Human safety data collection and evaluation for the approval of new animal drugs.

Journal of Toxicology and Environmental Health

ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19

Human safety data collection and evaluation for the approval of new animal drugs Maryln K. Perez To cite this article: Maryln K. Perez (1977) Human safety data collection and evaluation for the approval of new animal drugs, Journal of Toxicology and Environmental Health, 3:5-6, 837-857, DOI: 10.1080/15287397709529618 To link to this article: http://dx.doi.org/10.1080/15287397709529618

Published online: 20 Oct 2009.

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HUMAN SAFETY DATA COLLECTION AND EVALUATION FOR THE APPROVAL OF NEW ANIMAL DRUGS Maryln K. Perez

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Bureau of Foods, Food and Drug Administration, Washington, D.C.

Before a new drug is approved for use in food-producing animals, data are required which demonstrate that food derived from the animal does not contain unsafe residues. Also, an analytical method for residues must be provided which is practicable for government surveillance and enforcement activities. Residue information is derived by using radiolabeled drugs to study metabolism in the animal for which the drug is intended. Residues in the edible tissues are characterized, and the amount of residues and their rates of depletion from the different tissues after cessation of drug treatment are determined. Bioassays with laboratory animals are used to study the toxicity of the drug and its important metabolites and to establish tolerance limitations. From this information, the conditions for the analytical method are established—that is, the compound(s) measured (the marker), the tissue (target tissue) monitored to ensure control of "total residue," and the required sensitivity. The method is subjected to validation in government laboratories and must meet the Food and Drug Administration's standards of precision, accuracy, specificity, and practicability. Finally, the method is used in simulated field trials to establish the required withdrawal time after drug treatment before the animals can be marketed for their milk or for slaughter for food.

INTRODUCTION Before a new animal drug can be marketed, it must be cleared by the Food and Drug Administration (FDA) on the basis of evidence of efficacy and safety. The sponsor of the drug has the responsibility to provide this evidence. When the drug is for food-producing animals, not only must the safety of the animal be considered, but also the safety of food products derived from the treated animals that are intended for human consumption. The FDA reviewer for human safety must address the following considerations: The concepts presented here are the result of the evolution of regulatory policies in the Food and Drug Administration to which many scientists in food safety evaluation have contributed over the years. The author is grateful to Dr. Joseph Settepani and Dr. Charles Kokoski, from the Bureau of Foods, Food and Drug Administration, for their technical review and valuable comments, and to Dr. Jeffrey A. Staffa, Office of Science, Food and Drug Administration, for his special assistance in preparing the manuscript. Requests for reprints should be sent to Maryln K. Perez, Bureau of Foods, Food and Drug Administration, Washington, D.C. 20015.

837 Journal of Toxicology and Environmental Health, 3:837-857,1977 Copyright © 1977 by Hemisphere Publishing Corporation

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What residues are found in the edible products from the treated animals? What is their significance for human health? 2. Can safe conditions of use be established to ensure the integrity of food? What are these conditions? 3. Do the residues in food need to be monitored after drug use is approved? If so, is there an acceptable method of analysis that can be used for surveillance and enforcement to ensure that the established safe conditions of use are followed in practice?


1.

When an animal is treated with a drug, the recipient animal (target species) metabolizes the drug. The residues found in edible tissues depend on the particular metabolism of each target species. The amount and composition of the residue (parent drug and/or metabolites) left in tissues after drug treatment must be assessed for potential adverse effects on human health. Many drug uses require a withdrawal period—that is, a time for residues to be depleted to safe levels after drug treatment. During the withdrawal period, the animals may not be slaughtered for human food, and milk may not be marketed for human consumption. For eggs, drug usage must be such that no harmful residues will be present in the absence of a withdrawal period because any requirement for withdrawal is considered impractical for laying hens. Therefore, it is important to define residue depletion in the edible tissues so that proper withdrawal times can be specified in the directions for drug use on the product label. If the time required for residues to be depleted to a safe level is not practical, or would render the drug of little value, the drug cannot be approved. The amount of residue permitted in food may not exceed a safe level. However, tolerances for residues are frequently established at a level substantially below that considered safe when higher tolerances are not needed to achieve a beneficial use. There are strong reasons for this. Scientists recognize that absolute safety can never be ensured. The degree of one's confidence that a particular product presents no risk to human health is correlated with the tests and procedures used in the evaluation. Toxicology is not an exact science; it requires judgment. It generally depends on the use of experimental animals to predict the potential for effects in humans. Chemicals are tested in animal bioassays at levels that are much higher than those to which humans would be exposed by normal food consumption. A comfortable margin of safety is required between the level of exposure that does not affect the experimental animals (the established "no-effect" level) and the level of exposure permitted for humans. These margins of safety vary in magnitude, depending on the nature and degree of the toxicity and the extent of the testing that was required. The margins of safety and other conservative practices used to restrict residues in food also must take into account that humans are subject to


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many competing risks from chemical exposures in their environment. Because of practical constraints of animal testing, competing risks engendered by exposures to drugs, food and color additives, pesticides, and natural contaminants cannot be realistically determined. It is recognized that many factors involved in a cause-effect relationship are not necessarily evident in test results, and that the use of animal models in terms of relevance to humans suffers several deficiencies. While the conventions of testing drug residues at appropriate high doses, applying large safety factors, and further limiting residues to levels no higher than necessary for effective drug use may strike some observers as ultraconservative, the need for these conventions is clear when they are viewed in the universe of potential competing exposures and test limitations. Animal drugs comprise a wide spectrum of products. The individual nature and use of a product have a direct effect on the level and significance of the anticipated human exposure to residues in food. Drugs have varying degrees of toxicity and some may even be carcinogenic. Some may be metabolized rapidly and eliminated from the animal with little or no storage of residue in edible tissue; others essentially may not be absorbed. Some drugs may result in residues with a very short biological half-life in tissue, and others may result in persistent residues. For some drugs, the predominant site of residues is muscle tissue. However, for many drugs the less frequently consumed tissues, such as liver and kidney, may be the major sites for residue accumulation. Some drugs may have very limited use, such as anesthetics for surgery, while others may have widespread use, as in growth promotion, where a large percentage of meat destined for human consumption is derived from treated animals. Some drugs are effective for a specific disease in only one species of animal, while other drugs may be effective for a variety of diseases in many animal species—for instance, common antibiotics used for prophylaxis as well as therapy. Some drugs are given only as a single dose early in the animal's life, long before slaughter, to prevent disease or to treat afflictions unique to the young. Still other drugs are effective agents throughout life, engendering a higher potential for residues at the time of slaughter. All of these usage factors affect the levels of anticipated human exposure to drug residues. They are important considerations in deciding the kinds and amount of data required to support safe use. Therefore, the unique nature of each drug, as described by all these variables, is the overriding consideration in determining particular safety data requirements for each proposed drug use. However, certain general guidelines can be described for the key areas of consideration for the approval of a new animal drug application (NADA). A description of each of these basic areas will be prefaced by a brief discussion of investigational drug use and its role in drug development.

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INVESTIGATIONAL NEW ANIMAL DRUGS Clinical animal investigations are necessary to establish drug efficacy and to determine the best conditions of use for drug approval. These studies may require the use of a substantial number of animals. The FDA can permit the salvage of food for human consumption from investigational animals under conditions judged to be safe, including the provision that residues must be depleted to a safe level. Authorizations from the FDA are required for each specific trial, and the sponsor must provide follow-up reports for each investigation on completion. The FDA must be notified of the date and place of slaughter for each animal authorized, unless a waiver is granted on the basis of a provision that the animal will be held under observation for an additional 30 days beyond the approved withdrawal conditions for the study. Figure 1 reflects the rationale for handling requests to market food from animals used in investigational trials. The three triangles in Fig. 1 show the relationships among the amount of safety data available, the withdrawal period, and the number of animals involved in terms of anticipated human exposure. Data requirements are deliberately unconstrained in the first triangle because of the flexibility that may accompany the use of very extensive withdrawal times, particularly for large animals. During drug development, data on efficacy and human safety should progress concurrently. As the indications of efficacy grow and the sponsor feels justified in making larger research expenditures and in conducting

Data Provided by Sponsor

Assigned Withdrawal Time by FDA

Number of Animals Authorized by FDA

180 deys milkman * kmified by safety evaluation of structure (Torfm). d m of drag. proposed use. utiyiicocnomical propertm, «tc

. No effect I m l estabEshed m subdnnic ttufo rtd 2. Tine reqund for depletion of toad residues, to no effect level with e safety factor of t t hast 12000.

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F I G U R E 1 . Investigational new animal drug authorizations: investigational exemptions for salvage of food from animals used in efficacy studies, authorized on a sliding scale.



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larger trials, the required information on human safety must increase in parallel to support the expanded investigational use. As a practical matter for very large trials, the sponsor generally desires withdrawal times close to those that ultimately will be suitable for approval. Therefore, as the number of investigational animals increases, and the flexibility in the use of a withdrawal period to provide additional margins of safety diminishes, the safety data must increase correspondingly. At one extreme of the spectrum, food usage of a small number of investigational animals may be approved with a 6 month withdrawal period, based only on information about the nature of the structure of the drug and the proposed use. The major consideration is the probable biological half-life of residues compared to a 6 month withdrawal. Experience indicates that this very extended withdrawal time is usually ultraconservative, in view of the relatively short half-lives of most drugs. One cannot, of course, overlook chemicals such as polychlorinated biphenyls which have an unusual persistence. Lipid affinity is one of the FDA's primary considerations in identifying a drug with an extraordinarily long biological half-life. Data on partition coefficients in 1-octanol and water may be used as an indication of potential problem agents, which, if they have high lipid affinities, might not warrant approval without safety data, even with a 6 month withdrawal. As one moves down the triangles shown in Fig. 1, the amount of safety data provided by the sponsor must move closer to meeting the NADA approval requirements. In all cases, the withdrawal period for an investigational new animal drug (INAD) will usually be longer than that anticipated for NADA approval unless all of the NADA safety data requirements have already been met. Generally, to support very large field trials, information on both residues and toxicity is necessary. The most useful residue information for an INAD is obtained by measurement of total residues in the edible tissues of the target animal and residue depletion during withdrawal. It is preferred that such data be obtained by administration of the appropriate radiolabeled drug to target animals. The total radioactivity in each of the principal edible tissues (tissue and fluid combined), expressed as a concentration in parent drug equivalents, is taken to represent the amount of total residue in each edible tissue. Because the minimum toxicology data required for NADA approval of a drug used in food animals include a 90 day study of the parent drug in a rodent and a nonrodent mammalian species, all or part of this information is usually requested by the FDA before very large trials are authorized. Combinations of these two types of data are used to make conservative decisions on the withdrawal times necessary for investigational trials. Compensation for the remaining gaps in information is provided by appropriate adjustment of the required period of withdrawal.

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SIX-STEP EVALUATION PROCEDURE FOR NEW ANIMAL DRUG APPROVAL The NADA must contain information on the chemical characterization of the active drug ingredient, its purity and impurities, all components of the formulation, drug stability, and the drug manufacturing process. The proposed conditions of use and all efficacy claims must be disclosed. Collection and evaluation of human safety data then proceed stepwise. Various decision points are based on information as it is developed. The basic steps involved in achieving a determination of safety include the following. Step 1. Acquire information about the nature and amount of residues in the principal edible tissues and their depletion after treatment. Use this information to determine the probable human consumption of residues. (The principal edible tissues are muscle, liver, kidney, and fat. Data are also obtained on milk and eggs. Skin is added in the avian species.) Step 2, Determine the testing required in laboratory animals to assess toxicity and to estimate a safe level of exposure for humans, based on information about the residues and drug metabolism in both food animals and proposed laboratory test animals. (This includes the selection of appropriate animal species for toxicity testing and the selection of residue compounds to be tested.) Step 3. Acquire data in test animals to determine the toxic effects and dose response from dietary exposure. 1.

In the analysis of the test results, apply appropriate safety factors to determine the maximum acceptable human exposure to residues in food, or 2. If the drug is a carcinogen, perform a statistical extrapolation on the tumor data from lifetime studies to estimate a level of residue exposure that is considered virtually nonexistent and that poses a describable, yet mathematically low, carcinogenic risk. (The sole purpose is to define "no residue" operationally and thereby provide a basis for computing the lower limit of measurement required to accept a residue assay that meets the requirements of the anticancer provisions of the law.) Step 4. Determine the tissue(s) [target tissue(s)] that can be used reliably to monitor residues in all edible tissues. Determine the residue compound(s) [marker(s)] that can be used to monitor total residues in the edible tissues. Establish the lower limit of measurement of the target tissue marker that ensures that all residues in all edible tissues do not exceed a level established in step 3 as safe. Step 5. Develop a practicable method of analysis to meet the conditions established in step 4. Validate the method to ensure its reliability as a regulatory method. Step 6. Use the method (step 5) in residue studies conducted under


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simulated field-use conditions to establish that, under the proposed conditions of use including any necessary withdrawal period, residues will be within safe limits when label directions are followed. Each of the steps and the studies for deriving the information are described in greater detail in the following sections. This overview demonstrates that the information on metabolism and toxicology developed to fulfill steps 1, 2, and 3 forms the primary foundation for the remaining steps. The development of this information must proceed in parallel with all other data development because metabolism and toxicity are interdependent in determining the scope of the investigation required in each area. Target Species Metabolism Knowledge about the metabolism of a drug in the target species is used in steps 1, 2, and 4. This information may be developed in one comprehensive study designed to fulfill all requirements, or it may be obtained from two studies: the first designed to obtain sufficient information to establish toxicity testing requirements, and the second to fulfill the requirements of step 4. The use of a radiolabeled drug generally provides the most practical approach for determining the composition of residues in edible tissues and for estimating the initial level of total residue and residue depletion patterns in each edible tissue. However, the use of other techniques is not excluded. The label chosen and its position in the parent molecule are critical. The most important criterion is that all molecular species of potential toxicological significance will be traced through use of the label. This may require labeling in more than one position. Preliminary studies should be performed to ensure the stability of the label within the molecular species of interest. Generally, a l 4 C label in a stable position offers advantages over a tritium label. However, if a very stable proton can be labeled, tritium may provide increased sensitivity. Any isotope may be used if it allows the necessary data to be collected for completion of steps 1, 2, and 4 and does not contribute a significant isotope effect that alters the metabolic handling or interconversion of the drug in question. When a tritium label is used, evidence of the nonexchangeable nature of the tritium must be furnished. The proportion of tritiated water relative to the total radioactivity in each tissue including blood and urine must be determined by special techniques, such as lyophilization and azeotropic distillation. Not more than 10% of the total tritium in any tissue at zero withdrawal may be hydrolyzable tritium. Essentially no chemical exchange at physiological pH will be permitted. Purity of the radiolabeled drug is important because small amounts of impurities may confound the interpretation of residues in the tissues. A


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purity of at least 98% is generally required to obtain adequate data. The purity should be supported by radiochromatograms, with counting data taken from chromatograms where at least two solvent systems or two dimensional chromatography is used. The radiolabel should be prepared with as high a specific activity as possible to optimize the measurement of residues in the tissues. The required level of measurement is ultimately determined by the available toxicology data. However, the development of this information depends, in turn, on knowledge about the residues. Therefore, it is prudent to use a high initial specific activity so that whatever the outcome of the toxicology studies, the work will not have to be repeated because of the lack of sensitivity. Unless the drug is a carcinogen, a specific activity adequate for the measurement of residues in the low parts-per-billion range should be more than sufficient and is usually not too difficult to achieve. Sample size, quenching, and counting efficiency must be taken into account in projecting the specific activity necessary for the desired measurements. Data must be provided to substantiate the practical limit of residue measurement with the radiotracer used and to support the ability of the label to measure all residues of potential toxicological significance. The animals must be given the radiolabeled drug by the same route of administration proposed for the drug use (e.g., oral or intramuscular), although not necessarily in the same form. For example, if metabolism information has been provided for the approval of a drug as an additive in feed, this information is acceptable to fulfill the step 1 requirement for the use of the same drug in drinking water or as a bolus, provided the doses are equivalent. The maximum dose and duration of exposure proposed for the drug use must be followed with one exception. For drugs involving continuous, long-term administration, the duration of dosing required to achieve a steady-state level of residue in tissue may be determined and then used in the metabolism study. Alternatively, dosing for 5-7 days is usually acceptable, on the assumption that any effect on drug-metabolizing enzymes would be likely to stabilize in approximately 3-5 days. Slaughter times for residue determinations must be selected to provide enough data points to evaluate the amount of total residue in each of the principal edible tissues and the change in the residue composition or profile during depletion. The first determination should be made within the first 12 hr after the last dose, when maximum absorption is expected. The last slaughter time should be selected when total residues will be no higher than the level that the sponsor can demonstrate as safe. The intermediate slaughter times provide additional data from which depletion curves can be constructed. At each slaughter time the total radioactivity in each of the principal edible tissues must be determined. It is helpful to have some indication of



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the number of compounds comprising the residue in each tissue as well as the physicochemical properties of each residue. Metabolites that are important as residues, because of their amount, persistence, or potential for toxicity, must be structurally identified. Mass spectrometry is considered one of the most practical and powerful physical tools for the structural verification of metabolites at low levels. Various techniques can also be helpful, such as using standards of suspected metabolites for dilution with isolated components and recrystallization to constant specific activity as well as double reverse isotopic dilution. There are no rigid criteria for the degree of characterization required for residues. The criteria are dependent on the individual evaluation of each drug. Several factors are germane: theoretical pathways of metabolism, toxicological importance of the potential metabolites, and nature of the proposed use of the drug related to the degree of probable human exposure. The depth of investigation required will be more extensive for drugs classified as carcinogens and drugs for which relatively high levels of residues are sanctioned to provide for effectiveness. Criteria are flexible for the number of animals required in a metabolism study. As a guide based on FDA experience, three large animals or five or six small animals for the determinations at each slaughter time will generally provide adequate profiles of residue depletion and enough tissue for metabolite identification. Permitting the use of biopsy samples may increase the practicability of using more animals. However, data from biopsy samples have not been accepted because of lack of evidence on the degree to which such surgical procedures may drastically affect metabolism by causing stress in an animal. Initially, metabolism data are used to satisfy step 1 in determining the anticipated human exposure to residues. In addition, the data are an important element in determining the extent of toxicological testing required in step 3, as described in the next two sections. When all of the toxicological evaluations have been completed, the data on target species metabolism are used to complete step 4, as described below in sequence.

Toxicology Studies Regardless of the proposed use of a drug, an evaluation of toxicity must include, as a minimum, testing of the parent drug compound in both a rodent and a nonrodent mammalian species by daily, oral administration for at least 90 days. Generally, the rat and the dog are the preferred species because of the large base of reference data available for comparison. It is preferred that the rodent study follow an expanded protocol that includes exposure in utero, exposure during weaning through the mother's milk, and 90 day exposure after weaning, which is during the period of rapid growth. In each of the toxicity studies, one treatment level should be high


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enough to produce some toxicity. The lowest treatment level in each study is selected to demonstrate no adverse effect compared with the high test level and the unmedicated control group. A t least one intermediate treatment level is included so that a dose-response relationship may be assessed. Each test group must include enough animals to provide a meaningful comparison of effects with those in the unmedicated control group. Generally, 20-25 rodents per sex per dose level, or about four animals per sex per dose level for the nonrodent study, are adequate for this phase of toxicological evaluation. The data collected ordinarily must include the following: food consumption, weight gain, organ/body weight ratios, hematology, clinical chemistry determinations, and observations of change in the animal's state of health, general condition, or behavior. Gross pathology is reported for each animal. In rodent studies histopathology is required for all gross lesions as well as for about 28 tissues in each of the control animals and each of the animals in the high test level group. Animals at the next lower level are examined histologically, with emphasis on tissues seen to be adversely affected in the high test group, to determine the "no adverse effect" level. Histopathological examination of all tissues at all dose levels is preferred for all animals. However, for large studies, random selection of an adequate number of tissues and examination of all target tissues and gross lesions may be adequate to establish the no-effect level. In nonrodent studies, histopathology should be performed on all test and control animals. Special tests are frequently added to a protocol, depending on the types of effects that are anticipated for a particular drug or that may be of special concern for the drug class in general. Several factors determine whether additional testing of a parent drug is necessary beyond the minimum just described. First, if the compound or any residue in food is a suspect carcinogen, lifetime testing in at least two rodent species is required. A compound may be considered a suspect carcinogen if it is structurally related to known carcinogens or if it might be expected to be transformed to a known carcinogenic compound when ingested. The biological activity of a compound may also place it in this category—for instance, exhibition of estrogenic activity. In addition, a compound may have produced abnormal hyperplasia or cellular proliferation (e.g., thyroid hyperplasia or bile duct proliferation) in the subchronic studies, thus indicating a possible need for longer testing to demonstrate noncarcinogenicity. The FDA has not required tests for mutagenic activity (Kolbye, 1977). However, it has suggested that the sponsors test for mutagenicity early in the study of a compound to provide some guidance in planning additional toxicity study programs. It is hoped that in the not too distant future, sufficient information will become available to support the use of a battery of mutagenicity tests, to provide a sound adjunct to



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other criteria now being used to identify compounds that require testing for carcinogenicity. Meanwhile, if a drug sponsor has voluntarily conducted tests that show mutagenic activity, the resulting data are considered in weighing the possible need for further testing. In addition to suspected carcinogenesis, other very important factors determine whether additional testing of a parent drug is required. Both the nature of the effects observed in the subchronic studies and known effects for other chemicals possessing similar structures must be considered in relation to the limitations of a subchronic study. For example, if a compound is structurally related to known teratogens, special teratogenic studies would be required in at least two species. If effects were seen in the reproductive organs in subchronic studies, three-generation reproduction studies would be required to investigate further the involvement of the drug. It would be impossible to describe all occurrences that may trigger a need for additional investigation, but these few examples illustrate the evaluative process. One important area of consideration, aside from observed or suspected toxic effects, is related to the level of residue in food. There is an operational cutoff level for the maximum quantity of total residue that could be considered negligible, unless the drug is a suspect carcinogen. This is 0.1 ppm in meat and 10 ppb in milk and eggs. Residues above this level are considered finite. Regardless of the proposed use of a drug or the preliminary assessment of its toxicity, extensive testing is required to establish finite residue tolerances. This testing includes two lifetime studies in two different rodent species; at least a 6 month, or preferably longer, nonrodent mammalian study; a three-generation reproduction study incorporating a phase on teratology; and any other special tests that may be indicated by the information relevant to a particular compound. FDA staff usually participate in and follow closely the various study groups that have been involved in protocol design. We try to select the best features and developments in this area of design as they pertain to the FDA's particular needs. Therefore, protocol recommendations are not static, but are continually updated as new experience and information warrant. Several references (Nelson et al., 1970, 1971; Friedman, 1970) are helpful in the initial development of protocols. It is recommended that sponsors submit protocols to the FDA for comment before beginning studies. Lifetime studies are generally conducted in rats and mice. It is currently recommended that the protocol for one of the rodent studies be combined with a reproduction study, using one of the litters from the first-generation offspring for the chronic study to provide exposure beginning in utero. We recommend that lifetime studies include at least 50 animals per sex per dose level, and that whatever number of animals are planned for interim sacrifice or for special study be added to this.


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The results of the subchronic studies provide guidance for selecting dose levels for the lifetime studies. The high-dose level should be high enough to stress the animals and to produce some toxic response, but not so high as to cause early death or obvious undue stress and inanition. The lowest dose must be one that produces no adverse effect compared with the unmedicated control group, except in the case of carcinogens, where an experimentally determined level of no tumorigenic effect is of no practical significance. The data collection process previously discussed for subchronic studies remains generally the same for lifetime studies. Termination of each test group is usually recommended when only 20% of the starting number survive. Controls are sacrificed when the last dose group is terminated. If 20% survival is not reached by about 30 months in a rat study, termination is usually recommended at that time. The conditions and quality control under which animal studies are conducted must meet good laboratory practice standards. Certain additional indices are used to judge acceptability. For example, the maximum number of animals lost to autolysis or unsupervised death may not exceed 25%. A supervised death is one that takes place according to a predetermined sacrifice schedule or because of apparent illness or a moribund state. It is important that the control and test groups have a high survival rate for a period sufficient to ensure adequate evaluation of the potential for chronic effects, including carcinogenicity. For example, with rats a survival of at least 55% through 18 months is expected for an adequate lifetime study. Importance and Testing of Metabolites It is important to assess the safety of metabolic residues in food. A drug residue in food may contain only a few metabolites, whereas complex drug structures may contribute as many as 50 or more metabolites when major and minor pathways are considered. Many of these metabolites often cannot be isolated from the tissues by nondestructive techniques. Also, they often cannot be fully identified or produced in quantities sufficient for extensive testing. The metabolites of a drug may be more toxic or less toxic than the parent drug compound, and their structures may be closely related to or markedly different from that of the parent drug. The importance of any metabolite in terms of its level, persistence, structure, relationship to the parent drug, and anticipated human exposure must be taken into account in the subjective decisions as to the need for toxicity testing of particular metabolites. Metabolites in food may be ranked according to amount present. Those that can be isolated and identified could be ranked in terms of their structural similarity to the parent drug and, in some cases, in terms of their predicted toxicological significance. However, prediction of toxicity on the basis of available information on structure-activity correlations is not an established science at this time.



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To assist in reaching decisions about the need for additional testing, data may be required on the metabolism of a parent drug in the mammalian species proposed for the toxicity testing. The extent to which the test species is exposed through its own metabolism to the complement of residues found in the edible tissues from the target animal can then be determined. Relying on autoexposure to metabolites through testing of a parent drug is, of course, less reliable for the quantitation of toxicity than is direct testing of metabolites. However, this information is helpful for making trade-off decisions in attempting to provide adequate assurance of safety by utilizing reasonable testing requirements. It is also frequently necessary to deal with residues that cannot be characterized, synthesized, or tested by conventional procedures. One must ensure that the animal species or strains selected for toxicity evaluation are reasonably relevant to humans. Any information available on human relevancy is used in the test animal selection process. Testing is optimized by selecting, from among the relevant prospective species or strains, animals whose metabolic pathways result in autoexposure to residues that are qualitatively similar to those found in food consumed by humans. Other factors related to anticipated biological effects are also considered in the selection of test animals. Known sensitivity to a particular effect is important. For example, if the development of mammary tumors was an anticipated effect, one would consider the susceptibility to mammary tumor development of the candidate test animals to be an important selection factor. Special testing in the cat of dihydrostreptomycin for ototoxicity was requested because ototoxicity is a known human effect of acute high doses of this drug; the cat is known to be both a sensitive and an appropriate model in which to evaluate effects from extended low-dose exposure. Although test animals may be autoexposed to a metabolite in food through testing of a parent drug, additional separate testing of the metabolite may be required if it is considered to be a significant residue, based on a number of factors. These factors include the amount of the metabolite anticipated in food and the anticipated frequency of consumption of tissue in which it occurs, structural similarity or dissimilarity to the parent drug, anticipated absorption properties, and overall margins of safety available between the no-effect level established for the parent drug and the total residues to be permitted in food. Criteria applied to evaluation of potentially carcinogenic residues are more rigorous. These are described in the Federal Register (1977). When major amounts of residues elude characterization and the residues appear to be bound to macromolecules (intractable residues), various chemical approaches may be used to determine whether the residues are likely to include the intact parent drug molecule or a closely related structural species. In addition, it must be determined whether the


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residues result from incorporation of radiolabeled fragments into endogenous substances created by extensive metabolic breakdown of the parent drug, and whether they are of toxicological interest. Similarly, biological approaches may also be used to answer questions about the potential importance of intractable residues. For example, such residue fractions may be given to test animals to examine the degree to which they are absorbed and bioavailable. Tissues from target animals or composite tissue extracts containing residue, when possible, may be fed in short-term experiments (relay toxicity studies) to determine whether an inordinately toxic material is present. The relay toxicity approach is generally considered the last approach, used when all other approaches are not feasible. It is least desirable because it allows little, if any, application of safety factors in determining safe dose. A drug sponsor must consider the potential significance of metabolites. The sponsor must also present a reasonable analysis of the data that support the adequacy of testing to ensure sufficient margins of safety in establishing the acceptable total drug residue level to which humans are exposed through food. Establishment of Tolerances Tolerances are established for noncarcinogenic drugs by first selecting the level demonstrated to have no adverse effect in the most sensitive test species used in toxicity studies. This no-effect level is then adjusted to account for the differences between test animals and humans in food consumption versus body weight. The conversion is based on a 60 kg human with a total dietary exposure of 1,500 g of solid food or 1.5 liters of milk. This value becomes the base for acceptable levels of exposure. The safety factors to be applied to this value depend on whether finite or negligible tolerances are required. For finite tolerances the acceptable daily intake for humans is calculated by applying a safety factor usually of 1:100. Support of finite tolerances requires, at a minimum, lifetime studies in two rodent species, a 6 month or longer study in a nonrodent mammalian species, and a three-generation reproduction study as previously described. A smaller margin of safety would be considered adequate when direct information is available from chronic oral exposure in humans or when the no-effect level is based on a very sensitive parameter (e.g., cholinesterase inhibition) for which there is sound correlative information for the test animal and humans. Higher margins of safety are required in certain cases. For example, a teratogen requires a safety factor of at least 1:1,000. From the maximum acceptable daily intake calculation, tolerances are established for particular edible tissues after food consumption is estimated. Meat is generally considered to be one-third of the total daily diet. Therefore, the maximum acceptable concentration of residue permitted in meat, equivalent to the safe level of exposure, would be derived by



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multiplying the maximum acceptable daily intake level by three. Because milk may comprise the entire daily diet of infants, and because infants may be more susceptible than adults, tolerances in milk are generally established at 0.1 times the level established for meat. Tolerances in liver, kidney, skin, and fat may be established at levels higher than those in muscle tissue by using additional factors, provided the maximum daily intake is not exceeded. Table 1 presents consumption adjustment factors for these tissues relative to muscle tissue. Use of these additional factors depends on the amount of residue in that particular tissue in relation to the residue level in muscle tissue. Table 1 will be used as an interim guideline until better information is available on the amount and frequency distribution for human consumption of organ meats. It is important to stress that at no time will tolerances be established at a level higher than necessary for the effective use of a drug, even though the toxicity studies and the computations for the projected safe level appear to support higher levels. When lifetime testing has not been required and there is no reason to suspect that a drug may be carcinogenic, a negligible tolerance may be established on the basis of data from subchronic studies and any other special studies that may have been required for the drug. Negligible tolerances are computed in a manner analogous to that used to compute finite tolerances, except that a safety factor of at least 1:2,000 is applied and an upper allowable limit is imposed. The maximum approvable negligible tolerances in muscle and milk are 0.1 ppm and 10 ppb, respectively, even though the computed safe level with a safety factor of 1:2,000 may be above 0.1 ppm in muscle and 10 ppb in milk. Conversely, if the computed safe level results in a value less than 0.1 ppm in muscle tissue or 10 ppb in milk, then that lower value becomes the established negligible tolerance for that product. Usually, negligible tolerance is used to define the lower limit of reliable measurement required for a regulatory assay, which will be described in the section on selection of the regulatory assay conditions. When there are technological difficulties in meeting the analytical TABLE 1 . Consumption Factors Relative to Muscle Major species categories Tissue

Beef

Pork

Sheep

Poultry

Muscle Liver Kidney Skin

1 2 3 _o 4

1 3 4 4 4

1 5 5 _a 5

1 3 5 2 2

Fat

"Not used for human food.


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M. K. PEREZ

requirements for a particular tissue that is less important in the diet than muscle, special consideration will be given to a slight increase in the negligible limit in accordance with the ranking factors shown in Table 1. A tolerance is the allowable upper limit of total drug residues of potential significance. All residues (parent drug and metabolites) are considered potentially significant within the tolerance limitation unless part of the total residue has been specifically identified and shown to be safe. If this is done for any single component, the amount of the component may then be subtracted from the total residue, provided the component has no toxicological importance. If the component is less toxic than the parent drug and is the major component of the residue, it may be dealt with separately. In the latter case, special adjustment is made during establishment of control parameters in step 4 to ensure control of the safe levels of the component in question and the remaining total residue. In some cases a sponsor has sought a regulation on a negligible tolerance basis and has then found it necessary to develop a considerable amount of data beyond the subchronic testing stage, but short of the full complement of testing usually required for a finite tolerance and the use of a safety factor of 1:100. In such cases, it has been possible to establish a tolerance based on a safety factor slightly less than 1:2,000 but much greater than 1:100. Flexibility in the standard is maintained to maximize the use of reasonable scientific judgment. Waiver of Requirement for a Tolerance Some drugs, because of their special nature, may not require the establishment of a tolerance and the control of residues through monitoring. Approval of this type of drug requires assurance either that no withdrawal period is needed for residues to fall within safe limits or that an adequate withdrawal period is inherent in the proposed drug use. Also, there must be no concern about residues resulting from misuse or overdosing. Some drugs are very poorly absorbed, and some are metabolized rapidly and eliminated in such a way that no residues of toxicological interest occur in tissues, even from overdosing. The dosage form of a drug, prescription restrictions, and use as related to inherent withdrawal periods are all considered in assessing the need for control of residues and for potential violation of the approved conditions of use. Carcinogenic Drugs The Food, Drug, and Cosmetic Act permits the approval of a carcinogenic animal drug provided the use of the drug is efficacious, that it does not harm the animal, and that "no residue" will occur in food as determined by a method of analysis prescribed by the Secretary of Health, Education, and Welfare. The regulatory assay used to ensure no residue



853

will be the most sensitive, reliable procedure available. Because the FDA recognizes that the best available method may not be adequate to protect public health, minimum standards must be met for acceptability. For an operational definition of no residue, the tumor data from lifetime studies in rodents (where it has been shown that the drug is a carcinogen) are used in a modified Mantel-Bryan statistical extrapolation procedure. This procedure determines, with 99% confidence limits, that the theoretical level that represents a lifetime risk from cancer to a human following a daily exposure is no greater than 1 in 1 million. In the extrapolation, a dose-response slope of 1 is used unless the slope in the observable region indicates that a more conservative value (slope less than 1) is necessary. Proper selection of tumor data for use in the procedure is critical for maintaining the conservative intent of the process. The criteria that must be met are described in the Federal Register (1977). The extrapolated value is used as an operational definition of no residue for human exposure. This value is then translated to the appropriate values for specific tissues in a manner that ensures that the sum of the total residues (parent drug and metabolites) in all edible products does not exceed the level operationally defined as no residue in the total daily diet. A t this point, the procedures outlined for steps 4, 5, and 6 are applied to complete the requirements for approval. Selection of the Regulatory Assay Conditions (Step 4) After steps 1, 2, and 3 have been completed and the tolerance level has been determined, or the operational definition of no residue has been defined in the case of a carcinogen, the next step is to determine the conditions for selecting the regulatory assay. The residue depletion curves for the principal edible tissues, previously derived in a metabolism study, are used for this purpose. First, the tissue (target tissue) that can be used as a valid monitor for total drug residues in all tissues is selected. Usually, it is the tissue in which the total residues require the longest time to reach a safe level. However, there are situations in which another tissue is preferred and for which conditions exist to ensure its validity as the target tissue. Milk and eggs are always considered target tissues. In addition, one edible carcass tissue is selected to monitor all carcass tissues. Figure 2 illustrates how the metabolism data are used to select the residue compound(s) to be measured by the regulatory assay. Assume, for simplicity, that liver has been selected as the target tissue, and that total residues in all other edible tissues have depleted to the safe level in a shorter time than that required for depletion in liver tissue. Also, assume that the safe level for total residue in liver (SM) was established at 100 ppb in step 3. Metabolite A, if its structure had been identified, would be an excellent marker for control of total residues. A t all times after drug use,

M. K. PEREZ

854

900

„

500

Q.

a. •2 200

§ 100 Downloaded by [University of Cambridge] at 05:40 06 November 2015

c o o "35 0

= 100ppb

70 50 40 30

^Regulatory Assay Limit of Reliable Measurement

20

a. 10

1 2 3 4 5 6 7 8 9 10 11 12 Days (withdrawal)

FIGURE 2. Depletion curves for the parent drug (P) and its major metabolites (A, B, and C) in liver as target tissue; T is the amount of total residue derived from total radioactivity in the tissues, expressed as residue concentration in parent drug equivalents.

its level is relatively high and the slope is steep enough to provide a good discriminant for violative and nonviolative samples. The presence of metabolite A in liver at a concentration above 60 ppb is the indicator that the safe level (or tolerance) for total residues (100 ppb) has been violated. Conversely, if metabolite A is not present above 60 ppb, there is assurance that the total residues in all edible tissues will be below a safe level. In this manner, step 4 establishes the conditions for the regulatory assay: the compound to be measured (marker), the tissue on which the assay will be performed (target tissue), and the required lower limit of reliable measurement for the marker in the target tissue. Development and Validation of the Regulatory Method (Step 5) A regulatory assay must be developed in which the marker in the target tissue at the required lower concentration established in the preceding step is reliably measured. The drug sponsor must provide data demonstrating the accuracy and precision of the method, using control tissue samples "spiked" at various levels, including the required lower level of measurement. Data must also be provided to demonstrate the reliable determination of residues that result from drug treatment of the animal (dosed samples). The method must be specific for the marker, because it will be used on animal tissues with an unknown exposure history.


855


Frequently, a confirmatory test for residue identity is required when the assay proposed for surveillance is not inherently specific—for instance, a gas chromatographic method. In addition to being accurate, precise, and specific, the method must be practical for regulatory use in terms of time, equipment, and expertise. The validation data of the sponsor are statistically analyzed to determine whether the proposed procedure is ready for a validation trial in laboratories of the FDA and the U.S. Department of Agriculture (USDA). Before the drug is approved, the proposed regulatory assay must be validated successfully at the required lower limit of measurement and it must be acceptable to both the USDA and the FDA. Establishment of the Withdrawal Period (Step 6) The final step of the human safety evaluation process is to establish practical conditions of use which will ensure the depletion of residues to a safe level. Trials are conducted in target animals under simulated field use conditions according to the proposed conditions of use. The proposed regulatory assay is used for the residue measurements. These trials involve more animals, especially at or near the expected withdrawal time, than were required for the metabolism studies. The large number is used to provide a measure of the animal-to-animal variability in drug residue depletion and thereby, through the use of statistical techniques, to refine the withdrawal conditions necessary for safe use. The trials are conducted as a final step after a practical method of analysis has been established. The withdrawal time required to ensure safe use is extended, if necessary, to be compatible with conditions of animal husbandry normally expected to be followed. Withdrawal times pertaining to slaughter are specified in numbers of days. Withdrawal times for milk are specified in 12 hr milking intervals up to a maximum of eight intervals (96 hr). COMPOUNDS AFFECTING SUBSTANCES ENDOGENOUS TO TARGET ANIMALS Some endogenous substances in target animals are physiologically potent compounds; they include suspect or known carcinogens. The naturally occurring level of these substances in edible tissues may or may not be known. When there is reason to believe that a drug product may affect endogenous pools and present a potential risk to human health, such effects must be investigated. If there is an effect that increases the naturally occurring (normal) levels of potentially carcinogenic endogenous substances in a target animal, studies are required to unequivocally establish the normal levels for untreated target animals and to establish conditions under which these norms are restored in the drug-treated animals. The norm is established by measuring the affected endogenous sub-


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stances in edible tissues and by selecting a marker substance and target tissue in a manner analogous to that described in the preceding sections. An endogenous marker is then measured in untreated animals, using studies designed to account for differences related to factors such as breed, age, sex, state of estrus, and geographic location. These data are presented as a graph (99% confidence limits) of the cumulative frequency distribution versus the observed naturally occurring levels. This process defines the norm. The assay used must be sensitive enough to measure the endogenous marker in at least two-thirds of the sample population. This criterion is applied to ensure an adequate determination of the median and of the distribution pattern. Studies are then conducted to establish the postexposure decay of the endogenous marker in animals treated with the drug under the proposed conditions of use. These data are used to determine the withdrawal time for restoration of the norm. Restoration is achieved when the concentrations of the endogenous marker in the treated animals are the same as the norm previously described (99% confidence). When an endogenous substance is selected as the marker in step 4, the lower limit of required measurement for the regulatory assay corresponds to the 33rd percentile of the norm. Toxicity studies of affected endogenous substances need not be conducted if the above conditions are met. However, the drug sponsor retains the option of establishing safety through toxicity testing. USE OF THE REGULATORY ASSAY TO DETERMINE COMPLIANCE If a target tissue contains the marker residue at or above the approved level derived in step 4 , the carcass from which the target tissue was taken is considered adulterated and unsafe for human consumption. This evidence also suggests that the drug has been used in violation of the Food, Drug, and Cosmetic Act. Investigation of the circumstances leading to the violative residue may result in criminal prosecution by the FDA. There is, however, one exception. When drug residues are controlled through the measurement of an endogenous marker, the discriminant for a potential violation is measurement of the marker at the 99th percentile of the norm. Before regulatory action is initiated, an investigation will be conducted to determine whether the suspected drug was administered to the animals in question. It will also be determined whether the potentially violative sample originated from a group of animals whose median level of endogenous marker was greater than the norm established at the time of drug approval.


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REFERENCES Federal Register, February 22, 1977, p. 10412. Friedman, L. 1970. Symposium on the evaluation of the safety of food additives and chemical residues: I I . The role of the laboratory animal study of intermediate duration for evaluation of safety. Toxicol. Appl. Pharmacol. 16:498-506. Kolbye, A. C. 1977. Regulatory considerations concerning mutagenesis. Paper presented at the Annual Seminar of the Society of Cosmetic Chemists, Montreal, Canada, May 5. Nelson, N. et al. (Food and Drug Administration Advisory Committee on Protocols for Safety Evaluations: Panel on Reproduction). 1970. Report on reproduction studies in the safety evaluation of food additives and pesticide residues. Toxicol. Appl. Pharmacol. 16:264-296. Nelson, N. et al. (Food and Drug Administration Advisory Committee on Protocols for Safety Evaluation: Panel on Carcinogenesis). 1971. Report on cancer testing in the safety evaluation of food additives and pesticides. Toxicol. Appl. Pharmacol. 20:419-438. Received July 7, 1977 Accepted July 28, 1977

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