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Copv.-igh? arterial c;-j.ee Without Wssscn Environmental Health Perspectives Vol. 52, pp. 9-14, 1983 r &S 041094 Relevance of Experimental Studies to Human Risk by F. K. Dietz,** J. C. Ramsey* and P. G. Watanabe* Confidence in the extrapolation of animal toxicity data to humans can be enhanced by the application of pharmacokinetic concepts integrated with chronic toxicity data and knowledge of a chemical's mechanism(s) of toxicity. Basic pharmacokinetic concepts (including dose-dependent or Michaelis-Menten kinetics) and their relationship to the risk estimation process are discussed using vinyl chloride and styrene as specific exam ples. Species differences in metabolic rates must be considered in order to arrive at realistic estimates of human risk to vinyl chloride-induced liver angiosarcomas utilizing vinyl chloride toxicity data observed in rats. Because small animal species generally metabolize chemicals more rapidly than larger species on a body surface area basis, small animals should be more sensitive to chemicals (such as vinyl chloride) that exert their toxicities via the metabolic formation of toxic products. Inhaled styrene is a chemical whose clearance from the blood at low exposure levels in both rats and humans follows first-order kinetics. However, at higher exposure levels, the pharmacokinetic fate of styrene in rats is dose-dependent, suggesting a saturation of styrene metabolism. These data indicate that any extrapolation of observable toxicity at elevated exposure levels in rats to anticipated responses at lower levels in either rats or humans may be invalid. An integration of the foregoing concepts provides a sound scientific basis for the use of experimental animal data to predict the risk to humans from chemical exposure. Introduction to potentially harmful substances, interspecies variation in response may often preclude a simple Chemically induced carcinogenicity is one type extrapolation of animal toxicity to that antici of toxic response that has received primary atten pated in man. The purpose of this presentation is tion in recent years. The potential lethality of to review and emphasize the importance of ani cancer, combined with its generally irreversible mal pharmacokinetic studies in properly imple nature and long latent period are all characteris menting animal toxicity data to predict human tics that have placed carcinogenesis in the fore risk from chemical exposure. front of public concern. Many chemicals shown to be mutagenic in short-term in vitro studies and/or carcinogenic in long-term animal studies have consequently been considered as potential human Pharmacokinetic Concepts and Dynamics of Toxicity carcinogens. This conclusion is typically based on Pharmacokinetics is a study of the dynamics of studies of animaljnodels performed under tightly absorption, distribution, metabolism and excre controlled experimental conditions in which po tion of a chemical within the body. Pharmaco tentially interfering variables are kept to a mini kinetic studies of a chemical as a function of mum. Although a basic toxicological goal is to administered dose often provide valuable infor evaluate the risk to man associated with exposure mation on how a chemical's overall biological fate * may change in response to different amounts Toxicology Research Laboratory, Health and Environmen tal Sciences, USA 1803 Bldg., Dow Chemical USA, Midland, within the body. Since many toxic responses to chemical exposure are not only dependent on the MI 48640. amount of a chemical that reaches a target site ^Author to whom inquiries should be addressed. Present address: Health and Environmental Sciences--Texas, Lake Jackson Research Center, Dow Chemical USA, Freeport, TX 77541. but also on how long a sensitive site might be exposed, a comparison of the pharmacokinetic fate of a chemical between different species of 10 DIETZ, RAMSEY AND WATANABE animals can provide data about differential sus ceptibility relating to interspecies extrapolation of toxicity data. A classical approach to pharmacokinetic analy sis depicts the body as consisting of a system of compartments. An individual compartment gen erally refers to all tissues, organs, cells and/or fluids within the body for which the rate ofuptake and loss of a chemical is sufficiently similar as to preclude further kinetic resolution. Gehring et al. (1) have provided a detailed description of phar macokinetic compartments and their use in eval uating toxicity data, and consequently no de tailed discussion will be included here. While compartments do not always have direct physio logical or anatomical counterparts when ana lyzed kinetically, they constitute a basic tool by which quantitative expressions describing the fate of a chemical within the body can be derived. Dose-Independent Pharmacokinetics Over a range of selected dose levels, many chemicals exhibit first-order kinetics which can be referred to as being "linear." For these chemi cals, the rates of absorption, distribution, metabo lism and elimination from the body are propor tional to the concentration or amount of the chemical within the body. As a consequence of this proportionality, the rate constants of all these processes are thus independent of the ad ministered dose. In a simplified fashion, firstorder kinetics may be expressed by the equation: rate = --dCldt = kC \ in which C is the concentration of the chemical in the body at time t and k is the rate constant for the given process. As long as first-order kinetics apply and thus the rate constants for all processes responsible for a chemical's pharmacokinetic fate are independent of administered dose, tissue con centration and consequent toxicity should also be proportional to administered dose. Dose-Dependent Pharmacokinetics In actuality, many reactions that influence a chemical's pharmacokinetic fate are not indepen dent of administered dose, but are instead dosedependent. In this situation, saturable active transport systems or metabolic reactions that of ten play key roles in the prevention or enhance ment of chemical toxicity are not adequately de scribed by first-order kinetics. As a consequence, the administration of high dose levels, as fre quently done in long-term animal bioassays, may overwhelm these processes and result in a dispro portionate increase in blood and/or tissue concen tration and possibly elicit a toxic response. The rates of saturable processes are often de scribed by Michaelis-Menten or dose-dependent kinetics according to the equation: rate = -dC/dt = VmaxC/Km + C In this equation, -dCldt is the rate of change in the concentration of the chemical's concentration at time t, Vmu is the maximum velocity of the process and Km is the Michaelis constant or that concentration at which the rate of the process is at a value of one-half VmThere are two limiting situations to this equation. When the concentra tion (C) is much greater than Kmy the MichaelisMenten equation approaches a limit of: rate = - dCldt = Vmax CKm In this situation, the rate of the process is limited by the value Vmax, and, as C increases, the rate of the reaction remains constant. It is in this concen tration range (CKm) that the biological proc esses governed by this type of kinetic behavior have become overwhelmed and can be considered to be saturated. Conversely, if the concentration C is much less than Km, the rate of the processes described by the Michaelis-Menten equation can be approximated by: rate = -dCldt = kC CKm where k = Vmax/Km. Under these conditions, the rate of the process remains proportional to the chemical's concentration and all of the previously described concepts for first-order kinetics regard ing proportionality between blood and tissue con centration and toxicity apply. Use of Animal Studies for Predicting Human Toxicity Recent authors have suggested that there an at least five potential factors responsible for spe cies variations in response to chemical exposun (2). Collectively, these factors include absorption distribution, metabolism, site and mechanism o* action and excretion of the chemical from the body. It is of interest to note that an analysis of the dynamics of many, if not all, of these factors as a function of administered dose level consti tutes what has been previously described as a pharmacokinetic study. In utilizing animal studies to predict possible human toxicity, it is important to determine if the toxicity resulting from chemical exposure is due to the parent chemical itself or rather to an R&S 041095 EXPERIMENTAL STUDIES AND HUMAN RISK 11 R&S 041096 active metabolite generated via metabolic proc esses occurring within the animal. It is commonly accepted that metabolism can either lead to de toxification or activation, depending on the toxi cological potential of the parent compound and/or its possible metabolites. Consideration of this in formation is important for the extrapolation of results from animal toxicity studies to man, since there may be relative species differences in the activity of enzymes responsible for chemical me tabolism. As pointed out by Rail (3), it is often possible to estimate the relative sensitivity of different species to a chemical by using an ap proximation that the basal metabolic rate is roughly proportional to the body surface area. This suggests that if factors other than metabo lism are ignored, a large animal species is more sensitive to a directly toxic agent than a small animal species (Tbble 1). In contrast, a large ani mal species should then be less sensitive to toxic ity mediated by a metabolic activation than a smaller species (4). It should be emphasized, how ever, that for some types of metabolic activation the relative rates may not only be a simple func tion of body size. For example, the metabolic activation of 2-acetaminofluorene involves the formation of an active sulfate, and the species and organ sensitivity of the tumorigenicity of this agent correlates well with the level of sulfotransferase enzyme activity (5). Since the rat has a higher level ofsulfotransferase than the mouse, it develops more tumors when exposed to an equiva lent amount of 2-acetaminofiuorene. In this in stance, the larger animal species is more sensi tive to the effects of the metabolically activated agent than the smaller species. Thus, the reliabil ity of interspecies extrapolation depends to a large degree upon how much is known of the details of metabolism and how metabolism affects toxicity in the species of interest. Pharmacokinetic Concepts and Risk Estimation Many mathematical models are commonly used in extrapolating an observed carcinogenic re sponse in animal bioassays at relatively high dose levels (6). While these models differ from each other by the rapidity in which a zero response is approached as the dose level approaches zero, they are similar in that they usually assume that a zero response occurs only when the dose level equals zero. Another feature common to these models is their assumption that the concentration of the carcinogenic entity is directly proportional to the dose level of the administered chemical. Tkble 1. Predicted relative cancer risk from equival doses (mg/kg) calculated on the basis of (body weight).* Species Man (70 kg) Dog (20 kg) Rabbit (3 kg) Rat (0.5 kg) Mouse (0.03 kg) After Rail (5). Predicted relative cancer risk Directly toxic agents Metabolically activ; agents 1.00 0.66 0.35 0.18 0.08 1.00 1.52 2.85 5.58 13.20 This assumption applies whether the carcino genic entity is produced via a metabolic activa tion of the parent chemical or whether the parent chemical itself is the primary toxicant. An impor tant consequence of this assumption is that toxic ity or carcinogenicity is also expected to be pro portional to administered dose level. Recent studies of several chemicals, including vinyl chlo ride and styrene, illustrate how an understanding of the pharmacokinetic fate of these compounds as influenced by the magnitude of administered dose can be used to evaluate animal toxicity data in order to predict the hazard to man from expo sure to these agents. Vinyl Chloride An example of dose-dependent pharmaco kinetics that directly relates to carcinogenic risk estimation in man is that of inhaled vinyl chlo ride (7, 5). Vinyl chloride has been demonstrated to induce hepatic angiosarcomas in rats at expo sure levels ranging from 10 to 10,000 ppm, with an essentially flat dose-response curve at expo sure levels from 1,000 to 10,000 ppm (S). Numer ous studies have indicated that a reactive metab olite of vinyl chloride is likely to be the carcinogenic entity for this halogenated ethylene rather than the parent compound itself ilO-15). Other studies have shown that the bioactivation of vinyl chloride in rats is a saturable process that follows Michaelis-Menten kinetics becoming overwhelmed at high exposure levels, thereby limiting the in vivo production of the toxic metab olite (7, 16-19). As a consequence of this satura ble metabolic activation, Gehring et al. (7) have shown that the toxicity or carcinogenicity in rats resulting from vinyl chloride exposure is not di rectly proportional to all exposure concentra tions. Alternatively, these authors found it was possible to relate the observed carcinogenicity in rats to the amount of vinyl chloride metabolized after pharmacokinetic parameters describing the 12 DIETZ, RAMSEY AND WATANABE saturable bioactivation of vinyl chloride were de termined. In accordance with these observations of the kinetic behavior of vinyl chloride in rats, Gehring et al. (7, 8) utilized similar pharmacokinetic con cepts to estimate the amount of vinyl chloride metabolized in man. Their prediction incorporat ed the concepts that vinyl chloride bioactivation in man was a saturable process as observed in laboratory animals and that the metabolic proc ess responsible for vinyl chloride metabolism in mammals was related to differences in body sur face area. Using this methodology, these authors predicted that man's rate of vinyl chloride bioac tivation would be much less than that observed in rats, resulting in a decreased sensitivity of man to the tumorigenic effects of vinyl chloride (7, 8). Recent data from other laboratories on the rate of vinyl chloride metabolism have provided addi tional support for this conclusion. In studies of the pharmacokinetics of vinyl chloride in differ ent species (including man), Buchter et al. {20, 21) and Filser and Bolt (22) have confirmed that a marked species variation in vinyl chloride metab olism does indeed exist (Table 2). These investiga tors found that mice and rats metabolized vinyl chloride at a rate approximately 5-12 times that for man. In contrast to the results observed in these rodent species, rhesus monkeys were found to metabolize vinyl chloride at a rate that closely paralleled that seen in man. Collectively, these results are significant in that they confirm the predictions reached by Gehring et al. (7, 8), who used pharmacokinetic concepts to predict differ ences in the relative rates of vinyl chloride me tabolism in rats versus man. 1200 ppm the maximum blood concentration reached a value of 64 jig/mL. Thus, as exposure concentration increased by 15-fold, the maximum blood concentration increased over 80-fold, indi cating a dose dependency in the pharmacokinetic profile of styrene. As reviewed by the original authors (23), these and other data indicated that the capability of laboratory animals to metabolize styrene becomes overwhelmed at exposure con centrations somewhere between 200 and 600 ppm. These results indicate that any extrapola tion of animal toxicity data observed at exposure levels of 600 ppm and above to anticipated re sponses in animals at lower levels may be invalid. How do these results relate to the extrapolation of styrene toxicity data in laboratory animals to that in man? Such an extrapolation can be greatly facilitated by a direct pharmacokinetic comparison of the chemical in question between both species. Accordingly, Ramsey and Young Ihble 2. First-order metabolic clearance rates for vinyl chloride in man versus other species.* Species Clearances, L/hr/kg body weight Man Monkey Rat Mouse 2.02 3.55 11.00 25.60 Data from Buchter et al, (20,21) and Filser and Bolt (22). jig StyrwW ml Blood R&S 041097 Styrene Styrene is another example of a chemical whose metabolic elimination in experimental animals is dose-dependent. In an analysis of the pharmaco kinetic fate of styrene in rats following a 6-hr inhalation exposure to 80, 200, 600 or 1200 ppm, Ramsey and Young (23) observed that there was a marked dose dependency in the elimination of styrene from the. blood. Figure 1 depicts a plot of the blood styrene concentration versus time data in animals exposed to 80 or 1200 ppm for 6 hr. Other animals were left in the exposure chambers for periods of up to 24 hr in order to establish whether plateau blood levels were achieved. Note that a disproportionality exists between maxi mum blood concentration and exposure level. At an exposure level of 80 ppm, the maximum styrene concentration was 0.8 pg/mL, while at Figure 1. Blood styrene concentration in rats exposed to 80 or 1,200 ppm. Data from Ramsey and Young (23). jug Styrene/ ml Blood EXPERIMENTAL STUDIES AND HUMAN RISK 13 ity data obtained at high levels to predict possible risks to humans at lower exposure levels. Conclusions In summary, it should be noted that animal studies of pharmacokinetic behavior only repre sent one segment of the total data base of interre lated information required to make a rational extrapolation of toxicity data observed in labora tory animals to anticipated responses in man. Other authors have demonstrated that the risk estimation process is greatly enhanced when ani mal studies of pharmacokinetic behavior are inte grated with observations of chronic toxicity and a knowledge of mechanisms of toxicity such as the production of active metabolites that interact with critical macromolecular sites. Collectively, an evaluation of the relationships between these parameters and toxicity in experimental animals will improve the estimation of relative degrees nf risk to man associated with chemical exposu Figure 2. Blood styrene concentration in humans exposed to 80 ppm for 6 hr. Data from Ramsey and Young (23). (23) conducted a pharmacokinetic study of in haled styrene in human volunteers exposed to 80 ppm for 6 hr. Figure 2 depicts the blood styrene concentration during and after exposure in the four volunteers, Note that the blood styrene con centration rose to a maximum of 0.9 pg/mL at 6 hr and declined in a linear fashion. A comparison of these results with those of Figure 1-indicates a marked similarity between the pharmacokinetic fate of styrene in man and rats following exposure to 80 ppm. These authors concluded that this type of similarity lends confidence to the extrapolation of toxicity data observed in laboratory animals at levels below 80 ppm to that anticipated in man. In contrast, the demonstration of a saturable elimi nation of styrene from laboratory animals at higher exposure levels precludes the use of toxic 1. Gehring, P. J., Watanabe, P. G., and Blau. G. E. In: O Concepts in Safety Evaluation, Vol. 1, Part 1 (I ^ Mehlman, R. E. Shapiro and H. Blumenthal, Eds.), 1 q sphere Publishing Corp., New York, 1979, pp. 195-2 to 2. Reichsman, F. P., and Calabrese, E. J. Animal extra 00 tion in environmental health: its theoretical basis practical applications. Rev. Environ. Health 3: 5 (1979). 3. Rail, D. P. Difficulties in extrapolating the results of toxicity studies in laboratory animals to man. Environ. Res. 2: 360-367 (1969). 4. Reitz. R. H., Gehring, P. J., and Park. C. N. Carcinogenic risk estimation for chloroform: An alternative to EPA's procedures. Food Cosmet. Tbxicol. 16: 511-514 (1978). 5. Miller, E. C. Carcinogenesis by aromatic amines and amides. Reported at the symposium on environmental carcinogenesis. Michigan State University, East Lansing, MI, 1978. 6. Gaylor, D. W., and Shapiro, R. E. In: New Concepts in Safety Evaluation, Volume 1, Part 2 (M. A. Mehlman, R. E. Shapiro and H. Blumenthal, Eds.). Hemisphere Publishing Corp., New York, 1979, pp. 65-67. 7. Gehring, P. J.. Watanabe, P. G., and Park, C. N. Resolu tion of dose-response toxicity data for chemicals requiring metabolic activation: example--vinyl chloride. Toxicol. Appl. Pharmacol. 44: 581-591 (1978). 8. Gehring, P. J., Watanabe, P. G., and Park, C. N. Risk of angiosarcoma in workers exposed to vinyl chloride as predicted from studies in rats. Toxicol. Appl. Pharmacol. 49: 15-21 (1979). 9. Maltoni, C., and Lefemine, G. Carcinogenicity assays of vinyl chloride: current results. Ann. N.Y. Acad. Sci. 246: 195-224 (1975). 10. Barbin, A., Bresil, H., Croisy, A., Jacquignon, P., Malavetlle, C., Montesano, R., and Bartsch, H. Liver-microsome-mediated formation of alkylating agents from vinyl bromide and vinyl chloride. Biochem. Biophys. Res. Com* mun. 67; 596-603 (1975). 14 DIETZ, RAMSEY AND WATANABE 11. Bartsch, H., Malaveille, C., and Montesano, R. Human rat and mouse liver mediated mutagenicity of vinyl chloride in salmonella typhimurium strains. Int. J. Cancer 15: 429-437 (1975). 12. Bolt, H. M., Kappus, H., Kaufmann, R,, Appel, K. E., Buchter, A., and Bolt, W. Metabolism of 14C-vinyl chlo ride in vitro and in uivo. Inserm 52: 151-164 (1975). 13. Malaveille, C., Bartsch, H., Barbin, A., Camus, A. M., and Montesano, R. Mutagenicity of vinyl chloride, chloroethylene-oxide, chloroacetaldehyde and chloroethanol. Biochem. Biophys. Res. Commun. 63: 363-370 (1975). 14. Kappus, H., Bolt, H. M., Buchter, A., and Bolt, W. Liver microsomal uptake of (14C) vinyl chloride and transfor mation to protein alkylating metabolites in vitro. Tbxicol. Appl. Pharmacol. 37: 461^171 (1976). 15. Watanabe, P. G., Zempel, J. H., Pegg, D. G., and Gehring, P. J. Hepatic Macromolecular binding following exposure to vinyl chloride. Tbxicol. Appl. Pharmacol. 44: 571--579 (1978). 16. Bolt, H. M., Kappus, H., Buchter, A., and Bolt, W. Disposi tion of (1,2-140 vinyl chloride in the rat. Arch. Tbxicol. 35:153-162 (1976). 17. Watanabe, P. G., Hefner, R. E., Jr., and Gehring, P. J. Vinyl chloride induced depression of hepatic non-protein sulfhydryl content and effects on bromosulphthalein (BSP) clearance in rats. Tbxicology 6:1-8 (1976). 18. Watanabe, P. G., McGowan, G. R., and Gehring, P. J. Fate of 14C-vinyl chloride after single oral administration in rats. Tbxicol. Appl. Pharmacol. 36: 339-352 (1976). 19. Watanabe, P. G., McGowan, G. R., Madrid, E. 0., and Gehring, P. J. Fate of nC-vinyl chloride following inhala tion exposure in rats. Toxicol. Appl. Pharmacol. 37: 49-59 (1976). 20. Buchter, A., Bolt, H. M., Filser, J. G., Goergens, H. W., Laib, R. J., and Bolt, W. Pharmkokinetik und Karzinogenese von Vinylchlorid arbeitsmedizinische Risikobeurteilung. Verhandl. Deut. Gesell. Arbeitsmed. 18: 111-124 (1978). 21. Buchter, A., Filser, J. G., Peter, H., and Bolt, H. M. Pharmacokinetics of vinyl chloride in the rhesus monkey. Tbxicology Letters 6: 33--36 (1980). 22. Filser, J. G., and Bolt, H. M. Pharmacokinetics of halogenated ethylenes in rats. Arch. Tbxicol. 42: 123--136 (1979). 23. Ramsey, J. C., and Young, J. D. Pharmacokinetics of inhaled styrene in rats and humans. Scand. J. Work. Environ. Health 4: 84--91 (1978).