_l. , 1976b). Since radioactivity found in the carcass was non-volatile, this radioactivity represented the total amount of VC which was metabolized. AS I 000019474 5- - RESULTS Consistent with the results of previous studies (Watanabe, e't al. , 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: VS V K +S m (1) 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 Michaelis constant expressed as yg VC/% 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 = ~Kn'i l + Vm <2> ASI 000019475 -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 mm 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 860+159 (SD) yg VC/H 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 bhe 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 Vmanmd K , the value for Vm must be adjusted for the shorter exposure duration used by AS I 00001 9476 7- - Maltoni and Lefemine, 4 hours versus 6 hours. This adjust ment 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 : 5706 ^ , s,jja, v 860 (2, + S(H2) (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) ASI 000019477 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 pg VC metabolized/4 hours Using equation 3, the concentration of exposure to VC needed to give this value for v is 11.66 pg/f 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. 00001 9478 -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_ a_l., 1975; Malavielle, et ad., 1975; Rannug, 14 et ad., 1974) . Covalent binding of C to hepatic macro molecules in rats (Watanabe, et al^. , 1977) or in liver homogenates (Kappus, et. ad., 1976) exposed to ^C-VC also requires bioactivation. Covalent binding of electrophiles to DNA has been associated with tumorigenesis. AS 1 00001 9479 -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 arid the predicted level causing 0.01% hepatic angiosarcoma would have been even smaller. AS I 000019480 -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 efficientlv in rats exposed to concentrations of VC below 50 ppm (Watanabe, t al_. , ,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 Km will produce diminishingly smaller increments in the response; AS 1 0000) 9481 -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 4 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. AS I 000019482 -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. AS I 00001 9483 -14- REFERENCES Bartsch, H., Malavielle, C.f 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 o-f 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. ASI 0000.19484 -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. 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. Watanabe, P. G., McGowan, G. R., Madrid, E. 0., and Gehring, P. J., (1976a). Fate of "^C-vinyl chloride following inhalation exposure in rats. 37, 49-59. Toxicol.'Appl. Pharmacol., Watanabe, P. G., McGowan, G. R., and Gehring, P. J., (1976b). 14 Fate of C-vinyl chloride after single oral administra tion in rats, Toxicol. Appl. Pharmacol., 3_6, 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, , 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. AS] 000019485 -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 S (yg VC/i air)a 3.6 23.0 64.0 130.6 279.0 640.0 1308.2 2611.2 11776.0 . yg VC metabolized*3 6 hr 303C 242+26 557+42 1181+93 2406+173 3826+345 6263+355 4"257 + 76 5 9255+1467 v/S 8.33 10.52 8.70 9.04 8.62 5.98 4.79 1.63 0.79 1 ppm VC = 2.56 yg VC./i air Determined from the total radioactivity in the carcass Mean + standard deviation ASI 000019486 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 ,,pg VC metabolized13 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 vc = 2*56<FiTF> b Vm corrected for 4 hour exposure, 8558 (U9 VC metabolize^ _ 4/g = 5706 (ug VC metabolized, 5706 (42h?^) 860 (pgA) + S (jjg/) c From Maltoni and Lefemine {1975} . As i 000019487 -18LEGENDS 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. Figure 2 (a) Metabolism of vinyl chloride expressed as log v ,yg VC metabolized. .... ----------4"hr-----------------) 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. ASI 000019488 FIGURE 1 ASI 000019489 FIGURE 2 Percent Incidence Of Tumors Probit