- Email: [email protected]
Safety assessment of animal drugs
146-152(1982)
REGULATORYTOXICOLOGYANDPHARMACOLOGYZ,
Safety Assessment DAVID
of Animal Drugs’
KOBYLKA~
Divisionof Chemistry and Physics. Food and Drug Administration,
Washington, D. C. 20204
The Food and Drug Administration has published a proposal (Chemical Compounds in Food-Producing Animals: Criteria and Procedures for Evaluating Assays for Carcinogenic Residues, Federal Register 44, 17070-l 7114, March 20, 1979) to establish procedures and minimum criteria to ensure the absence of cancer-causing residues in edible products of foodproducing animals to which drugs, food additives, or color additives have been administered. Section III of the proposed rule (termed the Sensitivity of the Method Document or SOM) would require that metabolism and comparative metabolism studies be conducted in target (food-producing) and test (laboratory) animals, respectively. As a practical matter, current agency policy prescribes that information from such studies would be required whether the compound was considered to be a suspect carcinogen or a noncarcinogen. Metabolism studies to determine what drug-derived residues may be present in the tissues of food-producing animals at a particular withdrawal time as well as comparative metabolism studies to determine the appropriateness of the laboratory animal as a test species, and the relationship of the two studies to each other, are discussed in terms of the procedures used for the human safety assessment of animal drugs.
INTRODUCTION New animal drugs are used in food-producing animals for many reasons, ranging from increasing the rate of weight gain to therapeutic treatment of specific diseases. They may be administered once or fed over the entire lifetime of the animals. The fact that food-producing animals are capable of converting these drugs to a mixture of parent drug and biotransformation products (metabolites) requires that the human safety assessment take into account all residues of toxicological concern (parent drug and metabolites) before a new animal drug application (NADA) is approved. Unless it can be shown that certain residues have virtually no degree of toxicological concern associated with them, all metabolites and degradation products must be considered during an evaluation of drug residues in the edible tissues of foodproducing animals. Total drug residue can be defined as: All compounds present in edible tissues of the target animal that result from the use of the drug, including the drug, its metabolites, and any other substances formed in or on food because of the drug’s use.
The Food, Drug, and Cosmetic (FD&C)
Act was amended in 1958 to require
’ Presented at the Sixth Annual Spring Workshop, Association of Official Analytical Chemists, Ottawa, Ontario, Canada, May 12-14, 1981. ’ Send reprint requests to Dr. David Kobylka, Division of Chemistry and Physics, HFF-458, Food and Drug Administration, 200 C St., SW, Washington, D. C. 20204. 146 0273-2300/82/020146-07$02.00/O Copyright@ I982 by AcademicPrcss,Inc. All rightaofrcproduction in any form reserved.
SAFETY
ASSESSMENT
OF ANIMAL
DRUGS
147
premarket clearance for the use of food additives. However, it prohibited the use of carcinogens as food additives. This prohibition is known as the Delaney Clause and similar wording appears in the food (feed) additives section (409), color additives section (706), and the 1968 new animal drug section (5 12) of the Act. The anticancer proviso of the Clause reads as follows: . . . that no additive shall be deemed to be safe if it is found to induce cancer when ingested by man or animal, or if it is found, after tests which are appropriate for the evaluation of the safety of food additives, to induce cancer in man or animal. . . . Four years after its enactment, the Congress concluded that the anticancer proviso was too stringent as applied to food (feed) additives and amended it. Consequently, section 512(d)(l)(H) of the FD&C Act permits the use of carcinogens in food-producing animals: .
if the Secretary finds that, under the conditions of use specified in the proposed labeling and reasonably certain to be followed in practice, (1) such drug will not adversely affect the animals for which it is intended, and (2) no residue of such drug will be found (by methods of examination prescribed or approved by the Secretary by regulations) in any edible portion of such animals. . . .
In an effort to provide a scientific rationale for carrying out the above statutory mandate, the Food and Drug Administration has proposed criteria by which to evaluate new animal drugs under the Delaney Clause, including criteria for the analytical chemical methods to define the no-residue portion of the anticancer proviso amendment. Drafts of the proposed criteria have been published in the Federal Register (1973, 1977, 1979). The most recent version of the criteria employs a detailed scheme involving metabolism and pharmacokinetics in an integrated approach to human safety evaluation. The criteria include a six-step procedure that is to be used by the sponsor of a new animal drug in which the safe use of carcinogens and noncarcinogens in food-producing animals will be evaluated. The six steps are outlined: 1. A metabolic study in the target animal to characterize/identify residues of the drug in edible tissues and to establish their depletion profiles. 2. Comparative metabolism studies in laboratory animals (species/strains) to aid in evaluating/selecting test animals used for toxicity bioassays. 3. Toxicity/carcinogenicity testing to determine a safe level for residues of the sponsored drug. 4. A metabolic study in the target animal to identify a “marker residue” and “target tissue” which can be used to monitor total residues in the food supply. 5. Development of a regulatory assay to determine the “marker residue” in the “target tissue” at the required level of measurement. 6. Establishment of a premarket withdrawal period required for the safe use of the drug.
By inspection of the six-step procedure set forth above, it can be seen that metabolism and pharmacokinetics are major features of steps 1,2,4, and 6. Aspects related to these and other steps have been addressed by Weber (1981, 1982), Jackson (1980), and Perez (1977). METABOLISM
STUDIES
IN TARGET
ANIMAL
The Food and Drug Administration suggests that the most practical and sensitive means of obtaining significant and detailed knowledge of the metabolic fate of an
148
DAVID
KOBYLKA
administered compound is through the use of radiotracer techniques, whereby the amount and nature of the drug residues may be determined in the target animal as well as in the laboratory species/strains used in oncogenicity testing. The desirable characteristics of a radiolabeled parent drug are: 1. 2. level 3.
purity: equal to or greater than 98%; specific activity: sufficient to permit detection to low ppb total residue or a determined to be safe; site and stability: adequate to monitor all residues of toxicological concern.
Initially, the radiolabeled drug is used in a total residue depletion study in the target animal to permit the assignment of a tentative withdrawal time for investigational studies and the performance of a threshold assessment as discussed in the Sensitivity of the Method Document (SOM) (Federal Register, 1979) and Federal Register (1982). The outline for such a study is: 1. Administer radiolabeled parent drug to previously unmedicated animals at maximum anticipated level of use and, with the exception of therapeutic drugs, for sufficient duration to establish steady state equilibrium of total drug residues in edible tissues. 2. Sacrifice animals (male and female) in groups of three at intervals including and after zero time* which allow for the depletion and detection of total residues to the low ppb range or a level determined to be safe. 3. Determine radioactivity in muscle, liver, kidney and fat (skin with adhering fat in poultry). *For cattle, swine, etc., zero equals 6-12 hours after last dose. *For chickens, turkeys, etc. zero equals 6 hours after last dose. These times are predicated upon standard industry practices with respect to a practical zero-time withdrawal.
In step 1 of the six-step procedure, the sponsor continues experimentation based on the results from the total residue depletion study done in the target animal. The purpose of the target animal metabolism study is to obtain information regarding the total number, amount, relative percentage, character/identity, and persistence of the parent drug and its metabolites in the edible tissues. The basic concepts for this study are as previously outlined under the topics dealing with the radiotracer characteristics and the target animal total residue depletion study. It should be noted that, if the initial depletion study was adequately performed, the tissues from that study may be used for purposes of metabolite characterization/ identification provided that the stability of the radioactive residues in the frozen state, as a function of time, can be demonstrated. This aspect could be acceptably addressed by reanalyzing frozen tissue samples, dosed at representative levels, which were generated during the course of the depletion study and comparing their metabolite profiles to those obtained initially. Besides tissues, the examination of urinary and/or fecal drug residue profiles, with identification of the parent compound and those biotransformation products for which sufficient amounts of residue are available for identification purposes, is also suggested. Usually, mass spectrometry (MS), nuclear magnetic resonance spectrometry (NMR), and infrared spectrophotometry (IR) are employed for such identifications after thin-layer or high-pressure liquid chromatographic separation and purification from other components as outlined by Bakke et al. ( 1976). An additional source of such compounds could be from tissues of target animals dosed at 5 to 20 times the normal level. Although the composition of residues observed in tissues from overdosed animals may not accurately represent that observed at normal use levels, the technique might make it possible to identify residues that could not be identified in tissues from animals dosed at the proposed use level.
SAFETY
ASSESSMENT
OF ANXMAL
DRUGS
149
This technique has been used by Paulson and Struble (1980). Those residues which are too difficult to isolate and identify because of their low concentration or because of other problems such as covalent binding to macromolecules are defined as intractable and have been addressed by Weber (1980a,b). The above discussion is summarized below: 1. Determine the total residue present in the edible tissues at each depletion point. 2. Determine the percentage extractable versus nonextractable total residue in each tissue, using various solvents, pH levels, denaturing agents, and chemical and/ or enzymatic hydrolysis. 3. Characterize the metabolites present in the edible tissues and excreta (suggested), using chromatographic profiling techniques. 4. Identify the metabolites present in the edible tissues by MS, NMR, IR, and/ or rechromatography/recrystallization to constant specific activity. The tissues from several overdosed animals may be useful as a research aid. METABOLISM
STUDIES
IN TEST
ANIMAL
After the nature, amount, and persistence of the residue that is present in the edible tissues of food-producing animals are determined, the metabolic profile for the sponsored compound must be examined in the laboratory test species/strains in which the toxicity evaluation will be conducted. In the comparative metabolism study (step 2 of the six-step procedure), the emphasis is on comparing the laboratory test animal to the target animal to ascertain whether the test species produce the metabolites characterized/identified in edible food after administration of the parent drug. Minimal emphasis is given to the identification of metabolites appearing in the test animal which are not observed in the target animal. These concepts are noted as follows: 1. The same basic procedures and techniques regarding metabolite profiling, characterization, and identification of residues present in the tissues and excreta from the target animal are used for the test animal. 2. The test animals are sacrificed while on the drug (2-6 hours after last dose, i.e., at peak plasma levels). As indicated in the SOM document, all identified metabolites will be evaluated by a number of approaches, including structure-activity relationships, potential for genotoxicity, and other information that may be available in the literature as well as results from subchronic toxicity tests. Several tentative in vitro tests which are currently envisioned for use in determining the potential for causing mutations as indicators of oncogenicity as a human health hazard include: 1. point mutations in bacteria, e.g., McCann ef al. (1975), McCann and Ames (1976); 2. point mutations in the X-linked recessive lethal test in Drosophila, e.g., Wurgler et al. ( 1977); 3. point mutations in mammalian cells in culture, e.g., Huberman and Sachs ( 1976); 4. unscheduled DNA repair synthesis in mammalian cells in culture, e.g., Stich et al., (1976).
DAVID KOBYLKA
TL = LARGEST
VALUE
AMONG
T T , , TT2 or TT3
TIME
(APPROPRIATE
FIG.
UNITS, w.. HOURS, DAYS, ETCI
1. Residue depletion curves to be used in the determination of the target tissue (Federul Register,
1979).
SELECTION
OF MARKER
RESIDUE
Step 4 of the six-step procedure concerns the selection of a substance (marker residue) to monitor the total drug residue of toxicological concern and of the tissue (target tissue) in which the marker residue is to be monitored. After a safe level (S,,) has been calculated from the results of the appropriate toxicity bioassays by an extrapolation procedure such as the one described by Hoe1 et al. (1975), it is adjusted for the amount of meat in the diet. Consequently, a residue score in muscle tissue is calculated as 3 X So, which is considered to be the safe level in muscle (S,,,). This value may be further adjusted for other organ meats such as liver and kidney as well as for fat (Perez, 1977). The depletion of residues from each of the edible tissues is illustrated in Fig. 1. From this figure, tissue 1 can be selected as the target tissue in which total residues of toxicological concern take the most time to deplete to their respective safe level. Ordinarily, the metabolite depletion profile of the tissue that requires the longest time to deplete to its safe level is examined to determine which metabolite or parent drug residue (marker residue) can best monitor the total residue in that tissue, as shown in Fig. 2. The proportion of the marker residue to the total residue represents the apparent level (R,) for the marker residue, which the reguIatory assay (step 5 of the six-step procedure) must be capable of measuring.
SAFETY
ASSESSMENT
OF ANIMAL
DRUGS
151
DOh IF P2 ISSELECTED T O BE MARKER RESIDUE THENTHELEVELOFMARKEA RESIDUE THE REGULATORY ASSAY MUST BE CAPABLE OF MEASURING IS RY.
O-
OTOTAL RESIDUE IN TARGET TISSUE. TISSUE, i.e.. P. + P, + P2
O.
5-
I
2,
0 TIME (IN APPROPRIATE
FIG. 2. Selection of the marker assay (Federal Register, 1979).
MARKER
residue
and its R,
RESIDUE
UNITSI
level that
DEPLETION
must
be measured
by the regulatory
STUDY
The last facet of the six-step procedure (step 6), which utilizes kinetic data, is the depletion study of the marker residue in the target tissue. This study statistically estimates the time when the residue content of the edible tissues of 99% of the total projected population of animals does not exceed the required tolerance or safe level, i.e., the withdrawal time. The mathematical approach, which envisions the calculation of the 99th percentile statistical tolerance limit with 95% confidence, is based upon an article by Owen (1968). The intercept of the tolerance limit with the required R, for the regulatory assay is established as the preslaughter withdrawal time. CONCLUSION From the above discussion, it can be seen that current criteria for regulating new animal drugs depend substantially on analytical chemistry and its relationship to metabolism studies and pharmacokinetics. These studies in target and test animals
152
DAVID KOBYLKA
are used to characterize/identify and select compounds (parent drug and metabelites) for toxicity testing, to determine a safe level for the total drug residue of toxicological concern, and to set a required regulatory assay sensitivity. Finally, a statistical tolerance limit method based on data obtained from a marker residue depletion study using the regulatory analytical method is applied to establish a withdrawal period. All of these aspects, when viewed in context, comprise an integral portion of the procedures used for the human safety assessment of animal drugs. REFERENCES BAKKE, J. E., FEIL, V. J., PRICE, C. E., AND ZAYLSKIE, R. G. (1976). Metabolism of [%]crufomate (4-t-butyl-2-chlorophenyl methyl methylphosphoramidate) by the sheep. Biomed. Mass Spectrom. 3, 299-315. Federal Register (1973). Compounds used in food-producing animals. Procedures for determining acceptability of assay methods used for assuring the absence of residues in edible products of such animals. Fed. Reg. 38, 19226-19230. Federal Register (1977). Chemical compounds in food-producing animals. Criteria and procedures for evaluating assaysfor carcinogenic residues in edible products of animals. Fed. Reg. 42, 104 12- 10437. Federal Register (1979). Chemical compounds in food-producing animals. Criteria and procedures for evaluting assays for carcinogenic residues. Fed. Reg. 44, 17070- 17 114. Federal Register (1982). Chemical compounds in food-producing animals. Availability of new threshold assessment criteria for guideline. Fed. Reg. 47, 4972-4977. HOEL, D. G., GAYLOR, D. W., KIRSCHSTEIN, R. L., SAFFIOTTI, U., AND SCHNEIDERMAN, M. A. (1975). Estimation of risks of irreversible, delayed toxicity. J. Toxicol. Environ. Health 1, 133- 15 1. HUBERMAN, E., AND SACHS, L. (1976). Mutability of different genetic loci in mammalian cells by metabolically activated carcinogenic polycyclic hydrocarbons. Proc. Nat Acud. Sci. USA 73, 188192. JACKSON, B. A. ( 1980). Safety assessment of drug residues. J. Amer. Vet. Med. Assoc. 176, 1141- 1144. MCCANN, J., AND AMUIES,B. N. (1976). Detection of carcinogens as mutagens in the Salmonella/ microsome test: Assay of 300 chemicals: Discussion. Proc. Nut. Acad. Sci. USA 73, 950-954. MCCANN, J., CHOI, E., YAMASAKI, E., AND AMES, B. N. (1975). Detection of carcinogens as mutagens in the Sulmonellu/microsome test: Assay of 300 chemicals. Proc. Nut. Acud. Sci. USA 72, 51355139. OWEN, D. B. (1968). A survey of properties and applications of the noncentral t-distribution. Technometrics
IO, 445-478.
PAULSON, G., ANDSTRUBLE, C. (1980). I. A unique deaminated metabolite of sulfamethazine [CaminoN-(4,6-dimethyl-2-pyrimidinyl)benzenesulfonamide] in swine. Life Sci. 27, 18 11- 18 17. PEREZ, M. K. (1977). Human safety data collection and evaluation for the approval of new animal drugs. J. Toxicol. Environ. Health 3, 837-857. STICH, H. F., SAN, R. C. H., LAM, P. P. S., KOROPATNICK, D. J., Lo., L. W., AND LAISHES, B. A. (1976). DNA fragmentation and DNA repair as an in vitro and in vivo assay for chemical procarcinogens, carcinogens, and carcinogenic nitrosation products. In Screening Tests in Chemical Carcinogenesis (R. Montesana, H. Bartsch, and L. Tomatis, eds.), Scientific Pub. No. 12, pp. 617-638. IARC, Lyon. WEBER, N. E. (1980a). Bioavailability of hound residues. In Toxicological European Research: Proceedings, Symposium on Relay Toxicity and Residue Biouvuilubility. Paris, France, October 5. In press. WEBER, N. E. (1980b). Persistent residues: Interface with regulatory decisions. J. Environ. Pathol. Toxicol.
3, 35-43.
WEBER, N. E. (1981). Regulatory perspectives on chemical compounds used as animal drugs or feed additives. In Industriul und Environmental Xenobiotics (I. Gut, M. Cikrt, and G. L. Plaa, eds.), pp. 395-401. Springer-Verlag, Berlin. WEBER, N. E. (1982). Metabolism and kinetics in the regulation of animal drugs. J. Anim. Sci., in press. WURGLER, F. E., SOBELS, F., AND VOGEL, E. (1977). Drosophila as an assay system for detecting genetic changes. In Handbook ofMutagenicity (B. J. Kilbey, M. Legator, W. Nichols, and C. Ramel, eds.), pp. 335-373. Elsevier, New York.
REGULATORYTOXICOLOGYANDPHARMACOLOGYZ,
Safety Assessment DAVID
of Animal Drugs’
KOBYLKA~
Divisionof Chemistry and Physics. Food and Drug Administration,
Washington, D. C. 20204
The Food and Drug Administration has published a proposal (Chemical Compounds in Food-Producing Animals: Criteria and Procedures for Evaluating Assays for Carcinogenic Residues, Federal Register 44, 17070-l 7114, March 20, 1979) to establish procedures and minimum criteria to ensure the absence of cancer-causing residues in edible products of foodproducing animals to which drugs, food additives, or color additives have been administered. Section III of the proposed rule (termed the Sensitivity of the Method Document or SOM) would require that metabolism and comparative metabolism studies be conducted in target (food-producing) and test (laboratory) animals, respectively. As a practical matter, current agency policy prescribes that information from such studies would be required whether the compound was considered to be a suspect carcinogen or a noncarcinogen. Metabolism studies to determine what drug-derived residues may be present in the tissues of food-producing animals at a particular withdrawal time as well as comparative metabolism studies to determine the appropriateness of the laboratory animal as a test species, and the relationship of the two studies to each other, are discussed in terms of the procedures used for the human safety assessment of animal drugs.
INTRODUCTION New animal drugs are used in food-producing animals for many reasons, ranging from increasing the rate of weight gain to therapeutic treatment of specific diseases. They may be administered once or fed over the entire lifetime of the animals. The fact that food-producing animals are capable of converting these drugs to a mixture of parent drug and biotransformation products (metabolites) requires that the human safety assessment take into account all residues of toxicological concern (parent drug and metabolites) before a new animal drug application (NADA) is approved. Unless it can be shown that certain residues have virtually no degree of toxicological concern associated with them, all metabolites and degradation products must be considered during an evaluation of drug residues in the edible tissues of foodproducing animals. Total drug residue can be defined as: All compounds present in edible tissues of the target animal that result from the use of the drug, including the drug, its metabolites, and any other substances formed in or on food because of the drug’s use.
The Food, Drug, and Cosmetic (FD&C)
Act was amended in 1958 to require
’ Presented at the Sixth Annual Spring Workshop, Association of Official Analytical Chemists, Ottawa, Ontario, Canada, May 12-14, 1981. ’ Send reprint requests to Dr. David Kobylka, Division of Chemistry and Physics, HFF-458, Food and Drug Administration, 200 C St., SW, Washington, D. C. 20204. 146 0273-2300/82/020146-07$02.00/O Copyright@ I982 by AcademicPrcss,Inc. All rightaofrcproduction in any form reserved.
SAFETY
ASSESSMENT
OF ANIMAL
DRUGS
147
premarket clearance for the use of food additives. However, it prohibited the use of carcinogens as food additives. This prohibition is known as the Delaney Clause and similar wording appears in the food (feed) additives section (409), color additives section (706), and the 1968 new animal drug section (5 12) of the Act. The anticancer proviso of the Clause reads as follows: . . . that no additive shall be deemed to be safe if it is found to induce cancer when ingested by man or animal, or if it is found, after tests which are appropriate for the evaluation of the safety of food additives, to induce cancer in man or animal. . . . Four years after its enactment, the Congress concluded that the anticancer proviso was too stringent as applied to food (feed) additives and amended it. Consequently, section 512(d)(l)(H) of the FD&C Act permits the use of carcinogens in food-producing animals: .
if the Secretary finds that, under the conditions of use specified in the proposed labeling and reasonably certain to be followed in practice, (1) such drug will not adversely affect the animals for which it is intended, and (2) no residue of such drug will be found (by methods of examination prescribed or approved by the Secretary by regulations) in any edible portion of such animals. . . .
In an effort to provide a scientific rationale for carrying out the above statutory mandate, the Food and Drug Administration has proposed criteria by which to evaluate new animal drugs under the Delaney Clause, including criteria for the analytical chemical methods to define the no-residue portion of the anticancer proviso amendment. Drafts of the proposed criteria have been published in the Federal Register (1973, 1977, 1979). The most recent version of the criteria employs a detailed scheme involving metabolism and pharmacokinetics in an integrated approach to human safety evaluation. The criteria include a six-step procedure that is to be used by the sponsor of a new animal drug in which the safe use of carcinogens and noncarcinogens in food-producing animals will be evaluated. The six steps are outlined: 1. A metabolic study in the target animal to characterize/identify residues of the drug in edible tissues and to establish their depletion profiles. 2. Comparative metabolism studies in laboratory animals (species/strains) to aid in evaluating/selecting test animals used for toxicity bioassays. 3. Toxicity/carcinogenicity testing to determine a safe level for residues of the sponsored drug. 4. A metabolic study in the target animal to identify a “marker residue” and “target tissue” which can be used to monitor total residues in the food supply. 5. Development of a regulatory assay to determine the “marker residue” in the “target tissue” at the required level of measurement. 6. Establishment of a premarket withdrawal period required for the safe use of the drug.
By inspection of the six-step procedure set forth above, it can be seen that metabolism and pharmacokinetics are major features of steps 1,2,4, and 6. Aspects related to these and other steps have been addressed by Weber (1981, 1982), Jackson (1980), and Perez (1977). METABOLISM
STUDIES
IN TARGET
ANIMAL
The Food and Drug Administration suggests that the most practical and sensitive means of obtaining significant and detailed knowledge of the metabolic fate of an
148
DAVID
KOBYLKA
administered compound is through the use of radiotracer techniques, whereby the amount and nature of the drug residues may be determined in the target animal as well as in the laboratory species/strains used in oncogenicity testing. The desirable characteristics of a radiolabeled parent drug are: 1. 2. level 3.
purity: equal to or greater than 98%; specific activity: sufficient to permit detection to low ppb total residue or a determined to be safe; site and stability: adequate to monitor all residues of toxicological concern.
Initially, the radiolabeled drug is used in a total residue depletion study in the target animal to permit the assignment of a tentative withdrawal time for investigational studies and the performance of a threshold assessment as discussed in the Sensitivity of the Method Document (SOM) (Federal Register, 1979) and Federal Register (1982). The outline for such a study is: 1. Administer radiolabeled parent drug to previously unmedicated animals at maximum anticipated level of use and, with the exception of therapeutic drugs, for sufficient duration to establish steady state equilibrium of total drug residues in edible tissues. 2. Sacrifice animals (male and female) in groups of three at intervals including and after zero time* which allow for the depletion and detection of total residues to the low ppb range or a level determined to be safe. 3. Determine radioactivity in muscle, liver, kidney and fat (skin with adhering fat in poultry). *For cattle, swine, etc., zero equals 6-12 hours after last dose. *For chickens, turkeys, etc. zero equals 6 hours after last dose. These times are predicated upon standard industry practices with respect to a practical zero-time withdrawal.
In step 1 of the six-step procedure, the sponsor continues experimentation based on the results from the total residue depletion study done in the target animal. The purpose of the target animal metabolism study is to obtain information regarding the total number, amount, relative percentage, character/identity, and persistence of the parent drug and its metabolites in the edible tissues. The basic concepts for this study are as previously outlined under the topics dealing with the radiotracer characteristics and the target animal total residue depletion study. It should be noted that, if the initial depletion study was adequately performed, the tissues from that study may be used for purposes of metabolite characterization/ identification provided that the stability of the radioactive residues in the frozen state, as a function of time, can be demonstrated. This aspect could be acceptably addressed by reanalyzing frozen tissue samples, dosed at representative levels, which were generated during the course of the depletion study and comparing their metabolite profiles to those obtained initially. Besides tissues, the examination of urinary and/or fecal drug residue profiles, with identification of the parent compound and those biotransformation products for which sufficient amounts of residue are available for identification purposes, is also suggested. Usually, mass spectrometry (MS), nuclear magnetic resonance spectrometry (NMR), and infrared spectrophotometry (IR) are employed for such identifications after thin-layer or high-pressure liquid chromatographic separation and purification from other components as outlined by Bakke et al. ( 1976). An additional source of such compounds could be from tissues of target animals dosed at 5 to 20 times the normal level. Although the composition of residues observed in tissues from overdosed animals may not accurately represent that observed at normal use levels, the technique might make it possible to identify residues that could not be identified in tissues from animals dosed at the proposed use level.
SAFETY
ASSESSMENT
OF ANXMAL
DRUGS
149
This technique has been used by Paulson and Struble (1980). Those residues which are too difficult to isolate and identify because of their low concentration or because of other problems such as covalent binding to macromolecules are defined as intractable and have been addressed by Weber (1980a,b). The above discussion is summarized below: 1. Determine the total residue present in the edible tissues at each depletion point. 2. Determine the percentage extractable versus nonextractable total residue in each tissue, using various solvents, pH levels, denaturing agents, and chemical and/ or enzymatic hydrolysis. 3. Characterize the metabolites present in the edible tissues and excreta (suggested), using chromatographic profiling techniques. 4. Identify the metabolites present in the edible tissues by MS, NMR, IR, and/ or rechromatography/recrystallization to constant specific activity. The tissues from several overdosed animals may be useful as a research aid. METABOLISM
STUDIES
IN TEST
ANIMAL
After the nature, amount, and persistence of the residue that is present in the edible tissues of food-producing animals are determined, the metabolic profile for the sponsored compound must be examined in the laboratory test species/strains in which the toxicity evaluation will be conducted. In the comparative metabolism study (step 2 of the six-step procedure), the emphasis is on comparing the laboratory test animal to the target animal to ascertain whether the test species produce the metabolites characterized/identified in edible food after administration of the parent drug. Minimal emphasis is given to the identification of metabolites appearing in the test animal which are not observed in the target animal. These concepts are noted as follows: 1. The same basic procedures and techniques regarding metabolite profiling, characterization, and identification of residues present in the tissues and excreta from the target animal are used for the test animal. 2. The test animals are sacrificed while on the drug (2-6 hours after last dose, i.e., at peak plasma levels). As indicated in the SOM document, all identified metabolites will be evaluated by a number of approaches, including structure-activity relationships, potential for genotoxicity, and other information that may be available in the literature as well as results from subchronic toxicity tests. Several tentative in vitro tests which are currently envisioned for use in determining the potential for causing mutations as indicators of oncogenicity as a human health hazard include: 1. point mutations in bacteria, e.g., McCann ef al. (1975), McCann and Ames (1976); 2. point mutations in the X-linked recessive lethal test in Drosophila, e.g., Wurgler et al. ( 1977); 3. point mutations in mammalian cells in culture, e.g., Huberman and Sachs ( 1976); 4. unscheduled DNA repair synthesis in mammalian cells in culture, e.g., Stich et al., (1976).
DAVID KOBYLKA
TL = LARGEST
VALUE
AMONG
T T , , TT2 or TT3
TIME
(APPROPRIATE
FIG.
UNITS, w.. HOURS, DAYS, ETCI
1. Residue depletion curves to be used in the determination of the target tissue (Federul Register,
1979).
SELECTION
OF MARKER
RESIDUE
Step 4 of the six-step procedure concerns the selection of a substance (marker residue) to monitor the total drug residue of toxicological concern and of the tissue (target tissue) in which the marker residue is to be monitored. After a safe level (S,,) has been calculated from the results of the appropriate toxicity bioassays by an extrapolation procedure such as the one described by Hoe1 et al. (1975), it is adjusted for the amount of meat in the diet. Consequently, a residue score in muscle tissue is calculated as 3 X So, which is considered to be the safe level in muscle (S,,,). This value may be further adjusted for other organ meats such as liver and kidney as well as for fat (Perez, 1977). The depletion of residues from each of the edible tissues is illustrated in Fig. 1. From this figure, tissue 1 can be selected as the target tissue in which total residues of toxicological concern take the most time to deplete to their respective safe level. Ordinarily, the metabolite depletion profile of the tissue that requires the longest time to deplete to its safe level is examined to determine which metabolite or parent drug residue (marker residue) can best monitor the total residue in that tissue, as shown in Fig. 2. The proportion of the marker residue to the total residue represents the apparent level (R,) for the marker residue, which the reguIatory assay (step 5 of the six-step procedure) must be capable of measuring.
SAFETY
ASSESSMENT
OF ANIMAL
DRUGS
151
DOh IF P2 ISSELECTED T O BE MARKER RESIDUE THENTHELEVELOFMARKEA RESIDUE THE REGULATORY ASSAY MUST BE CAPABLE OF MEASURING IS RY.
O-
OTOTAL RESIDUE IN TARGET TISSUE. TISSUE, i.e.. P. + P, + P2
O.
5-
I
2,
0 TIME (IN APPROPRIATE
FIG. 2. Selection of the marker assay (Federal Register, 1979).
MARKER
residue
and its R,
RESIDUE
UNITSI
level that
DEPLETION
must
be measured
by the regulatory
STUDY
The last facet of the six-step procedure (step 6), which utilizes kinetic data, is the depletion study of the marker residue in the target tissue. This study statistically estimates the time when the residue content of the edible tissues of 99% of the total projected population of animals does not exceed the required tolerance or safe level, i.e., the withdrawal time. The mathematical approach, which envisions the calculation of the 99th percentile statistical tolerance limit with 95% confidence, is based upon an article by Owen (1968). The intercept of the tolerance limit with the required R, for the regulatory assay is established as the preslaughter withdrawal time. CONCLUSION From the above discussion, it can be seen that current criteria for regulating new animal drugs depend substantially on analytical chemistry and its relationship to metabolism studies and pharmacokinetics. These studies in target and test animals
152
DAVID KOBYLKA
are used to characterize/identify and select compounds (parent drug and metabelites) for toxicity testing, to determine a safe level for the total drug residue of toxicological concern, and to set a required regulatory assay sensitivity. Finally, a statistical tolerance limit method based on data obtained from a marker residue depletion study using the regulatory analytical method is applied to establish a withdrawal period. All of these aspects, when viewed in context, comprise an integral portion of the procedures used for the human safety assessment of animal drugs. REFERENCES BAKKE, J. E., FEIL, V. J., PRICE, C. E., AND ZAYLSKIE, R. G. (1976). Metabolism of [%]crufomate (4-t-butyl-2-chlorophenyl methyl methylphosphoramidate) by the sheep. Biomed. Mass Spectrom. 3, 299-315. Federal Register (1973). Compounds used in food-producing animals. Procedures for determining acceptability of assay methods used for assuring the absence of residues in edible products of such animals. Fed. Reg. 38, 19226-19230. Federal Register (1977). Chemical compounds in food-producing animals. Criteria and procedures for evaluating assaysfor carcinogenic residues in edible products of animals. Fed. Reg. 42, 104 12- 10437. Federal Register (1979). Chemical compounds in food-producing animals. Criteria and procedures for evaluting assays for carcinogenic residues. Fed. Reg. 44, 17070- 17 114. Federal Register (1982). Chemical compounds in food-producing animals. Availability of new threshold assessment criteria for guideline. Fed. Reg. 47, 4972-4977. HOEL, D. G., GAYLOR, D. W., KIRSCHSTEIN, R. L., SAFFIOTTI, U., AND SCHNEIDERMAN, M. A. (1975). Estimation of risks of irreversible, delayed toxicity. J. Toxicol. Environ. Health 1, 133- 15 1. HUBERMAN, E., AND SACHS, L. (1976). Mutability of different genetic loci in mammalian cells by metabolically activated carcinogenic polycyclic hydrocarbons. Proc. Nat Acud. Sci. USA 73, 188192. JACKSON, B. A. ( 1980). Safety assessment of drug residues. J. Amer. Vet. Med. Assoc. 176, 1141- 1144. MCCANN, J., AND AMUIES,B. N. (1976). Detection of carcinogens as mutagens in the Salmonella/ microsome test: Assay of 300 chemicals: Discussion. Proc. Nut. Acad. Sci. USA 73, 950-954. MCCANN, J., CHOI, E., YAMASAKI, E., AND AMES, B. N. (1975). Detection of carcinogens as mutagens in the Sulmonellu/microsome test: Assay of 300 chemicals. Proc. Nut. Acud. Sci. USA 72, 51355139. OWEN, D. B. (1968). A survey of properties and applications of the noncentral t-distribution. Technometrics
IO, 445-478.
PAULSON, G., ANDSTRUBLE, C. (1980). I. A unique deaminated metabolite of sulfamethazine [CaminoN-(4,6-dimethyl-2-pyrimidinyl)benzenesulfonamide] in swine. Life Sci. 27, 18 11- 18 17. PEREZ, M. K. (1977). Human safety data collection and evaluation for the approval of new animal drugs. J. Toxicol. Environ. Health 3, 837-857. STICH, H. F., SAN, R. C. H., LAM, P. P. S., KOROPATNICK, D. J., Lo., L. W., AND LAISHES, B. A. (1976). DNA fragmentation and DNA repair as an in vitro and in vivo assay for chemical procarcinogens, carcinogens, and carcinogenic nitrosation products. In Screening Tests in Chemical Carcinogenesis (R. Montesana, H. Bartsch, and L. Tomatis, eds.), Scientific Pub. No. 12, pp. 617-638. IARC, Lyon. WEBER, N. E. (1980a). Bioavailability of hound residues. In Toxicological European Research: Proceedings, Symposium on Relay Toxicity and Residue Biouvuilubility. Paris, France, October 5. In press. WEBER, N. E. (1980b). Persistent residues: Interface with regulatory decisions. J. Environ. Pathol. Toxicol.
3, 35-43.
WEBER, N. E. (1981). Regulatory perspectives on chemical compounds used as animal drugs or feed additives. In Industriul und Environmental Xenobiotics (I. Gut, M. Cikrt, and G. L. Plaa, eds.), pp. 395-401. Springer-Verlag, Berlin. WEBER, N. E. (1982). Metabolism and kinetics in the regulation of animal drugs. J. Anim. Sci., in press. WURGLER, F. E., SOBELS, F., AND VOGEL, E. (1977). Drosophila as an assay system for detecting genetic changes. In Handbook ofMutagenicity (B. J. Kilbey, M. Legator, W. Nichols, and C. Ramel, eds.), pp. 335-373. Elsevier, New York.