Document DvXXGjmr6J0LG0e3rVdyeomLQ

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 July 12, 1977 Toxicology Research Laboratory Health and Environmental Research Dow Chemical, U.S.A. Midland, Michigan 48640 This study ivas funded by the companies supporting the vinyl chloride, projects being administered by the Manufacturing Chemists Association, Washington, V.C. CMA 001158 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. 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 inspite 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 vinyl chloride. Theoretical extention of the extrapolation to humans after adjusting for metabolic and body mass differences was under taken. The theoretical extrapolation for man exposed daily for 3 hours to 1 ppm suggests an incidence of 1.5 per 1 ,300. This theoretical incidence, although a likely CMA 001159 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 spontane ously. It is concluded that pharmacokinetic parameters must be elucidated before designing toxicological experiments or before interpreting the results therefrom. CMA 001160 1- - IMTRODUCTION There exists a great deal of uncertainty in predicting the potential response of exposure tc chemicals at concentra tions 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 less 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 projection with a slope of one is utilized. A flaw innate to all of these methods is that the dose-response 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. Tne use of high doses to reveal the chronic toxicity incurred with exposure to a chemical is a common, scientifically CMA 001161 defensible practice if judgment and scientific rationale is used in designing the experiments and in assessing the resulting data. Such doses overwhelm frequently 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. CMA 001162 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 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 14C-VC for 6 hours. Exposure and Procedure. The rats were exposed by inhalation under dynamic conditions in a 30 2, 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, e_t al. , (1976a) . Immediately following CMA 001163 the 6-hou.r exposure to various concentrations of ^C-VC (1.4-4600 ppm) the rats were killed bv 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 metabolized. CMA 001164 RESULTS Consistent with the results of previous studies (Watanabe, et a_l. , 1976a and 1976b) , 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 metabolized during 6 hours of exposure to various concentrations of VC appeared to be in accordance with Michaelis-Menten kinetics as described bv the eauation: v Km + S (1) In this eauation, v and Vm , are the velocity and maximum velocity respectively for the biotransformation of VC expressed as ug equivalents VC metabolized per 6 hours. S and are the concentration of VC being inhaled and the Michaelis constant expressed as pg VC/7 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 ^o + Vm (2) CMA 001165 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. 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 bv fitting the model directly are 85581147 (SD) ug VC metabolized and 860159 (SD) yg VC/l air for V and K respective.lv. 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. 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 V and K , the value for V mm m nu.it be adjusted for the shorter exposure duration used bv CMA 001166 Maltoni and Lefemine, 4 hours versus 6 hours. This adjust ment is accomplished by multiplying bv 4/6. Thus, the amount of VC biotransformed dailv by rats exposed to the various concentrations used in the experiment of Maltoni and Lefemine can be calculated from the equation: 860 (H2) + S(^f-) (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 observed in rats bv Maltoni and Lefemine (1975) versus the amount of VC biotransformed for 4 hours of exposure, v, or the exposure concentration, S. 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 (Figure 2) was deter mined 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) CMA 001167 8- - Using -the foregoing equation, a projection below the levels of exposure producing an experimentally discernible response has been made (dashed line). Assuming no 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 percent representing an incidence of 0.01s is 1.28. Substitution of this value into the equation (4) yields: log v = 1.8827 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/2, or 4.6 ppm (approximate 95% confidence limits obtained by substituting the upper and lower 95% confidence limits for v in equation 3 and solving for S are 0.03-8.7 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. CMA 001168 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 a 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 biotransfornation as a function of dose or exposure before a meaning ful 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 (3artsch, et al., 1975; Malavielle, et al., 1975; Rannug, et al., 1974). Covalent binding of 14 C to hepatic macromolecules in rats (Watanabe, et al., 1977) exposed to 14C-VC also requires bioactivation. Covalent binding of electrochi .cs to DNA has been associated with tumorigenesis. CMA 001169 -10- For vinyl ehloride-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. After arbitrarily excluding data acquired fro'm rats exposed to concentrations of VC exceeding 500 ppm in the experiment of Maltoni and Lefemine (1975), Schneiderman, et al (1975) extrapolated the remaining data to predict an incidence of 0.01% hepatic angiosarcoma in rats exposed to 1 ppm VC. This number is reasonably close to our prediction of 4.6 ppm VC for the same incidence. If Schneiderman, et al_ had used all of the data, a dose-response curve with an unrealistically shallow slope would have resulted and the predicted level causing 0.01% hepatic angiosarcoma would have been much snallcr, on the order of 0.00001 ppm. CMA 001170 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 in rats is 4.6 ppm. Aside from interpreting toxicity data for chemicals requiring activation to a toxic form, there are some practical implica tions of the concepts presented herein for designing experi- ' ments to assess the toxicity, including carcinogenicity, of such chemicals. For these chemicals, increasing the concen tration above the apparent will produce diminishinglv smaller increments in the response; no increase in the response is to be expected when the exposure concentration is 2 or 3 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 Equation 3, the only parameter influence by exoosure time is Vm , which is increased linearly with time. Therefore, after the concentration to which the animals are exD* osed becomes 2 to 3 times Km , the amount metabolized, v, will increase linearly with increasing exposure time. For CMA 001171 -12' this reason, the gradation of incidence of angiosarcoma in rats exposed to high concentrations of VC will become a function of exnosure 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 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 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 scientifi cally unsound if the objective is to assess the potential toxicity of exposure to much lower doses or exposures. For some chemicals detoxification 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 dispropor tionately larger rather than smaller (see Gehring and Blau, '-j7T) . CMA 001172 In the foregoing analysis of the dose-response data for the induction of angiosarcoma in the rat, no threshold for the response was assumed. As indicated extrapolation below the range of doses causing an observable response may over estimate 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 al, 1976c). Indeed, further analysis of the data reported by Maltoni and Lefemine (1975) also provides an indication of a practical threshold. In a subsequent presentation of these data, it was revealed that the latency for the development of hepatic angiosarcoma was respectively 64, 70, 78, 81, 79 and 135 weeks for rats exposed to 10,000, 6,000, 2,500, 500, 250 and 50 ppm VC (Maltoni, 1975). These results indicate that at the low level of exposure the time required for induction exceeds considerably the mean life expectancy for rats of approximately 104 weeks. This is consistent with the work of others suggesting that multiples of a lifetime may be required for expression of cancer in response to low doses of a carcinogen (Druckney, 1967 and 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 or^'^cted incidence. CMA 001173 -14- The ultimate objective of a toxicological study is to develop data which can be used to assess the potential risk in 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: 1) Induction of angiosarcoma is related to the amount of reactive metabolite of VC per unit of mass. 2) Exposure of rats for 12 months approximates exposure of workers for their working life. 3) There is no threshold for the induction of angiosarcoma in either rats or man which probably overestimates the assumption of risk as discussed above. 4) The efficiency of the metabolic processes involved in the conversion of VC to the reactive form is propor tional to the body surface area. Since data for the biotransformation 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. CMA 001174 -15- 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 (see Schmidt-Nielsen, 1970 and Pinkel, 1958). For this reason, administration of biolo gically 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 relation ship 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 mass 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 transfromed must then be normalized for mass to estimate an equivalent response. CMA 001175 -16- Using aforementioned rationale, the maximum velocity, V , for a 70 kg man can be estimated by calculation using the V value obtained for a 0.250 kg rat. The Vm of man for VC will be : Vm (man) or Vm (man) = (8558 yg/6 hr)( 1.85 0.045 sgsqmm) = 351829 yg/6 hr where the values 1.85 and 0.045 sq m are the body surface areas of a 70 kg man and a 0.250 kg rat, respectively (Pinkel, 1958) . For an 8 hour exposure, the value is 469105 eg/8 hr. In order to use this number to theoreti cally 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 V^ for man on a mass equivalent basis to that of rats is 1675 yg/8 hr. Using this value, the amount of VC transformed to a reactive form by man on a mass equiva lent basis to rat is given by the equation: 1675 ug/8 hr . S \ig/l V eg/8 hr = 860 yg+ S eg/l (5) CMA 001176 -17- 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 angio sarcoma estimated from equation 4 (Table 3). For men exposed to greater than 200 ppm 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, angiosarcoma observed in rats exposed to 50 ppm occurred only in a rat that lived 135 weeks. 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. CMA 001177 -18- Inspite 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 angiosarcoma in rats is relatable not to the concentration of exposure but rather to the amount of VC biotransformed. Biotransformation of VC by rats is a dosedependent process characterized by Michaelis-Menten type kinetics. 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. Having characterized the dose-response for induction 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 CMA 001178 incidence "is likely an over-estimate 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 extrapo lation of data outside the range of doses causing experi mentally 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. CMA 001179 REFERENCES Albert, R. E. and Altschuler, B. (1973). Considerations relating to the formutation of limits for unavailable population exposures to environmental carcinogens. Radionuclide Carcinogenesis, Proceedings of the 12th Annual Hanford Biology Symposium at Richland, Washington, 234-253. 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. Committee on Safe Drinking Water (1977). Summary Report: Drinking Water and Health, Advisory Center on Toxicology, Assembly of Life Sciences, Washington, D.C. Druckney, H. (1967). Quantitative aspects in chemical carcinogenesis. In Potential Carcinogenic Hazards From Drugs. Evaluation of Risks, R. Trubant, Ed., UICC Monograph Series, Vol. 7, Springer-Verlag, Berlin, 60-78. Food and Drug Administration Advisory Committee on Protocols for Safety Evaluation (1971) Panel on Carcinogenesis Report on Cancer Testing in Safety Evaluation of Food Additives and Pesticides, Tox. Appl. Pharmacol., 20: 419-438 . CMA 001180 -21- Fox, J._ and Collier, P. F. (1977) . Mortality experience of workers exposed to vinyl chloride monomer in the manufacture of polyvinyl chloride in Great Britain, Brit. J. Ind. Med., 34:1-10. 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. Makk, L., Delmore, F., Creech, J. L., Ogden, L. L., Fadell, E. H., Songster, C. L., Clanton, J., Johnson, M. N. and Christopherson, W. H. (1976). Clinical and morphologic effects of hepatic angiosarcoma in vinyl chloride workers. Cancer, 37:149-163. Malavielle, C., Bartsch, H., Barbin, A., Camus, A. M., and Montesano, R. (1975) . Mutagenicity of vinyl chloride chloroethyleneoxide, chloroacetaldehvde and chloroethanol. Biochem. Biophys. Res. Comm., 63, 363-370. Maltoni, C. (1975) . The value of predictive experimental environmental carcinogenesis. An example: vinyl chloride. Ambio, 4_:18-23. Malconi, C. and Lefemine, G. (1975). Carcinogenicity assays of vinyl chloride: Current results. Ann. N.Y. Acad. Sci., 246, 195-224. CMA OOliai -22- Mantel, N^ and Bryan, W. R. (1961) . "Safety" testing of carcinogenic agents. J. Nat. Cancer Inst., 27, 455-470. Pinkel, D. (1958). The use of body surface area as a criterion of drug dosage in cancer chemotherapy. Res. , 18:853-856. Cancer Rannug, U., Johansson, A., Hamel, C. and Wachtmeister, C. A. (1974) . The mutagenicity of vinyl chloride after metabolic activation, Ambio, 3, 194-197. Schmidt-Nielsen, K. (1970). Energy metabolism body size, and problems of scaling, Fed. Proc., 29:1524-1532. Schneiderman, M. A., Mantel, N. and Brown, C. C. (1975). From mouse to man - or how to get from the laboratorv 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 ^C-labeled vinyl chloride, J. Labeled Compounds, 11, 535-542. CMA 001182 -23- Watanabe, P-. G., McGowan, G. R., Madrid, E. 0., and Gehring, P. J. (1976a). Fate of 14 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 14C-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 001183 -24- LEGENDS Figure 1. Metabolism of vinyl chloride analyzed in accordance 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 (,u"g---V--C----m--^etharb--o--l-i-z--e---d- ,) versus percen.t i.ncid. e. nce 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. The solid line is the best fit for experimentally observed responses while the dashed line represents extrapolation below those doses producing an observable response assuming no threshold. CMA 001184 FIGURE 001185 FIGURE 00X186 (a) o Per tent Incuicnce 01 Tumors (b) Prutnt 600 > 4.00 3.00 2.00 1.2B -27- 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 (ug VC/2, air)3 3.6 23.0 64.0 130.6 279.0 640.0 1308.2 2611.2 11776.0 _yg VC metabolized 6 hr 303C 242+26 557142 1181193 24061173 38261345 62631355 42571765 925511467 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 yg VC/9, air b Determined from the total radioactivity in the carcass c Mean i standard deviation CMA 001187 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/Z air)a 25,600 15,360 6,400 1,280 640 128 ug VC metabolized 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 b,, . , ,, ,, occo ,pg VC metabolized* ,, Vm corrected for 4 hour exposure, 8558 (2---------gThr-------------- * * 4'6 5706(42hF') * S^/!L) v - ------------------------------------- ---- 860 (pg/JL) + S(pg/) ,pg VC metabolized* i/Ub (--2--------4"hr--------------- c From Maltoni and Lefemine (1975). 001188 TABLE 3 Theoretical Amounts of Reactive Product Formed From VC Bv a 70 kq Man Exposed | Continuously For 8 Hours and the Corresponding Expected Incidence of ,, Angiosarcoma as Predicted From Data Collected in Rats Assuming No Threshold Exposure Concentration EETM \ig/la 200 512 50 128 5 12.8 1 2.56 yig VC metabolized13 v 8 hr 625 217 24.6 4.97 Log v 2.79 2.34 1.39 0.70 Probit rj Response Theoretical Percent Incidence of Angiosarcoma0 2.68 1.98 0.52 -0.54 1.02 0.11 3.74 x 10-4 1.5 x 10~6 NiJ kO I a 1 ppm = 2.56 ug/Jl. b v has been calculated using the Michaelis-Menten equation after calculating the V for man HlclX from the Vmax determined for rats. This calculation was made by assuming Vmax is proportional to body surface area snd subsequently adjusting it to a mass equivalent to that of rats. See text. The expected probit response was calculated from probit equation 3 (text). Subsequently the theoretical percent incidence of angiosarcoma was determined from the respective probit. CMA 001189