Chapter 7 Metabolism and pharmacokinetics

Chapter 7 Metabolism And Pharmacokinetics HISTORY AND IMPORTANCE Over a period of about 30 years, increasing emphasis has been placed upon the need for metabolic studies as an integral part of the safety evaluation of a compound. As long ago as 1859, Buchheim expressed the following view: "In order to understand the actions of drugs, it is an absolute necessity to have knowledge of the transformations they undergo in the body. It is obvious that we must not judge drugs according to the form and amount administered, but rather according to the form and amount which actually elicits the action." (Conti & Bickel, 1977.) General acceptance of the value of such metabolic studies became more widespread as methodology improved and regulatory requirements for classes of chemical products such as food additives or agricultural chemicals made the provision of metabolic information mandatory. Increased understanding of the value and applicability of pharmacokinetics has led to the recognition that metabolic and pharmacokinetic data are not merely ancillary information but constitute vital mainstream characteristics of the compound, to be used in planning both the strategy and tactics of the approaches to safety evaluation. Metabolic and pharmacokinetic information is central to the design of protocols, leading to proper species selection, definition of dose regimens and the appropriate conduct of both in vivo and in vitro tests. Intelligent application of metabolic and pharmacokinetic information is one of the principal weapons that permits the selection of appropriate tests and conditions specifically tailored to the characteristics of the individual compound under study. Thus, in the plan of investigation developed herein, metabolic comparability between man and the animal species proposed for use in a long-term test will be established before any such test is initiated.

OBJECTIVES The general account that follows is not intended to be all-inclusive. It seeks to highlight investigations in vitro and in experimental animals. The nature of the human data to be developed, and the manner in which they will be related to the results from animals, will be discussed in the later section on human studies. The study of the metabolism and pharmacokinetics of an ingested substance is carried out for the following purposes: A. To gain a general understanding of the absorption, biotransformation, disposition, and elimination of the ingested substance after a single dose and after repeated doses. B. To provide information about the rates at which these processes occur, and the temporal relationships between dose and tissue levels reached so that the following information may be derived: 1. Whether steady-state tissue concentrations are achieved. 2. Time required to achieve a steady state with a particular dosage regimen. 3. Total metabolite load at the steady state. 4. How the metabolic and pharmacokinetic characteristics change with time, dose, and dosage regimen. 5. Under what circumstances the metabolic and/or excretory systems become overloaded. This need implies that pharmacokinetic studies should bracket the toxic doses for the species investigated, i.e., should cover " n o r m a l " metabolism as well as metabolism under extreme stress levels of the compound. In accordance with this separation in the degrees of required sophistication, the study of metabolism and pharmacokinetics may be divided into two stages as follows: Stage/--scheduled to follow a series of acute toxicity and mutagenicity evaluations and to precede subchronic tests. The purpose is to provide a general overview of the fate of an ingested substance in the body. The information obtained should help to determine the doses to be used, to arrive at 65

the method and frequency of administration, species to be selected, and determine the duration of the subchronic studies in animals. Stage//--follows a decision to embark upon long-term studies and involves a more detailed examination intended to yield the central information for planning the dose regimen and other features of the long-term investigations. Stage I aims at fulfilling objective A. above and, to a limited extent, providing some of the items of information (for example, B. 1. and 2.) listed under objective B. By the time stage II is undertaken, the main emphasis will be directed to the outstanding questions arising from the findings to date, as they relate to objective A., but more particularly to objective B. Any attempt to specify precisely the nature of the investigations and their protocols in stages I and II would be contrary to the general policy laid down for this report, namely the maintenance of flexibility to permit the overall approach to be tailored to the particular compound under investigation. The relevance of these considerations is exemplified by the description below of a wide range of preliminary investigations, as well as ancillary metabolic studies that can be carried out in vitro. Judicious selection of appropriate experimental procedures will often prove helpful. To attempt to weave all these approaches into a rigid sequence of mandated studies would result in much wasted effort; conversely, to omit them altogether would deprive the innovative investigator of valuable techniques. INTERSPECIES COMPARISONS Until quite recently, the principal justification for developing a knowledge of the metabolism of a compound was attributed to the value of interspecies comparison, which made possible the selection for toxicity tests of one or more species that resembled man most closely in regard to the metabolic pathways of the compound. Such an outlook appears superficially attractive and rational, but in reality encompasses three potential fallacies. These are: A. The assumption that human metabolism will be studied as part of the interspecies comparison. While this presumption is usually applicable to potential therapeutic drugs, it is much less often possible for food additives and pesticides to be studied in this way. A later section addresses the need for, and problems attendant upon, the investigation of human metabolism of foreign compounds. Use of one or more primates, such as macaques (especially rhesus monkeys, Macaca mulatta) may be sufficient to establish some comparison between primate and nonprimate metabolism. In many instances, metabolic pathways in man and monkey may show great similarity; for example, in the case of 3,4,4'-trichlorocarbanilide (Birch, Hiles, Eichhold, Jeffcoat, Handy, Hill, Willis, Hess & Wall, 1978; Hiles & Birch, 1978). Baboons (Papio sp.) and, to a greater degree, chimpanzees (Pan troglodytes) may afford an even closer comparison to man. Thus, study of metabolism in nonhuman primates is obviously useful, insofar as it broadens the scope of our understanding of possible metabolic pathways for a test compound, and may well provide an indication of their relative importance; but in most cases, such studies are not a complete substitute for parallel investigations in human volunteers. B. The assumption that qualitative similarities in regard to the nature of the metabolites formed reflect similar rates of formation and generally comparable pharmacokinetics in different species. C. The assumption that valid conclusions can be drawn from comparisons of results derived from tests carried out under different dosage regimens. As closely as possible, similar doses, routes and conditions of administration should be used. The search for a species "comparable" to man has been in many instances lip service to a seemingly unattainable ideal, the pursuit of the philosophers' stone. In keeping with this situation, metabolic information was at one time regarded as ancillary to the toxicity tests carried out, unless such information could be used to establish some advantageous characteristics of the compound such as nonabsorption from the gastrointestinal tract or rapid and complete elimination from the body. Experience of interspecies metabolic comparisons, carried out with the same compound, has led to acceptance of the value of such studies as indicators of likely handling of the material in man. Recent years have also brought a realization that species differences in metabolism may be attributable to a variety of mechanisms (Gillette, 1977). The majority of these differences are genetically determined, as for 66

instance in the metabolism of purines to uric acid a n d / o r allantoin, or the lack of gulonic acid oxidase in man and guinea pig, for whom ascorbic acid is an essential nutrient. In various species, different pathways may predominate, for example 7-hydroxylation of coumarin in man (Shilling, Crampton and Longland, 1969), in contrast to 3-hydroxylation and formation of o-hydroxyphenylacetic acid as the major metabolites in rats and rabbits (Kaighen and Williams, 1961). Species differences in the kinetics of formation of a particular metabolite may be reflected in slower production, as when the apparent Vma x is lower, the apparent K m is higher and the intrinsic clearance of the substrate by the enzyme involved (Vmax/(K m + S)) is depressed (see Michaelis-Menten kinetics discussion). A case of this sort is the formation of the reactive metabolite of acetaminophen in rats and mice (Davis, Potter, Jollow and Mitchell, 1974). Species differences in metabolism also arise as a consequence of the different responses to multiplicative encounters with xenobiotics. A striking example is the stimulation of individual metabolic capacities by phenobarbital or other mixed function oxidase inducers. A realistic evaluation of the impact of such inducers can only be arrived at in vivo, with respect to the totality of the enzymes affected, rather than by the study of individual processes in vitro. PROCEDURES: OVERALL STRATEGY The order and extent of the studies to be carried out is determined by (a) the chemical and physical nature of the compound, (b) its anticipated behavior and expected biotransformation, and (c) the availability of, or feasibility of developing accurate, sensitive, selective and reproducible assay procedures for the compound (and, ideally, at least some of its possible metabolites) in biological fluids. Technical difficulties are substantially reduced if the compound is available, labeled in an appropriate position with a suitable isotopic label, and with an adequate specific activity. Where the labeled compound can be made available, obvious advantages thus accrue. In other circumstances, for example with a complex mixture, the availability of a good analytical method permits Stage I studies to be carried out. Even without a radiolabeled product or an adequate assay procedure, for example with an extract prepared from natural sources, some Stage I studies can still be devised that will provide useful qualitative data for use in designing subchronic tests. The account of available procedures provided below does not attempt to distinguish which approaches are most suitable in each of the instances cited above, i.e., for individual compounds or complex mixtures. Nor is it implied that each compound should be put through the whole gamut of investigations described. Availability of procedures does not signify that they are necessary or useful, or even appropriate for any given compound. Judgment is needed for purposes of informed selection. PRELIMINARY EXPLORATION (STAGE) In Vitro Studies. Depending on the nature of the compound, a variety of in vitro studies may well prove to be helpful in preparing for more far-reaching investigations. Examples are exposure of a compound to acid, alkali, enzyme hydrolysis (esterase, protease, etc.), and possibly the use of perfused organs in vitro. One assumes that at least some of this information will be available from preliminary work done to investigate the characteristics and the stability of the compound in food. Data derived from in vitro tests should be developed preparatory to undertaking animal studies. Information about enzyme hydrolysis in the intestinal tract, or by the action of esterases in tissues, is usually readily developed for various classes of compounds, including simple esters, which can readily be hydrolyzed into the component acids and alcohols. For any such esters, one or other or both of these hydrolytic components may be a common foodstuff or intermediary metabolite normally present in human tissues. Depending on the levels of anticipated exposure, it may be decided that further toxicological investigation of the compound, the acid or the alcohol is to be considered unnecessary; one must guard against a misguided conclusion, for experience with sucrose esters, for example, has demonstrated that hydrolysis in the intestine may not be sufficiently rapid nor complete for purposes of absorption. Interactions. In the case of possible interaction of the test compound with food components, two situations may be envisaged. In the first, the food additive may be used for a functional purpose with known reactivity, exerting its technical effect by interaction with food components. Here it is obviously necessary to know something about the nature of the processes involved (Chapter 3). The chemical reactivity of the compound, coupled with an informed estimate of the importance of the information likely to be gained, will determine the extent and depth of the investigation carried out. Where distinct interaction products are shown

67

to be formed, and they can be separated or synthesized, it may be possible to subject them to some of the short-term mutagenicity tests as part of the safety evaluation of the parent substance. Also, the test compound may undergo changes while it is on or in the food (for example, the photodegradation undergone by pesticide residues remaining on the surface of food crops). Where the technical effect produced by a compound does not depend on reaction with food, information about the chemical nature, stability, reactivity and other properties of the material, will give some clues as to the reactions likely to occur with food components. If animal experiments are to be done with the compound added to food, it is necessary to check the stability of the test compound in the animal diet. Further study of interaction with food is generally indicated if the compound disappears on standing, other than by volatilisation, elution, transesterification, or degradation (see Chapter 3). Degradation. The test compound may undergo degradation in the gastrointestinal tract. Besides a possible acid hydrolysis or proteolytic degradation in the stomach, interaction with nitrite either in the stomach or the intestine is possible, particularly in the case of secondary or tertiary amines, quaternary ammonium compounds, substituted ureas, N-containing heterocyclics or other nitrogenous compounds. Degradation may occur in the gut by physicochemical means, or, in the case of some polymeric materials, depolymerization may occur by an unknown mechanism. Enzymes present in the intestinal tract may depolymerize macromolecular materials and may be responsible for hydrolytic reactions or other effects resulting in degradation of the compound. Biodegradation in the gut commonly comes about through the actions of bacteria, which are capable of bringing about a vast range of reactions, from the reductive rupture of azo links, or reduction of nitro groups, to the rupture of nitrogen-sulphur bonds through the utilization of the sulphur (for example, in the conversion of cyclamate to cyclohexylamine). Under these circumstances, individual variations are often noted within groups of animals; some of the test animals metabolize the compound while others do not. The activities of nitro reductases of intestinal bacteria tend to be much weaker than those of the corresponding enzymes in some of the microorganisms used for bacterial mutagenesis testing. Hence, the latter may yield misleading positive results, even taking into account the mammalian nitro reductases present within certain organs such as the liver. In vitro tests with gut contents (cecum or large intestine), or with cultures of isolated anaerobic gut bacteria may be useful indicators of potential degradative capacity that may be manifested when the test compound is administered by the oral route in vivo. INTERLINKED A P P L I C A T I O N OF MUTAGENESIS TESTS TO METABOLISM Attention is drawn to the fact that the Decision Tree has mutagenesis tests separate from metabolic studies. The purpose of this section is to indicate that there may be a close interrelationship between the two areas of investigation. Donahue, McCann & Ames (1978) have demonstrated the value of bacterial mutagenesis test for the detection of those impurities which are responsible for the mutagenic properties of noncarcinogens. This approach is equally applicable to the pursuit of mutagenic metabolites. Every opportunity should be grasped to utilize bacterial mutagenesis tests and other readily applicable short-term tests in the study of the metabolic fate of the compound. The metabolites formed may be present in the exhaled breath, in body fluids including blood, saliva and milk, or in the excreta. Mutagenicity tests may be useful to act as a guide in the separation and purification of individual metabolites, as well as in the determination of the biological characteristics of these metabolites. Thus, although the Food Safety Decision Tree shows the investigation of mutagenesis as separate from that of metabolism, the intention is that these investigations be interlinked as far as possible, in a manner consistent with intelligent utilization of the information forthcoming from such studies. Conversely, metabolic studies can help guide mutagenesis testing by determining the "genetically active dose"; that is, the dose that is effective in causing germ-cell mutations. In the course of disposition studies it may be apparent that the substance a n d / o r its metabolites are segregated by passage across the blood-gonad barrier, transport across the membranes of gonadal cells and interaction with cell components. Evidence of selective cumulation of radioactive label in the gonads is a signal for further investigation. CIRCUMSTANCES T H A T PRECLUDE THE NECESSITY FOR METABOLIC AND P H A R M A C O K I N E T I C STUDIES Situations may exist in which consideration of the properties of the test compound should lead to the 68

decision not to proceed with metabolic studies. An example is the case where a product is not absorbed after ingestion. Stringent evidence in support of this supposition or claim should be at hand before a decision is made not to do metabolic studies. For example, claims that there are, or could be, polymeric food additives which are not absorbed require careful scrutiny. One has to take into account the polydisperse nature of such polymeric materials, and the fact that even when it appears that almost 100% of the administered substance is recovered in the feces, there may be significant amounts stored in the reticuloendothelial system. Moreover, the capacity for persorption, though usually limited, must not be overlooked since this may also result in the uptake of polymeric material into the body from the intestine. The decision not to proceed with metabolic studies may be reached on the basis of preliminary (even in vitro) study of biodegradation. For example, a highly toxic metabolic product may be identified, such as the cyanide ion, rendering the parent compound unacceptable for use. Data indicating the formation of a physiological handling of the metabolite, constitute encouraging but not conclusive evidence of safety. Even in such circumstances, care must be exercised to ascertain that the intact molecules of undegraded material are not absorbed as such, even though the great bulk of the substance may undergo hydrolysis or other degradation in the intestine. These considerations apply to short-chain peptides, oligosaccharides and similar materials.

ANCILLARY METABOLIC STUDIES IN VITRO

Commonly Used Systems. The procedures to be described are not a formal part of metabolic studies, but may offer an opportunity to check the range, variety and nature of metabolites formed. Preparations from human liver and other human tissues can be used in metabolic studies, as appropriate. One of the most commonly used systems affords access to the microsomal mixed function oxidase enzymes from liver and other organs. The purity can range from the crude post-mitochrondrial supernatant fraction obtained from homogenized liver all the way to a preparation of purified cytochrome P-450, NADPH-cytochrome c reductase and necessary cofactors for mixed function oxidase activity. Since liver is most commonly the main site of metabolism of a test compound, these preparations are usually derived from rat liver. However, it may be advantageous to study the metabolism of a compound in kidney tubules, lung, intestine and possibly other tissues separately, as well as species other than rats. lsolated Liver Cells. Isolated liver cells are coming into increasing use for metabolic studies. The availability of good techniques for separation of liver cells in a functional condition (Tager, Soling & Williamson, 1976) offers attractive possibilities for the study of metabolism of a compound in vitro under a variety of conditions. The isolated liver cells may be derived from different species, including man. The isolated ceils may also be studied under conditions of perifusion, i.e., superfusion, involving a flow-through perfusion technique that provides an extracellular environment containing physiological levels of substrates maintained at a constant concentration (van der Meer, de Haan & Tager, 1976). The animal from which the cells are derived may be pretreated with phenobarbital or other mixed function oxidase inducers which serve to increase the metabolic capacity of the liver cells in bringing about the biotransformation of test chemicals. It is possible to separate centrilobular and periportal cells. Moreover, the isolated liver cells can be maintained in short-term culture, or even in longer-term culture. However, the profile of microsomal mixed function oxidases in such long-term culture cells is reported to undergo considerable changes as a result of the process of adaptation of the hepatocytes (Guzelian, Bissell & Meyer, 1977). The recommendations concerning in vitro systems are state-of-the-art but for this reason deserve some cautionary statements. The use of isolated liver cells is a developing science. The main advantage of such systems is that they potentially offer the full metabolic capabilities (including conjugation pathways) of the intact liver and can also be utilized for such studies as macromolecular binding and DNA repair. Thus, while the isolation techniques may be straightforward, the use of such ceils as a metabolic bridge between in vitro and in vivo studies is still in the development stages and the use of microsomal systems is perhaps more realistic at the present time. Perfused Organs. The use of perfused liver, lung, kidney and other organs affords an opportunity to check the range and variety of metabolites formed, without the complications that are inherent in the use of the intact animal. As with all in vitro preparations, the results are not identical with those which are obtainable in the whole animal. Nevertheless, useful preliminary interspecies comparisons can be made from in vitro studies. I'.C.T. 16 SUPPL. 2

F

69

NEWER SYSTEMS

Whole-Body and Other Forms o f Radioautography. The scope and application of radioautographic techniques for use in toxicology has been developed through the pioneering work of Ullberg. Although applied successfully in the pharmaceutical industry, these approaches have not yet gained general acceptance. The necessary equipment is readily available for work with a wide range of species, from mice to small monkeys. A recent issue o f A c t a Pharmacol. et ToxicoL (Vol. 41. Supplement 1, 1977) is wholly devoted to the applications of radioautography in pharmacology and toxicology, illustrating the versatility of this approach as an indicator of the distribution of radiolabeled compound in the whole body of an animal, including fetal tissues. Ideally, the method provides a means for surveying the organs and tissues of the body as a guide to quantitation of the distribution of the compound. A variety of unexpected storage sites have been revealed that would not normally be considered; for example, nasal mucosa is one of the first sites of localization for nicotine, and the fetal eye for methylmercury. In view of its ability to reveal possible toxic consequences of sustained selective secretion (for instance, of a metabolite of acrylonitrile into the stomach) or of specific affinity for a target organ, this technique should not be neglected. The specific organ effect--perhaps involving the local conversion of a very minor proportion of the compound to a chemically-reactive metabolite--will not be revealed by the usual kinetic analysis. Radioautography provides a ready means of following changes in distribution with time and dose. This is turn should permit a more appropriate design of quantitative distribution studies that focus attention on a particular organ. A systematic account of the potentialities of radioautographic methods which has been published recently (Liss & Kensler, 1976) should encourage more widespread use of these techniques. Such techniques can also be effectively utilized in the isolated cell systems for quantitation of DNA repair induction (Chapter 6). Electrophilic Reactants. The capacity of the compound under investigation to form chemically-reactive metabolites (CRM) is a piece of key information that has not usually emerged from metabolic studies. Knowledge of the formation of CRM provides insight into the toxic potentialities of the test compound (with particular reference to "irreversible" effects), and gives clues to the understanding of species and strain differences in toxicity, contributing especially to the extrapolation of test results in animals to man. Depending on their reactivity, CRM bind covalently with a variety of tissue macromolecules such as proteins, the various classes of RNA, DNA and glycogen, as well as interacting with lipids and other small molecules such as glutathione. The often transient existence of such CRM means that they are not detectable as such in body fluids or excreta but must be sought for at their nucleophilic binding sites. Evidence of the formation of a CRM may be forthcoming from ligand absorption spectra obtained by incubation of the test compound with hepatic or other microsomes in vitro. Such spectral indication has been used effectively by Uehleke, Taparelli-Poplawski, Bonse & Henschler (1977) to establish the formation of 2,2,3-trichlorooxirane in the course of microsomal oxidation of trichloroethylene. Within the category of covaiently-bound CRM, evidence may be developed from examination of blood and urine, as described below. The study of covalently-bound derivatives of test materials should seek to provide answers to the following questions: A. What is the evidence for the formation of (one or more) CRM? B. What is the tissue in which it is formed, and at which site? C. What is the enzymatic mechanism of CRM production? D. Where are the adducting sites and what are the adducting materials? E. What is the fate of the adducting moiety: If excision repair occurs in the course of DNA repair, with release of adducted products, can these be identified? If the excised products are excreted, can exposure to the test compound be gauged by the excretory pattern? F. What factors influence the formation and binding of CRM, for instance by affecting enzymatic activity or competing for adducting sites? G. Can species (and strain) differences in the toxic action of the test compound, or in susceptibility to such action, be accounted for on the basis of the information obtained concerning CRM? H. What are the time-dose relationships for CRM formation and covalent binding to genetic and nongenetic material? I. To what extent does CRM formation proceed at low doses? Is there a definable dose-response relationship? J. What proportion of genetic material covalently bound to CRM remains unrepaired at various doses and times? 70

At present, answers to the questions above are available for relatively few compounds. Certainly, the study of electrophilic reactants is not in the category of routine tests. Looking to the future, however, one must accept the fact that the methods are available, and the desired data fulfill more than a research objective in the "nice to know" catesory--on the contrary, in ~¢l¢¢ted ¢a~¢~, the information can help materially in the process of safety evaluation. The decision on how much of this work to pursue will depend on the nature of the test compound and other technical considerations. In discussing available methodology, an outline will be provided without recapitulating in detail the procedures and findings of Gillette and his colleagues (Gillette, Mitchell and Brodie, 1974; Gillette and Pohl, 1977) or the recent investigations on CRM derived from halogenated alkenes. In view of the fact that the CRM formed often represents only a very small proportion of the dose of test compound administered, work in this area usually necessitates the use of appropriately radiolabeled (most often 14C or 3H) compound. The studies of covalently-bound radioactivity may be conducted in vivo, for example with liver or other microsomes, as dictated by the observations in Stage I metabolic studies and the findings in subchronic toxicity tests. The analysis of species and tissue specificity of CRM formation and binding may be supplemented by studies on the influence of sex, age (e.g. weanling vs. young adult) and transplacental exposure. The reactions that lead to irreversible binding may be brought about by constitutive microsomal enzymes (i.e., the enzymatic activities present in the normal, "uninduced" animal) as well as by induced microsomal enzymes; both categories of mixed function oxidases usually follow Michaelis-Menten kinetics, at least up to substrate concentrations that achieve maximum reaction velocity. Attempts to detect the formation of an electrophilic reactant include a check for metabolic formation of free radicals or for ability of the compound, or its metabolites formed in vitro or in vivo, to react with nucleophilic acceptors (trapping agents such as nucleic acids, oligonucleotides, guanosine, etc.). The detection of carcinogen-DNA adducts by radioimmunoassay (Poirier, Yuspa, Weinstein & Blobstein, 1977) opens up the possibility of studying covalent binding to human tissues by this means. A variety of indirect approaches may be cited for the study of CRM and their irreversible binding. An indication of the formation and structure of CRM is derived by extrapolation back from stable metabolites. For example, reactive epoxide metabolites are the chemically reactive carcinogenic form of the polynuclear aromatic hydrocarbons, aflatoxin and other carcinogens. While the epoxide is the species which binds to macromolecules, stable dihydrodiol metabolites are isolated. Since the epoxides open enzymatically to dihydrodiols, the measurement of this type of stable secondary metabolite provides good evidence for the formation of CRM and, for the case of dihydrodiols, identifies the bond on the molecule across which the epoxide forms. The presence of lipid peroxidation may also be an indicator; this change may be monitored by various means, among which ethane evolution in the expired air is a useful non-invasive technique (Hafeman & Hoekstra, 1977 a,b). CRM may form adducts with proteins, attacking groups with relatively high nucleophilicity such as thiol, carboxyl and histidine residues. A search for such adducts was successfully conducted by Ehrenberg and his colleagues, measuring quantitatively the alkylation of histidine residues in hemoglobin of man and animals as an index of exposure (Osterman-Golkar, Ehrenberg, Segerback & Hallstrom, 1976; Osterman-Golkar, Hultmark, Segerback, Calleman, Gothe, Ehrenberg & Wachtmeister, 1977). The process has been taken a step further by Truong & Legator (1977), who have used an amino-acid analyzer to measure alkylated amino acid residues in hemoglobin and among the urinary amino acids excreted by rats. These techniques not requiring the use of labeled compound and readily applied to man have great potential. Ultimately, this approach may be extended to the excision products resulting from repair of DNA damage, as illustrated by the measurement of urinary excretion of methylated purines following exposure to dimethyl sulfate (L~froth, Osterman-Golkar & Wennerberg, 1974). Perhaps it would be prudent to point out that covalent binding of chemicals to proteins and RNA is not equivalent to binding to DNA and there are examples in the literature demonstrating that such differential specificity is correlated with carcinogenic potency. Indeed, as pointed out above, differential binding to specific sites of bases in nucleic acids has been correlated with the persistence of that binding and carcinogenic potency. Investigators are encouraged to recognize these differences, although the existence of any electrophilic properties of a potential food additive calls for careful investigation. One of the advantages that may be expected to accrue from measurement of the formation of CRM in man and animals stems from the application of pharmacokinetic principles to these data (Gillette, 1976). More specifically, the relationships between CRM formation on the one hand, and dose and conditions of administration on the other, is a principal consideration in those circumstances where CRM formation constitutes a "spillover" pathway of metabolism that is opened up once the reserve capacities of the normal metabolic and other defensive and adaptive mechanisms are exceeded. 71

STAGE I METABOLISM STUDIES General Aspects. The term metabolism refers to the study of the processes of biotransformation undergone by the test compound in the human or animal body, or in any other living organism. For any given chemical, the pathways of biotransformation may be numerous and diverse, giving rise to many metabolites. In many instances, some of the likely metabolites of a particular compound can be predicted. It is far more difficult to predict the relative metabolism by various animal species. Usually unknown are the effects of age, sex, diet, enzyme inducers and inhibitors and the influence of dose upon the pathways of metabolism and rates of formation, distribution and excretion of metabolites. To put all these matters to the test usually involves a substantial effort, which can be justified only to the extent that it contributes to risk assessment and to the design of long-term tests. An attempt will therefore be made to set limits to the scope of the recommended investigations, recognizing that the experienced toxicologist must guide decisions on the amount of work required for reliable and effective safety evaluation. Metabolic studies are usually conducted with radioisotope-labeled compound, although improvements in instrumentation are making possible more widespread use of stable isotopes such as 2H, 13C, lSN and 180. Non-radioactive labeled compounds have obvious advantages for studies in human volunteers. (One must not, however, overlook the operation of isotope effects, especially with deuterated compounds.) While one of the objectives of metabolic studies is to identify the main metabolites and their conjugated derivatives, there are two other major objectives, whose attainment is not particularly dependent on individual metabolite identification: 1. Quantitative and temporal relationships of the uptake, distribution, retention and elimination of radioactivity associated with the test compound. 2. Changes of metabolite "fingerprint" and of the relationships observed under 1., above, with various doses of the test compound. At least in those species of animals to be utilized for longterm feeding experiments, a range of doses should be studied from radio-tracer amounts, through the anticipated human exposure level (on mg/kg basis), to a dose approximating about 10-100 times this level, and a highest dose close to the maximum tolerated dose (Chapter 10) for the particular species (age, sex, diet, etc.) under study. Where enzyme induction or other forms of adaptation are known to occur, the high dose, or both doses, should be studied in adapted as well as in untreated animals (by an " a d a p t e d " animal is meant one that has undergone metabolic or other physiologic changes in response to exposure to the test chemical). Comparative Metabolism. Data on comparative metabolism are essential in the exploratory phase of safety assessment. The early studies should be conducted in those species likely to be used for the short-term toxicity tests, usually the rat and a non-rodent species. The strain of rat most frequently used is the randombred Sprague-Dawley, but for long-term studies this strain suffers from serious disadvantages and is increasingly being replaced by the inbred Fischer 344 rat (Chapter 10). These and other common rat strains demonstrate some striking differences in metabolic capacity, for the most part quantitatively. There are even greater differences among random-bred and inbred mouse strains, and, unfortunately, variations among the same mouse strains from different suppliers (Litterst, 1978). Thus, species and strains for metabolic studies must be chosen with the prospective toxicity tests very much in mind. The aim is to approach as closely as possible the qualitative and quantitative characteristics of human metabolism of the test compound; but, since these characteristics are usually unknown at the time testing begins, arbitrary decisions must be made. A suggested approach is to start with rats (Sprague Dawley or Fischer 344 strains) and also to do some experiments in mice (for example, Swiss Webster strain). For purposes of extrapolation to man, confidence in the results increases with the number of species found to be similar with regard to the metabolites formed. To rely exclusively on rodents, however, has led to errors. Hence, some metabolic work with nonrodents, preferably dogs or non-human primates, is desirable. The guinea pig, hamster, cat and pig have also been used for particular reasons relating to various categories of toxicity tests for which these species have been specially selected. "Major vs. "Minor" Metabolites. Weiner and Newberne (1977) argue against the current tendency of regulatory authorities to require separate toxicity studies of " m a j o r " metabolites. They point out that the biologically-important metabolites of concern from a safety standpoint are usually not the " m a j o r " metabolites, but often " m a j o r " chemically-reactive metabolites, that may not be detected at all unless evidence of their formation and covalent binding to cellular and other macromolecules is specifically sought for. As far as food components are concerned, what one is studying is the chemical entity. If this material is shown to be safe under conditions of intended use, there is little reason for concern about the possible toxicity 72

of some of the metabolites when tested at high doses. Moreover, a " m i n o r " metabolite may under certain conditions become a " m a j o r " metabolite and vice versa. There are circumstances in which a minor metabolite or degradation product is known to be toxic or carcinogenic; for example, ethylenethiourea derived from bisdithiocarbamate pesticides. Thus, special tests are justified where suspicion exists that a major or minor metabolite may present a hazard. Pharmacokinetics. Ideally, pharmacokinetic information should be developed as soon as tests involving subchronic repeated administration are contemplated. Much depends on the availability of either a sensitive and specific assay for the test compound or of the radiolabeled compound. If neither approach is possible, or really involves a major effort, the pharmacokinetic component of Stage I may be postponed to Stage II. Whether the early pharmacokinetic studies are conducted in Stage I or Stage II, the object is the same, namely the production of data for which, if possible, a simple mathematical model can be developed. Data should be gathered from which the apparent volume of distribution, and rates of absorption, distribution and elimination can be determined. The conventional approach, especially with candidate drugs, is to administer a single bolus dose of the compound by rapid intravenous injection and to collect blood, urine, feces, bile and possibly cerebrospinal fluid at predetermined intervals for a period of 96 or 120 hours. This procedure offers some advantages, but it will probably prove inappropriate for some food-related materials or other compounds under consideration here. For such substances, it may be necessary to use a single oral dose, preferably given by gastric intubation, and to carry out a simple balance study that provides a general view of the intake-output relationships of the compound.

STAGE II METABOLIC AND PHARMACOKINETIC STUDIES General Aspects. The importance of pharmacokinetic studies in toxicology has been the subject of numerous reviews in recent years (Gehring, Watanabe & Blau, 1976; Withey, 1977; Gehring & Young, 1977). The objectives of Stage II studies are to gain an understanding of the effects of dose and other factors on the ability of the animal to metabolize and otherwise handle the test compound. It is particularly necessary to gauge the effects of very high doses on these processes, so as to gain an idea of the abnormalities introduced should the body's metabolic a n d / o r excretory resources be swamped. Investigations of this kind, carried out on the actual species to be used in the long-term tests, greatly facilitate the extrapolation of results obtained in those long-term studies from one species to another, from high dose to low dose, and ultimately to the assessment of hazard for man himself. The Stage II studies also provide an opportunity to characterize the major metabolites formed in the two species and strains of animals selected for prospective use in the long-term tests, as well as to determine the relative proportions of these metabolites. Stage II studies should provide information on the tissue distribution of the parent compound and its metabolites. These data should help to elucidate target organ toxicity and thus possibly the nature of the toxic mechanisms. Investigation of the pharmacokinetics is intended to provide information on the dose-related absorption, distribution, metabolism and elimination after single and repeated doses of the test compound by the oral or other routes. In preparation for long-term tests, the data derived from animals of various ages (4-6 weeks, 10 months, and 20 months old) is instructive but not essential. The pharmacokinetic data after single as well as multiple exposures provide a means of calculating the rate constants for absorption (ka ), and elimination (k e), the half-life for elimination ( t ~ ) a n d the apparent volume of distribution (Vd). A basis is thus provided for the application of one or several mathematical models to the data. Understanding of the pharmacokinetic characteristics of the compound should lead to prediction of the equilibrium (or steady state) concentrations to be achieved in the long-term study, and the assurance that the dosages selected for the long-term tests will lead to different equilibrium concentrations. It should be stressed that the mathematical model is not intended to be a physiological model, i.e., it does not represent distinct tissue distributions nor mechanisms of disposal o f the compound. Procedural Aspects. Irrespective of the route by which a compound is to be administered, the first step in the pharmacokinetic evaluation should be the monitoring of blood levels, at properly selected times, depending upon the compound under investigation, after an intravenous bolus administration. Five geometrically-spaced doses should be used starting with the lowest dose that is capable of generating measurable blood levels. The results of this first investigation should give answers to the following questions: A. Is the compound eliminated (by metabolism, distribution and excretion) according to a first order (monoexponential) rate or is the elimination better described by a sum of two or three (or 73

more) exponentials (which is indicative of the involvement of more than one rate process proceeding at different rates)? B. Is there a change in the pharmacokinetic mechanism with dose? This may be reflected in the semilogarithmic plot of blood concentration against time, by a change in the slope of the linear terminal phase or the resolved (by means of residuals) initial phase of the curve. A change in the volume of distribution (calculated by dividing the known administered dose by the blood concentration at t =0) with dose can also indicate a change in the pharmacokinetic and physiological disposition. Further investigations should seek to apply this information to the conditions selected for the proposed long-term toxicity studies by using the same route of administration, the same doses and animal species. Subsequently, the experiments are repeated in fresh groups of animals that have been exposed to multiple administration of the unlabled compound in order to permit these animals to undergo whatever enzyme induction or other adaptive process the compound is capable of bringing about. A comparison of the results obtained in naive and in the multiply-exposed animals serves to indicate likely changes in the pharmacokinetics that might occur during the course of the long-term study. Distinct differences in the pharmacokinetic parameters obtainable after single and after multiple exposures suggest either that the compound can induce its own metabolism; or that sufficient body burdens can accumulate to saturate essential pathways of elimination or detoxication; or that the compound or its metabolites bring about cellular toxicity and tissue damage capable of affecting organ-specific metabolism or excretion, and hence altering the pharmacokinetic profile. Studies in Pregnant Animals. Dose-related studies of absorption, distribution, metabolism and elimination should also be carried out in pregnant animals. Studies of reproduction and teratogenesis clearly depend upon selection of appropriate doses, not only in regard to the maternal animal but also with respect to the amount of test compound reaching the conceptus. Pharmacokinetic analysis makes it possible to assess the amount of placental transfer of the parent compound and its metabolites at critical periods of organogenesis in relation to maternal exposure and handling. It is advisable to use timed-pregnancy animals strictly comparable with those intended for study in reproduction and teratogenesis experiments. In studies involving embryofetal pharmacokinetics and metabolism, whole embryos, fetuses, or selected fetal tissues may need to be pooled within litters (Chapter 8). The remarkable metabolic versatility of the placenta (Juchau, 1973), as well as the special characteristics of the placental barrier, need to be taken into account in studies involving exposure of the fetus in utero. Numerous unresolved questions surround carcinogenesis bioassays that begin with exposure of the parent generation, followed by lifetime exposure of the offspring. For example, doubts persist regarding inequality of distribution among the fetuses in utero of materials such as hexachlorobenzene (Andrews & Courtney, 1976), that have been administered to the mother. Basic issues such as the following should be resolved, if technically feasible, before undertaking studies spanning two generations: A. Qualitative and quantitative definition of the materials reaching the fetus, in relation to dose administered to the mother. B. Extent to which there is even distribution of such materials--qualitatively, quantitatively and temporally--among the fetuses within a single litter and between different litters. C. The metabolism and retention within the fetus of the test compound, and/or its metabolites, as well as the subsequent fate of any such retained materials. In this situation, distorted pharmacokinetics may reveal cumulative toxicity of retained products to the conceptus. Lack of data under headings A.-C. will necessitate the use of much larger groups of randomly-distributed pups for the 2-generation study. Man and rhesus monkey have a similar placental structure and circulation. The rat does not have a chorioallantoic placenta, yet placental transfer studies of some compounds have demonstrated a similarity to man.

Analysis o f Pharmacokinetic Data. The procedures for analysis of the pharmacokinetic data have been described in several texts (Goldstein, Aronow & Kalman, 1974; Gehring et al., 1976). The object is to develop a biologically meaningful pharmacokinetic model to which the data will fit. The log-linear portions of the pharmacokinetic data curves are fitted by linear regression analysis, using the method of least squares. Analysis of variance is one technique which has been used for comparing pharmacokinetic and metabolic data obtained at different dose levels and under different conditions.

74

For the situation in which the compound is administered rapidly as an intravenous bolus and is eliminated by first-order kinetics (i.e., at a rate proportional to its concentration), the interrelationships of the various critical parameters are described by the following equation applicable to the one-compartment open model: Ct = Co e-ket or

where

in Ct = in CO -ket

Co = initial concentration Ct = concentration at time t ke = rate constant for elimination

ke is the slope of the straight line plotted on semilogarithmic paper and In Co is obtained as the intercept on the ordinate axis. For first-order (exponential) elimination, the half-time for elimination ty~ is given by solving the equation In (½) = -kety~; hence, t,/: = 0.693/k e. The apparent volume of distribution Vd (given by dose/Co, the ratio of the amount of compound administered to the concentration of compound in the blood at t = 0) is that volume of fluid in which the compound appears to distribute with a concentration equal to that in the blood, assuming that the body acts as a single homogeneous container or compartment with respect to the compound. A common one-compartment model comprising an absorption phase, for a compound given orally, with consequent relatively slow absorption into the blood compartment, is described by the following equation: -FD Ct = Vd

ka (e -ket-e-kat) ka - k e

.

where D is the dose, F the fraction of the dose absorbed, Vd the apparent volume of distribution and k a and ke the rate constants for absorption and elimination, respectively (assuming first-order kinetics for both processes). For repetitive bolus administrations at time intervals ~, Wagner et al. (1965) have derived the following expression for the average concentration of compound in plasma or tissues, C ~ , when a plateau (steady state) is reached: FD Cao~ v - Vdke./. whence the corresponding amount of compound in the body, A ~ = 1.44t~FD/r. More complex situations are discussed by Gehring et al. (1976). It is important to recognize the assumptions inherent in these mathematical models (Wagner et al., 1965). Complications are introduced by continuous or intermittent dosing (cf. non-dosing at weekends); stimulation or inhibition of the metabolism of the compound by itself or some other agent; first-pass effects (metabolic transformation of the compound, or a substantial portion of it, during the first pass through the liver, so that it fails to reach the systemic circulation); protein binding, especially if the plasma level of the compound exceeds the protein-binding capacity; saturation of metabolic pathways or exhaustion of conjugating agents; renal reabsorption; enterohepatic circulation; decrease in F at higher values of D. Michaelis-Menten Kinetics. A special and important case is that of Michaelis-Menten kinetics giving rise to apparent zero-order kinetics. This situation comes about as follows. The simple linear one-compartment model provides an adequate description of the kinetics of compounds that distribute readily into the blood and tissues before substantial elimination of the compound has occurred. In addition to the more complex, but still linear, multicompartment models, it is necessary to consider the existence of non-linear systems. A common source of non-linearity is saturation of a key system, whether it be a metabolic, tissue-binding, distributive or excretory system. Thus, non-linear models describe situations such as those arising from saturation of mixed function oxidase capacity, for example as a consequence of the administration of high doses of a test compound having low toxicity. Such a saturable process is often effectively described by Michaelis-Menten kinetics, so that the rate of change of concentration C of a compound at time t is given by: ~C

VmC

~t

Km+ C

75

where V m is the maximal velocity of the saturable system and the Michaelis constant K m represents the concentration of the compound at which the rate equals 0.5 V m . At low doses or concentrations, approaching real-life situations, C < < K r n , s o t h a t 6(2 _ VmC 6t Km

that is, first-order kinetics apply. At the other extreme, for instance when a "Maximum Tolerated Dose" is applied,

C > > K m , s o t h a t --6C = Vm 8t a constant or zero order rate. There are serious practical implications of this difference between the pharmacokinetics of high and low doses. The application of this concept is particularly important where repair of damage to genetic material is involved. The work of Rajewsky, Kleihues and others (Goth & Rajewsky, 1974; Kleihues & Margison, 1974; Margison & Kleihues, 1975; Nicoll, Swann & Pegg, 1975; Kleihues, Lantos & Magee, 1976; Buecheler & Kleihues, 1977) on the excision of O~-alkylguanines has left little doubt that the repair of damage involving such bound reactive metabolites in brain DNA is a process that can readily be saturated. Hence, it is reasonable to regard this repair process as a non-linear system and to suppose that Michaelis-Menten kinetics are applicable. At high doses, or frequently repeated exposures, the capacity of the repair mechanism is clearly inadequate, so that progressive accumulation of unrepaired damage to the genetic material follows inexorably. Further Considerations. Nelson (1976) has emphasized the usefulness of initial graphic analysis of pharmacokinetic data prior to computer fitting to a system of differential equations that define the best multicompartment model. He makes the point that in this way comparison among several species of test animals is facilitated, and subsequent statistical analysis permits an assessment of closeness with which the model fits the observed data. In a perceptive exploration of pharmacokinetic prediction of tissue residues of drugs in foodproducing animals, Dittert (1977) emphasizes the importance of correlating data on excreta with plasma values when constructing a pharmacokinetic model, using a digital computer and curve-fitting program that permits any number of compartments. This precaution protects against at least two major errors: mistaking the slowest process observed as the overall elimination of the compound when what is really happening is sequestration in, or return from a deep compartment; and attributing to redistribution or elimination an increase in Vd associated with decreased plasma and tissue concentrations that are the result of growth of the animal. Problems of change in animal size are important in long-term pharmacokinetic studies. Yet, long-term studies are essential for compounds which, by virtue of their lipophilicity, resistance to metabolic degradation, ready sequestration or other properties, have a long biological half-life. The pharmacokinetic studies should be continued for a sufficient length of time to achieve steady-state tissue levels; for only under steadystate conditions can one assess the clinical effects, pathological consequences and other potentialities of exposure to the compound. An alternative to prolonged pharmacokinetic studies has been suggested by Hammer & Bozler (1977). These authors have administered a dose of radio-labeled compound to animals at the end of a long-term toxicity study and compared the resulting pharmacokinetic profile with that following the same single dose given to the control animals. Differences in pharmacokinetic profiles provide indications of enzyme induction or subtle damage to excretory organs, for example, that may not be evident morphologically. While there may be value in this suggestion as far as long-lived species are concerned, it is doubtful whether the condition of senile rats, mice or hamsters would lend itself to meaningful studies for purposes of extrapolation to man. If this stage of pharmacokinetics evaluation were set at one year, this would avoid defeating the main object of such investigations, which would be to provide confirmation or otherwise of the conclusions drawn from pharmacokinetic studies that preceded the long-term test. In effect, the pharmacokinetic profile would be added to the other investigations performed at the one-year interval sacrifice. 76

HUMAN METABOLIC AND P H A R M A C O K I N E T I C H A N D L I N G OF T H E COMPOUND Reference to human studies has been made at various places in this chapter. There is increasing use of human cells and tissues for in vitro studies of metabolism, including comparison of the activities and, occasionally, divergent pathways displayed by various human tissues, intact cells and microsomal preparations. Interindividual variations in activity amounting to 30-fold or more have been reported (Harris, Saffiotti & Trump, 1978). The issues involved in carrying out biomedical research in human subjects have been debated with increasing intensity in recent years. The use of prisoners is now effectively prohibited (Research Involving Prisoners, 1976; Protection of Human Subjects, 1977). The options that remain open in the development of human data through surveillance of exposed populations or by means of controlled exposure studies in volunteers are set forth in reports published by the National Academy of Sciences (1965, 1967, 1975a, 1975b). The procedures to be followed in order to comply with the legal requirements as well as ethical and moral obligations, and to insure truly "informed consent", are described in these and other publications (References on Use of Human Subjects). One alternative to the use of human volunteers for metabolic studies involves auto-experimentation by the investigator(s), an approach that Altman (1972) has termed " a n unappreciated tradition in medical science". Indeed, as Conti & Bickel (1977) have recounted, much of the early research on the metabolism of xenobiotics was based on auto-experimentation. There may be circumstances in which human exposure to a compound occurs inadvertently, as in research or during a spill, or originates as part of manufacture of the compound. These situations may provide opportunities for investigation of the presence and levels of the compound and its metabolites in excreta and body fluids. For most new compounds, however, normal volunteers are the most effective source of human metabolic information. The object of this section is to indicate (1) the potential contribution that work in human volunteers can make towards the achievement of the goals spelled out earlier in this chapter (Golberg, Milby & Davies, 1969); (2) the information to be regarded as an essential prerequisite to planning of human metabolic studies; and (3) the nature of the studies suggested as necessary, or merely useful, to attain the intended purpose. Potential Contribution. The importance of interspecies comparisons with regard to metabolism and pharmacokinetics of a test compound has been stressed. Obviously, since the ultimate concern is with health effects in man, knowledge of human metabolism of the compound provides information for which there is no adequate substitute. While the use of human volunteers is fully accepted in Phase I of clinical drug investigation, it is not always considered appropriate for other categories of chemicals, despite the fact that, in the latter instance, far more people may ultimately be exposed when each product comes into use. In vitro studies with human tissues (from surgery or autopsy) contribute useful data, but cannot replace experience of human metabolism in vivo. The prospect of widespread exposure of the human population to a compound in food makes it desirable to provide, at the very least, some human metabolic and pharmacokinetic da~ta as a prudent measure of protection against potential hazard. Such data make possible a comparison with the corresponding findings in animals, thereby indicating the degree to which each of the animal results is representative of human handling of the compound. Information of this kind is valuable in planning further and more elaborate, as well as more meaningful, investigations in animals (Golberg, 1975). Essential Prerequisites. Timing of the investigations in man would most appropriately be set after the completion of the subchronic studies in animals. At that point, short-term tests for mutagenic/carcinogenic potential will have been carried out, and the information forthcoming from Stage I metabolic studies in animals will be available. Evaluation of these data should indicate whether the compound is sufficiently readily metabolized a n d / o r cleared from the animal body, especially the degree and duration of retention, if any, of radioactivity derived from the labeled compound. The nature, dose-dependence and reversibility of any toxic effects seen in animals should be known, at least for the duration of the short-term studies. A dose level eliciting no observable adverse effect should have been determined. Availability of analytical methods for the compounc[ and for at least the major metabolites is desirable, though probably too much to ask for at this stage in many instances. Nonetheless, if labeled compound cannot be used for reasons of lack of safety or unavailability, procedures satisfying the necessary criteria for adequate analytical methods used in detection and measurement at low levels in body fluids and excreta will have to be developed before proceeding further. Nature o f Suggested Studies. Essentially, what is proposed is the equivalent of the studies carried out in Phase I of clinical drug evaluation; that is to say, cautious and limited administration of the compound under carefully-controlled conditions for the purpose of determining the time course and major pathways of 77

metabolism. Based on estimates of likely human daily exposure, a comparable single oral dose is used in each of two volunteers. If the compound is radiolabeled, the necessary safeguards are incorporated in the protocol. The nature, timing and number of the measurements to be carried out will be determined by the knowledge of the properties gained in the animal studies. The salient similarities or differences in the body's handling of the compound should be defined as clearly as possible. The end result should thus indicate the degree to which the rodent species, and any larger animals used in subchronic tests, are representative of human metabolism and pharmacokinetics of the compound. In further studies, dependent on the findings of this first investigation, and the results in the animal metabolic and pharmacokinetic studies, it may be considered advisable to administer 4 daily doses of the unlabeled compound, followed by a test dose of labeled material on the fifth day. The number of volunteers participating in the investigation may be increased and the dose raised to a small multiple of that used initially, in order to have some inkling of possible effects of dose on metabolic and pharmacokinetic patterns. Conclusions. Metabolic patterns with respect to the same compound may differ in man and animals. There is a need for metabolic data in man in order to select an appropriate animal model for long-term studies. Present law prohibits the use of prisoners but not volunteers. Even preliminary tests in a few volunteers of the metabolism of a compound in man will guide the selection of the proper animal species. Such a preliminary test can be conducted without undue hazard to the subjects, after completion of the subchronic tests, and before the start of chronic toxicity testing.

RESIDUES OF DRUGS USED IN FOOD-PRODUCING ANIMALS The discussion of metabolism and pharmacokinetics has been concerned with food additives or other food components. It hardly does justice to the complexity of the issues arising from the presence in food of residues of drugs used in food-producing animals. This subject has been discussed by Perez (1977) and other authors in an issue of the Journal of Toxicology and Environmental Health (Vol. 2, Number 4, March 1977), devoted to the topic of animal drug residues. As far as metabolism is concerned, the main complications arise from (1) the extension of test species for comparative metabolism to include the "target" species, usually farm animals; (2) the complex nature of the total residue problem in meat, milk, eggs and other animal food products. Not only is one dealing with a wide variety of extractable metabolites of the original drug, but also with nonextractable " b o u n d " residues, assessment of whose bioavailability and toxicity requires special methods of investigation (Jagland, Glenn & Neff, 1977). In essence, animals of target species are treated with the labeled drug and blood and tissues collected at appropriate times are, in turn, administered to laboratory rats or mice for follow-up of the fate of the label, both qualitatively and quantitatively. Concern with drugs used in food-producing animals is far more complex than those considerations ordinarily associated with evaluating a food additive. One has to consider how the food animal will metabolize a drug and what the biological half-life of the various metabolites will be that may appear as residues in the food derived from these animals. This in itself is a complex problem because most of the techniques involve radiolabels and then one has a secondary problem determining whether remaining radioactivity is associated with a biologically active fragment of the drug or, in the alternative, whether the drug has been degraded into components that have become incorporated in normal body tissues as normal biochemical components of tissue. Then one has to proceed to determine the most appropriate way to test the parent drug and related metabolites for toxicological and carcinogenic effects. If rats or mice are found to metabolize the parent drug in a manner similar to the way in which the food animal metabolizes it, this simplifies the problem. However, if rodent metabolism differs markedly from that of the food animal, then one may have to test certain metabolites much more extensively in rodents. Thus, issues of comparative metabolism assume great importance. A drug approved for use in one species of animal may have to be completely re-examined if proposed for use in another species of animal, whose metabolic patterns are markedly different. The whole subject of metabolism and metabolites assumes an added dimension because of the regulatory aspects of the establishment of legal "tolerances" and the need to monitor residues, which may involve the measurement of " m a r k e r " residues, i.e. of a major metabolite rather then of the parent compound. Further reading of the cited literature should provide insight into the standard approaches to the problems in this specialized field.

78

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Pharmacokinetic prediction of tissue residues. J. Tox. env. Hlth. 2,735. Donahue, E. V., McCann, J. & Ames, B. N. (1978). Detection of mutagenic impurities in carcinogens and noncarcinogens by high-pressure liquid chromatography and the Salmonella/microsome test. Cancer Res. 38, 431. Gehring, P. J., Watanabe, P. G. & Blau, G. E. (1976). Pharmacokinetic studies in evaluation of the toxicological and environmental hazard of chemicals. In New Concepts o f Safety Evaluation. Edited by M. A. Mehlman, R. E. Shapiro and H. Blumenthal. Hemisphere Publishing Corporation, Washington, D. C., p. 195. Gehring, P. J. & Young, J. D. (1977). Application of pharmacokinetic principles in practice. Proc. 1st Internat. Cong. Toxicol. In press. Gillette, J. R. (1976). Application of pharmacokinetic principles in the extrapolation of animal data to humans. Clin. Toxicol. 9, 709. Gillette, J. R. (1977). The phenomenon of species variations; problems and opportunities. 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Variations in hepatic microsomal metabolism in C57B1/6 mice from three different suppliers. Pharmacology 16, 131. LiSfroth, G., Osterman-Golkar, S. & Wennerberg, R. (1974). Urinary excretion of methylated purines following inhalation of dimethyl sulphate. Experientia 30, 641. Margison, G. P. & Kleihues, P. (1975). Chemical carcinogenesis in the nervous system. Preferential accumulation of O6-methylguanine in rat brain deoxyribonucleic acid during repetitive administration of N-methyl-N-nitrosourea. Biochem. J. 148, 521. Nelson, R. L. (1976). The utility of pharmacokinetics to the pharmaceutical industry. J. Clin. Pharmacol. 16, 565. Nicoll, J. N., Swarm, P. F. & Pegg, A. E. (1975). Effect of dimethylnitrosamine on persistence of methylated guanines in rat liver and kidney DNA. Nature, Lond. 254, 61. 79

Osterman-Golkar, S., Ehrenberg, L., Segerback, D. & Hallstrom, I. (1976). Evaluation of genetic risks of alkylating agents. II. Hemoglobin as dose monitor. Mutation Research 34, 1. Osterman-Golkar, S., Hultmark, D., Segerback, D., Calleman, D. J., Goethe, R., Ehrenberg, L. & Wachtmeister, C. A. (1977). Alkylation of DNA and proteins in mice exposed to vinyl chloride. Biochem. Biophys. Res. Commun. 76, 259. Perez, M. K. (1977). Human safety data collection and evaluation for the approval of new animal drugs. J. Tox. Env. Hlth. 2, 837. Poirier, M. C., Yuspa, S. H., Weinstein, I. B. & Blobstein, S. (1977). Detection of carcinogen-DNA adducts by radioimmunoassay. Nature 270, 186. Shilling, W. H., Crampton, R. F. & Longland, R. C. (1969). Metabolism of coumarin in man. Nature, Lond. 221, 664. Tager, J. M., Soling, H. D. & Williamson, J. R., Editors. (1976). Use o f Isolated Liver Cells andKidney Tubules in Metabolic Studies. North Holland Publishing Company, Amsterdam. Truong, L. & Legator, M. S. (1977). Alkylation of macromolecules. Draft manuscript. Uehleke, H., Tabarelli-Poplawski, S., Bonse, G. & Henschler, D. (1977). Spectral evidence for 2,2,3-trichloroxirane formation during microsomal trichloroethylene oxidation. Arch. Tox. 37, 95. van der Meet, R., de Haan, E. J. & Tager, J. M. (1976). Perifusion of isolated rat liver cells. In Use o f Isolated Liver Cells and Kidney Tubules in Metabolic Studies. Edited by J. M. Tager, H. D. Soling and J. R. Williamson. North Holland Publishing Company, Amsterdam, p. 159. Wagner, J. G., Northam, J. I., Alway, C. D. & Carpenter, D. S. (1965). Blood levels of drug at the equilibrium state after multiple dosing. Nature207, 1301. Wagner, J. G. (1971). Biopharmaceutics and Relevant Pharmacokinetics. Ed. 1. Drug Intelligence Publications, Hamilton, Ill. Weiner, M. & Newberne, J. W. (1977). Drug metabolites in the toxicologic evaluation of drug safety. Tox. Appl. Pharmac. 41, 231. Withey, J. R. (1977). Pharmacokinetic principles. Proc. 1st. Internat. Cong. Toxicol. In press.

REFERENCES ON USE OF HUMAN SUBJECTS Barber, B., Lally, J., Makarushka, J. L. & Sullivan, D. (1973). Research on Human Subjects. Russell Sage Foundation, New York. Bloom, J. J. (1973). Non-therapeutic medical research involving human subjects. Syracuse Law Review 24, 1067. Butler, J. K. (1978). Is it ethical to conduct volunteer studies within the pharmaceutical industry? Lancet i, 816. Code of Federal Regulations. Title 45, Pt. 46, Protection of Human Subjects. (Revised as of January 11, 1978). Subpart B (1978)--Additional protections pertaining to research, development and related activities involving fetuses, pregnant women and in vitro fertilization. Fed. Reg. 43, 1758. Etzioni, A. (1973). Genetic Fix. Macmillan, New York. Freedman, B. (Aug. 1975). A moral theory of informed consent. Hastings Center Report Studies, 32. Frost, N. C. (1975). A surrogate system for informed consent. J. Amer. Med. Assoc. 233,800. Harris, C. C., Saffiotti, U. & Trump, B. F. (1978). Carcinogenesis studies in human cells and tissues. Cancer Res. 38, 474. Informed consent--a proposed standard for medical disclosure (1973). New York University Law Review 48, 548. Katz, J., Editor (1972). Experimentation with Human Beings. Russell Sage Foundation, New York. Levine, R. J. (1975). In Comment. J. Amer. Med. Assoc. 232,259. McCormick, R. A. (1974). Proxy consent in the experimentation situation. Perspectives in Biology and Medicine 18, 2. National Academy of Sciences--National Research Council (1965). Some Considerations in the Use of Human Subjects in Safety Evaluation of Pesticides and Food Chemicals. Publication 1270, Washington, D.C. National Academy of Sciences--National Research Council (1967). Use of Human Subjects in Safety Evaluation of Food Chemicals. Publication 1491, Washington, D.C. National Academy of Sciences--National Research Council (1975a). Experiments and Research with Humans: Values in Conflict. National Academy of Sciences--National Research Council (1975b). Principles for Evaluating Chemicals in the Environment, p. 126. Page, I. H. (1975). Experiments on people. J. Amer. Med. Assoc. 232, 257. Proceedings of the International Conference on the Role of the Individual and the Community in the Research, Development, and Use of Biologicals. (1977). World Health Organization. Protection of Human Subjects. Research Involving Prisoners. The National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. Report and Recommendations. (1977). Fed. Reg. 42, 3076. Research Involving Prisoners. The National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. Appendix to Report and Recommendations. (1976). DHEW Publication No. (OS) 76-132. Toole, J. F. (1973). Informed consent. Circulation 48, 1. U. S. Department of Health, Education and Welfare. Public Health Service. National Institutes of Health. (December 1, 1971). The Institutional Guide to DHEW Policy on Protection of Human Subjects. DHEW Publication No. (NIH) 72-102. 80

GENERAL REFERENCES Baty, J. D. (1977). Species, strain, and sex differences in metabolism, Foreign Compound Metabolism in Mammals 4, 347. Bonse, G. & Metzler, M. (1978). Biotransformationen organischer Fremdsubstanzen. Georg Thieme Verlag, Stuttgart. Bridges, J. W. & Chasseaud, L. F., Editors (1976). Progress in Drug Metabolism, Volume 1. John Wiley & Sons, London. Davies, D. S. (1977). Drug metabolism in man. In Drug Metabolism--Microbe Man, Syrup. 1976, 357. Edited by Parke, D. V., Smith, R. L., Taylor & Francis, London. Dring, L. G. (1977). Species variation in pre-conjugation reactions of non-primate mammals. In Drug Metabolism-Microbe Man, Syrup. 1976. 281. Edited by Parke, D. V., Smith, R. L., Taylor & Francis, London. Goldstein, A., Aronow, L. & Kalman, S. (1974). Principles of Drug Action: The Basis of Pharmacology. Second Edition. John Wiley & Sons, New York. (Chapters on "The Absorption, Distribution and Elimination of Drugs", "Drug Metabolism", and "The Time Course of Drug Action"). Hottendorf, G. H., Van Harken, D. R., Madissoo, H. & Cabana, B. E. (1976). Pharmacokinetic considerations in toxicology. Proc. Eur. Soc. Toxicol. 17, 255. Jerina, D. M., Editor (1977). Drug Metabolism Concepts. ACS Symposium 44. American Chemical Society, Washington, D.C. Krueger-Thiemer, E. (1977). Pharmacokinetics. Kinetic aspects of absorption, distribution, and elimination of drugs. Handb. Exp. Pharmakol. 47, 63. Mazel, P. & Pessayre, D. (1976). Significance of metabolite-mediated toxicities in the safety evaluation of drugs and chemicals. Adv. Mod. Toxicol. 1, Pt. 1 (New Concepts in Safety Evaluation), 307. Quinn, G. P., Hurwic, M. J. & Perel, J. H. (1976). Interspecies differences in drug metabolism. Psychopharmacology 2, 605. Rowland, M. (1977). Pharmacokinetics. In Drug Metabolism--Microbe Man, Syrup. 1976, 123. Edited by Parke, D. V., Smith, R. L., Taylor & Francis, London. Van Rossum, J. M., Van Ginneken, C.A.M., Henderson, P. T., Ketelaars, H.C.J. & Vree, T. B. (1977). Pharmacokinetics of biotransformation. Handb. Exp. Pharmakol. 47, 125. Wagner, J. G. (1971). Biopharmaceutics and Relevant Pharmacokinetics. First Edition. Drug Intelligence Publications, Hamilton, Ill. Welling, P. G. (1977). Drug kinetics. Foreign Compound Metabolism in Mammals4, 1.

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