Document NEB59Ov91gM5aygGNYYjL8eJy

R&S 021514 r K-/V//'d'35) TOXIl-OLOCV ANDAPPUt'll PHARMACOLOGY 44, J8I-JH < IVTSl Resolution of Dose-Response Toxicity Data for Chemicals Requiring Metabolic Activation: Example--Vinyl Chloride* P. J. Gehring,2 P, G. Watanabe.2 and C. N. Park' Toxicofafir Research Laboratory. Health and Environmental Research, and Physical Research l.aboratorv. Math Applications, Dow Chemical L\SA., Midland, Michigan J8640 Received August JO, 1977: aecepted Sovember 4. 197? Resolution of Dose-Response Toxicity Data for Chemicals Requiring Metabolic Actuation: Example--Vinyl Chloride. GiHKiMi, P, J.. Waianaiik P. G.. a\i> Park. C. N. <1478). Toxicol. Appl. Pharmacol. 44. 581-591. The toxicity of many chemicals results from hiotr&nsfnrmation products formed from the chemical rather than from the chemical per se. In such cases, the incremental response may become diminishing)}' smaller with increasing dose or exposure because activation of the chemical to the toxic form follows apparent MichaclisMcntcn 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 Michaclis-Mcnten 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 Tor man exposed daily for 8 hr to I 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 (hat 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. 1 Health and environmental Research. Dow Chemical U.S.A.. Midland. Michigan 48640. manuscript No. B 600-176-77. * Physical Research Laboratory, Dow Chemical U.S.A., Midland. Michigan 48640. 581 <KMl-O0tX/7ft/<U4M><StSO2.ttV0 Copyright <0 I9?g tty Acedcmic Ptn*. Inc. All rights ofrtproJgcttnn in ny form reserved. Frttued in Grcti Brium "3.1V R&S 021515 GliHHlNO, XVATANAHE. AND park jcction with a slope of I 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 arc used in designing the experiments and in assessing the resulting data. However, such doses frequently overwhelm the enzymatic processes lor activation ofthe chemical to the toxic form or for deactivation of the toxic form to an innocuous form. In this paper, it is demonstrated how the dosc-depcndcnt activation of vinyl chloride to an oncogenic product must be considered in resolving the dose-response or rats exposed via inhalation to vinyl chloride. METHODS Material. Vinyl chloride (,4C-labclcd) was synthesized from l,2-dichIoro( 1.2HClethanc (New England Nuclear. Lots. No. 819-221 and 819-292. 5.0 and 4.8 mCi/mmol, respectively) directly prior to use (Wagner el a!.. 1975). Nonlabelcd VC (Maiheson Gas Products) of 99.9% purity was mixed with the 14C-labeled material to obtain the desired specific activity. Animals. Male Spraguc-Dawlcy 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 1JC-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 31 Lppm. Details of this exposure and the method of analytical determinations have been reported previously (Watanabc et al.. 1976a). Immediately following the 6-hr exposure to various concentrations of l4C-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 UC-VC per sc during 72 hr following a 6-hr inhalation exposure (Watanabc et al., 1976a). A large proportion of the 12% is comprised of 14CO, 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 or metabolized VC. u ; * ' ? |- RESULTS " --; , Consistent with the results of previous studies (Watanabc et at., 1976a,b)t 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 -f-, . metabolized during f accordance with Mic In this equation, r aij biotransformalion of| hr. .5' and arc ih expressed ns microgrj I1 All AMI 11 KS I OK l)l ! Exposure concentration S (ppm of VC) " I ppm of VC = 2..SL * Determined from die] r Mean + SD, To ascertain whct| were analyzed in formation of the Mic It can be seen from i verifying, at least vis by the ordinate intcil fitting the nonlinear [ estimates. The estin of VC metabolize^ respectively. Once a means is I function of exposurj VC exposure to the! VC per se incurred I . .:: r R&S 021516 [l 1.2H 4.8 e vc jial to hr. (tannic ms of 1} 1.4. nf this [fously arious head, evious Jtcd as posure `CO, ng the after bolized b), the -_{}' \ Fcasing DOSE response: vinyl chloride 583 metabolized during 6 hr of exposure to various concentrations of VC appeared to be in accordance with Michaclis-Mcnten kinetics as described by the equation: I'S A' + .5 U) In this equation, r and Fm. are (he velocity and maximum velocity, respectively, for the biotransformation of VC expressed as microgram equivalents of VC metabolized per 6 hr. 5 and Km arc the concentration of VC being inhaled and the Michaclis constant expressed as micrograms of VC per liter of air. respectively. TAULI: I Parameters eor Dfscrihino Till' Mitahoi.ism of IMIAIFD Vinyl ClIIOKIDI: (VC) USIMJ Miciiaeus-Mfnten Kim.iics Exposure concentration 5 (ppm of VC) 1.4 9 25 51 109 250 511 1020 4600 Stug or VC/liter of air)" 3.6 23.0 64.0 130.6 279.0 640.0 1308.2 2611.2 11776.0 r(pg of VC mctaboli?ctl*/'6 hr) 3o z y 242 * 26 557 i 42 1181 r 93 2406 :7 3826 345 6263 355 4257 + 765 9255 1467 v/S 8.33 10.52 8.70 9.04 8.62 5.98 4.79 1.63 0.79 " | ppm of VC -- 2.56 pc of VC/liler of air. * Determined from the total radioactivity in the carcass. f Mean + SD. To ascertain whether Michaelis-Menten kinetics were applicable, the data in Table I were analyzed in accordance with the linear Woolf-Augustinson-Hofstce trans formation of the Michaelis-Menten equation (Segel, 1976), (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. 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) /tg of VC metabolized and 860 + 159 (SD) /ig of VC/liter of air for 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. Eg MS /"^' ' ,< k *j*f; -1 V'5 * 1 Sy, ' `v *-fi- * V:> -y' "Mi y.&3s& 584 GKHRING. WAfANAIlE. AND PARK must be adjusted versus 6 hr. Thi amount of VC hit in the experiment 1-10. I. Metabolism of vinyl chloride analyzed in accordance with the Wooll'-Augustinson-Hofslcc linearized form of ihc Michnclis-Mcmcn equation. Values or r and i /S were taken from Table I. The line was fit by linear regression analysis. The correlation coefficient, fi. 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/wcck 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 or Vinyl Chloride. Metabolism and Induction of Hepatic Angiosarcoma in Rats Exposure concentration S(jtg of VC/ (ppm of VC) liter of airY 10.000 6.000 2.500 500 250 50 25,600 15,360 6,400 1,280 640 128 v(jig of VC metabolized*/! 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 Percentage incidence or hepatic angiosarcoma' 15 22 22 12 7 .2 I ppm of VC . :.ii ( ---- V \lilerofair/ Rg of VC metabolizedl'\ * V,, corrccled for 4-hr exposure, 8558 5hr~ 1 -4/6 - 5706 (ligoTVC metabolized) /mo(VC\ 1/ \ 4hr -------15706 I ^ ^--I Stug/litcr) / |860(vg/liter) + 5(ug/lilcr)|. c From Maltoni and Lefemine (1915). : 'N -7 w^ b.t i j i. gustinson-Hofslcc i Tabic I. The line Sarcoma in rats 12 months and lpting to relate (the Michaclis- hc value for Vm HOLISM AND Percentage incidence of hepatic angiosarcoma7 flag/liter)). DOST KliSFONSli; VINYL CIll.OKIIli: 585 must he adjusted for the shorter exposure duration used by Maltoni and Lcfeminc. 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 Lcfeminc can be calculated from the equation: ,, Vji of VC\ 5706 --------- S.. \ 4 hr / tiller/ 860 4 5 (3) The resulting values for v arc 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 Lcfeminc (1975) versus the amount of VC biotransformed for 4 hr of exposure, v. or the exposure concentration. S. The incidence of hepatic angiosarcoma in rats is linear with respect to log r 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 Prc*t Inttd Of Ttmr fi.OO - 40 - 30 1 // - *.00 / /f / / -1 XQ0 / ;/ / !' i________l- i.i v ' Uf* 30 10 * , 1 at _ aj at au - * >* J/ ` // / " tilt UaS Fio. 2. (A) Metabolism of vinyl chloride expressed as logo (Micrograms of VC mctabolixcd/4 hr) versus percentage incidence or hepatic angiosarcoma (probability scale). (B) Exposure concentration expressed as log S (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 (It for experimentally observed responses while the dashed line represents extrapolation below those doses producing an observable response assuming no threshold. 586 GI IIKISG. WATANAMK. AND PARK threshold for the induction of angiosarcoma in rats exposed to VC. the exposure, concentration producing one angiosarcoma in 10.000 rats can he calculated. The probit percentage representing an incidence of 0.01% is 1.28. Substitution of this value into the Eq. t4) yields: Logo - 1.8827. r -- 76.23 .ug of VC mctaboli/.ed/4 hr Using Eq. (3). the concentration of exposure to VC needed to give this value for v is 11.66 //g/lilcr or 4.6 ppm (approximate 95% confidence limits obtained by substituting the upper and lower 95% confidence limits for p in Eq. (3) and solving for 5 are 0.038.7 ppm I. Hence, exposure of rats to 4.6 ppm of VC for 4 hr daily. 5 dnys/wcck 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 manv chemicals, toxicity may not be a function of exposure to the chemical per sc. hut rather to a biotransformation product of the chemical. Frequently, production of a toxic metabolite is dependent upon envzmatically mediated reactions which arc classically described by Michaelis-Mcntcn kinetics. Since enzymatically mediated reactions arc 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 el al.. 1975: Malavielle et al., 1975; Rannug el a!.. 1974). Covalent binding of UC to hepatic mncromolcculcs in rats (Watanabc el al., 1978) exposed to UC-VC also requires bioactivation. Covalent binding of electrophiles to DNA has been associated with tumorigencsis. 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 gencsis. Furthermore, a more reasonable evaluation of the dose-response data for vinyl chloride-induced tumorigencsis 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 Lefcmine (1975), Schncidcrman 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 close to our prediction of 4.6 ppm of VC for the same incidence. If Schneidcrman et al. (1975) had used all of the data, a dose-response curve with an J j unrealistically hepatic angiol The conccf Lefcmine (I9| Assuming thi the experime produce an ir Aside fron form, there designing cxI chemicals. F'l will produccl response is Since total dl to determine f parameter ir Therefore, a) three times exposure tin exposed to than conccntj well as expd logical studio Unless thd resolved pril value for cF Thus, the cul fractions the toxicity of cl tion of the cl dose-dependl incremental | larger rather In the fo sarcoma in i potation held the response of VC may I ppm (Watad and Lefemir quent presc^ of hepatic exposed to results india exceed cond others suggd 31 So CO fh R&S 021520 r |./he exposure |ted. The probit i value into the s value for r is bv substituting nr J ore 0.03ays/week for I dose-response Ic experimental c chemical per production of ons which arc ally mediated Ton^xposures to Ifil^^amount xposurc before ion to produce exposed to VC ilent binding of 0 *`C-VC also >een associated nic probability metabolized. o. :al straight line n plotted as a >ort further the ite for tumori- data for vinyl ) activated as a ats exposed to 1 and Lefemine , to predict an t of VC. This lame incidence. curve with an jV DOSU-KESPONSI-:: VINYL CIILOKIOI; 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 arc some practical implications of the concepts presented herein for designing experiments to assess (he toxocity, including carcinogenicity, or 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 A'n. 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 which is increased linearly with time. Therefore, after the concentration to which the animals arc exposed becomes two to three times Km, the amount metabolized, i>, 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 or epidemio logical studies of people exposed to high concentrations of VC in the work environment. Unless the dose-dependent. Michaelis-Mcnten type pharmacokinetic parameters arc 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 sc 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 el al,, 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 or 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 ofcancer in .!J>" ' Asi? cyVi'-S1' ' f ,Vv. R&S 021521 588 GEHKJNC, WATANABE. AND PARK response to low doses of a carcinogen (Druckrcy, 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 or 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 arc: (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 ot man which likely overestimates the assumption of risk as d'seussed 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 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. The last assumption deserves comment. There are considerable data in the literature showing that metabolism in general and other physiological parameters as well arc relatablc directly to the surface area of the body (Schm'dt-Niclscn, 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 efTect 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, for a 70 kg man can be estimated by calculation using the Vm value obtained for a 0.250-kg rat. The of man for VC will be: / 1.85 m2 \ (man) = Vm (rat) \0.045 m2)I or I 1.85 m2 \ (man) = (8558 fig/6 hr) 351829 fig/6 hr. \0.045 m2/ where the values 1 .f 0.250-kg rat. rcspcc Ug/8 hr. In order to data collected in rat rats since toxicity i divided by 70 kg/0.! to that of rats is reactive form by ma Using this equation, to rats was calculi incidence or angioss For men exposet has been reported calculated incident approximately 50 f induction of angin induction of angios that r on a mass number lies belowJ sarcoma observed il (Maltoni. 1975). Fff killed. Thus, as ind range of exposure? Theoretical AmoI Exposed CoNTtNca Angiosarcoma as | Exposure concentration | (ppm) 200 50 5 1 (ug/litcr)" 512 128 12.8 2.56 | * I ppm - 2.S6 pg/lil *r has been calcul/ the determined forL area and subsequent!^ rThe expected pro! theoretical percentage r (V; -'y r I* Altshuler, 1973). posures causing a pdence. hich can be used oncepts presented bn is fraught with labolite of VC per s for their working kits or man which fion of VC to the |[lata for the biosis for translation Knee area. in the literature peters as well are ?0; Pinkel, 1958). ^various species i proportion to Surface (Pinkel. [ incurred by man Drinking Water, bust, however, be ally active agents, jification are more fper unit of mass vhile the dose per ncmical requiring gnsformed will be function of the ned must then be to kg man can be It. The V- of man [hr, . V dose response; vinyl chloride 589 where the values 1.85 and 0.045 m1 are the body surface areas of a 70-kg man and a 0.250-kg rat. respective;/ (Pinkel. 1958). For an 8-hr exposure, the value is 469.105 //g/8 hr. In order to use this number to theoretically estimate the response in man using data collected in rats, the 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 f'm for inan on a mass equivalent basis to that of rats is 1675 /rg/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; ,W8hr)=l675/,g/8hl`50;8/lilCf) 860 /rg/litcr + 5(/rg/litcr) v* 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 i-f 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 f 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. A; 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'S Theoretical Amounts op Reactive Product Formed from VC ay a 70-kg Man Exposed Continuously for 8 hr and tiie Corresponding Expected Incidence of Angiosarcoma as Predicted from Data Collected in Rats Assuming no Threshold Exposure concentration r0/g of VC metabolized*/? hr) Theoretical percentage Probit incidence of response* angiosarcoma* (ppm) 200 50 5 1 (Aig/literf 512 128 12.8 2.56 625 2.79 2.68 1.02 217 2.34 1.98 0.11 24.6 1.39 0.52 3.74 x 10-* 4.97 0.70 -0.54 1.5 x I0-* * I ppm = 2.J6 jig/litcr. *r has been calculated using ih.- Michaclis-Mentcn equation alter calculating the Vm for man from the determined for rats. This calculation was made by assuming I', is proportional to body surface area and subsequently adjusting i; to a mass equivalent to that of rats. See test. *The expected probit resp nse was calculated from probit Eq. (3) (see lextk Subsequently the theoretical percentage inetdee.e of angiosarcoma was determined from the respective protow. R&S 021522 590 GEHRING, WATANABF,. AND PARK In spite of the likelihood of overestimating the incidence cf 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 eases in the U.S. annually (Makk ei at.. 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 or VC by rats is a dose-dependent process characterized by Michaclis-Mcnten type kinetics. The concepts evolved from this analysis reveal why pharmacokinetics must be considered in designing toxicology experiments as well ns 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 I ppm because there is some evidence for at least a practical threshold in both rats and man. There arc 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 Altscmuu.h, B. (1973). 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