Guideline 3
I. Guideline For Metabolism Studies And For Selection Of Residues For Toxicological Testing
A. INTRODUCTION
Section 512(d)(2) of the act explicitly provides that FDA consider the safety of any substance formed in or on food by a sponsored compound before approving its use. The compound administered to food-producing animals (target animals) is not necessarily the substance present in the edible products from these target animals. The enzymatic systems or physiological fluids of an animal can act upon a compound administered to the animal and produce new substances (metabolites and degradation products of the sponsored compound). The amount of these substances in edible animal products will be a complex function of the rate and extent of absorption of the parent compound, the rate and extent of the metabolism of the absorbed parent compound, and the rate of excretion of parent compound and metabolites.
The total residue of a sponsored compound in treated animals will consist of parent compound, free metabolites, and metabolites that are covalently bound to endogenous molecules. The relative and absolute amounts of each residue will vary among the tissues with the amount of the compound given and the time following the last administration of the sponsored compound to the animal. Because different components of the total residue may possess dissimilar toxicological potentials, the sponsor should develop information on the amount, persistence, and chemical nature of the-total residue in the edible products of treated target animals. The sponsor should also develop information on the metabolism of the compound in the species of laboratory animal used for the toxicological testing.
FDA needs the same type of information on metabolism of the sponsored compound in target and laboratory animals for both suspect and non-suspect carcinogens. However, after a chronic bioassay demonstrates that a compound is a carcinogen, FDA may ask the sponsor to obtain more information on the carcinogenic potential of individual metabolites. Such information could include more complete structural elucidation, in vitro genetic toxicity testing, and chronic bioassays for carcinogenicity.
B. TOTAL RESIDUE DEPLETION STUDY
The sponsor should measure the depletion of total drug-related residue in edible tissues of target animals at times after the last administration of the compound. For large animals, the edible tissues are muscle, liver, kidney, fat, and, where appropriate, milk. For poultry, the edible tissues are muscle, liver, skin with adhering fat, and, where appropriate, eggs. For an injectable compound, the sponsor should also measure the depletion of residues remaining at the injection site. To facilitate this determination, the sponsor should shave and permanently mark a circular area on the hide and inject the compound at the center of this area. At sacrifice, the sponsor should homogenize approximately 500 g of tissue from the area and measure its residue concentration. (The dimensions for a cylinder containing 500 g of tissue are, for an intramuscular injection, 10 cm in diameter and 6 cm in depth, and, for a subcutaneous injection, 15 cm in diameter and 2.5 cm in depth.)
The sponsor may use these tissues for the identification and quantification of parent drug and individual metabolites as described in Section C below. FDA may use these data to establish a withdrawal period for INAD purposes. FDA will use other experimental data, as discussed in another guideline, to establish a withdrawal period for NADA purposes.
Radiotracer methodology is currently the most useful technique for determining the total drug-related residue. Carbon-14 is the isotope most widely used because there is usually no problem with intermolecular exchange of the label. Ordinarily, the sponsor performs the depletion study by administering radiolabeled drug to a sufficient number of previously unmedicated animals to permit the serial sacrifice of groups of animals at intervals after the last treatment.
The administered compound should have a high radiopurity because radiolabeled contaminants may result in artifacts that could give the appearance of persistent drug residues. The sponsor should choose the site(s) of the radiolabel to assure that portions of the parent compound that are likely to be of toxicological concern are adequately labeled. For example, in the situation where two chemical moieties of the parent compound are likely to be of toxicological concern and a metabolic process may chemically separate the two moieties, then the sponsor should determine the depletion of both moieties. The sponsor could conduct a single study by administering equimolar amounts of the compound with each moiety individually labeled, two studies by separately administering the compound with each moiety individually labeled, or a single study by administering a dual-labeled (for example, Carbon-14 and Tritium) compound.
The sponsor should choose the specific activity for the radiolabeled compound high enough to demonstrate that the concentration of total residue at the last sacrifice time is below the expected permitted concentration. Ordinarily for non-carcinogens, this concentration is near 100 parts per billion (ppb), but for a carcinogen, the concentration may be below 1 ppb. The sponsor is urged to consult with FDA on the appropriate specific activity before designing a total residue depletion study. The specific activity of the administered compound need not be the same for each time point. For example, the sponsor could use low specific activity material in animals sacrificed at short withdrawal times and higher specific activity material in animals sacrificed at long withdrawal times.
The sponsor should conduct the total residue depletion study in previously unmedicated animals that are representative of the proposed target population. The metabolism of a compound may vary according to a number of parameters including species, sex, age, dose, and duration of treatment. The differences observed due to these parameters any include the rate of production and excretion of metabolites and their chemical nature. For this reason FDA will generally require a radiotracer study in each distinct species for which the sponsor is seeking approval. If a product is intended for use in both male and female animals, then the sponsor should use animals of both sexes in the study because males and females of a species can metabolize chemicals at a different rate. Because the enzyme type and concentration in young animals may not be representative of the adult animal, the sponsor should conduct the study in animals that are representative of the target population, that is, in neonates, prepubertal animals, or sexually mature animals. In those instances when a drug's use will be extended from one production class to another within a species, FDA generally will accept an abbreviated total residue depletion study to support approval in the new production class. Sponsors should discuss plans for the abbreviated study with FDA.
The dose should be the highest intended treatment level and should model exposure received by target animals. For example, if a drug is given once to an animal for a specific therapeutic effect, then a single dose of radiolabeled drug is appropriate exposure. When the sponsor is requesting prolonged treatment and is also requesting a zero withdrawal period, the sponsor should supply data to demonstrate that the residue concentration has approximately reached steady state. In other cases, FDA will generally accept that a seven day dosing regimen is adequate for the purposes of the residue depletion studies. However, the sponsor should use a twelve day dosing regimen for laying hens to approximate the time required for complete development of the yolk. Where the data do not demonstrate that a steady state concentration is attained, the sponsor should show that there is no new metabolite being formed as a result of prolonged treatment. For example, the sponsor could demonstrate that the metabolite pattern and relative proportions of metabolites in tissues have stabilized.
When measuring the depletion of the total residue in edible tissue, the sponsor should sacrifice groups of at least three animals at zero withdrawal and usually at three later times. If the sponsor is requesting a zero withdrawal period, the sponsor should 'dose six animals for a sufficient time previously demonstrated to achieve a steady state concentration of residue and then sacrifice the animals at zero time. For other special cases, the sponsor is urged to consult with FDA before designing a study. If the sponsor intends to use the dosed tissue for metabolite identification, the sponsor may wish to include additional animals in the study. For purposes of the residue depletion study, zero withdrawal for tissues of large animals is considered eight to twelve hours after the last treatment; for lactating animals, zero withdrawal for milk is considered twelve hours after the last treatment. For tissues of poultry, zero withdrawal is considered six hours after the last treatment.
The sponsor should present the results of these experiments in a format that will facilitate FDA review and should include supporting raw data. The sponsor should provide:
The specific activity of parent drug.
The radiopurity of parent drug (98% or greater is recommended) with supporting chromatograms from at least two different chromatographic systems and a determination of microbiological activity if appropriate.
A full description of methodology and of the statistical and/or mathematical approach including sample calculations.
A practical demonstration of the limit of detection of parent drug through fortification of each of the edible tissues. In the absence of this demonstration, FDA will use twice the background counting rate as a nominal limit of detection.
The sampling procedure and sample size. FDA recommends that the whole organ or representative portions be homogenized and the analysis be done on an aliquot.
Data from duplicate analyses of each tissue sample derived from treated animals. For each experimentally derived data point, results should be identified with an individual male or female animal and be presented in CPM, in DPM, and in parent drug equivalents per gram of wet tissue.
C. METABOLISM STUDIES IN TARGET ANIMALS
The sponsor should provide information on the metabolic fate of the compound in the edible tissues of the target animal dosed at the maximum use level requested. Often the published literature contains information on the metabolism of a closely related chemical. Such information is useful but usually cannot replace the experimental observation of the metabolic fate of the sponsored chemical in target
The sponsor may use the tissues from the total residue depletion study for these studies. If the tissues are stored frozen for long periods of time, FDA may ask the sponsor to demonstrate that the metabolites are stable in the frozen state, such as by demonstrating that metabolite profiles do not change. If suitable tissue is not available, the sponsor should conduct a total residue depletion study as discussed above in Section B. The sponsor should develop procedures for extraction, fractionation, separation, and isolation of metabolites that will facilitate the comparison of metabolite profiles for each tissue and for later comparison of these profiles with the metabolite profiles derived from laboratory animals. Unless human food safety questions arise requiring examination of the other edible tissues, the sponsor will need to collect data only in the target tissue.
1. Structural Identification of Metabolites
Structural identification of major metabolites may be necessary depending on the degree of toxicological concern for the parent compound and its potential metabolites. FDA will consider a metabolite to be a major metabolite if, at the time the concentration of total residue peaks (normally zero withdrawal), either (a) it is present in an amount greater than 10% of the total residue in an edible tissue, or (b) its concentration exceeds 0.1 ppm. In some cases, chemical characterization rather than unequivocal structural identification for a major metabolite will be sufficient. For example, if chromatographic evidence demonstrates that a metabolite and its conjugate are present in tissue, then FDA will not consider structural identification of the particular conjugate to be necessary for evaluating the safety of the compound. Similarly, the sponsor may use chromatographic evidence to demonstrate that the same major metabolite occurs in more than one tissue. FDA will normally not require structural identification or chemical characterization of minor metabolites.
The sponsor may isolate sufficient quantities of metabolites for structural identification by a variety of techniques. The sponsor may isolate metabolites from excreta. Another example of an acceptable approach is the research procedure described by Paulson and Struble (Ref. 1) and Bakke et al. Ref. 2), summarized below.
The sponsor treats a group of animals with cold drug at or near the use level for a period of time, then administers from one to several large doses (5x to 20x) of radiolabeled drug, and slaughters the animals six to twelve hours after the final dose. The sponsor then uses purification and separation techniques to obtain individual metabolites for structural determination.
2. Persistence of Metabolites
The sponsor should determine the concentration and relative percent of the parent compound and individual metabolites in edible tissue(s) at a series of times after the last administration of the compound. Because the residues that persist to the expected withdrawal period will be consumed by people, these are the residues of toxicological concern. FDA will normally require an evaluation of their toxicological potentials in laboratory animals (see sections D and E below).
3. Covalently Bound Residues
In some cases the sponsor may be unable to extract the total radiolabeled residue. The non-extractable material usually represents two general classes of compounds, endogenous components derived from a portion or the radiolabeled compound of covalently bound residues derived from the reaction between a metabolite of the compound and cellular macromolecules. If the sponsor shows that a portion of the total residue results from the incorporation of radiolabel into endogenous compounds (for example, amino acids, fatty acids, carbohydrates, nucleotides), FDA will subtract that portion from the total residue because it is not of toxicological concern. FDA will consider the covalently bound residue to be of toxicological concern, but will not consider it to be more toxic than the parent compound. Unless the covalently bound residue represents a major portion of the residue and the information is needed for analytical methodology, FDA will not require that the sponsor attempt structural identification.
If the total residue concentration (that is, free metabolites plus covalently bound residue) is below the permitted concentration of residue in edible tissue at the requested withdrawal time, then this residue is shown to be safe within the meaning of the act, and FDA will not ask for additional safety testing on the covalently bound residue. However, if the concentration of covalently bound residue exceeds the permitted concentration of residue, then FDA cannot approve the compound until the sponsor provides additional data to demonstrate safety.
If the parent compound is not a carcinogen, FDA will discount from the residue of toxicological concern that portion of the covalently bound residue that the sponsor demonstrates is not bioavailable, provided that a substantial portion (for example, 50%) of the covalently bound residue is not bioavailable. FDA will adjust the total residue based on the relative bioavailability of the parent compound and the covalently bound residue. The experimental technique described by Gallo-Torres (Ref. 3) is an example of an acceptable protocol. However, if the parent compound is a demonstrated carcinogen, FDA will normally not accept bioavailability data alone to discount the covalently bound residue from carcinogenic concern. In any specific case the sponsor has the option of proposing to FDA an experimental approach to demonstrate that the covalently bound residue is not of carcinogenic concern.
4. Reporting of Data
The sponsor should submit data from the metabolism studies in a format that will facilitate FDA review and should include supporting raw data. A flow chart of known or postulated metabolic pathways is usually helpful. The sponsor should provide:
D. METABOLISM STUDIES IN LABORATORY ANIMALS
The purpose of these studies is to determine whether the metabolites that people will consume from tissues of target animals are also produced by metabolism in the laboratory animals used for the toxicological testing. The sponsor should conduct these studies for compounds being tested for carcinogenicity as well as those being tested for other toxicological endpoints. To facilitate evaluation of the data, the sponsor should use the same procedures for chromatography and chemical characterization as those employed in the metabolism study in the target animals. Qualitative information on metabolites is sufficient. FDA will use this information to determine what metabolites, if any, need separate toxicological testing (see section E below).
The sponsor should give laboratory animals a sufficient number of daily doses of the radiolabeled test compound to ensure that it undergoes all relevant metabolic events, including those associated with enzyme induction. The sponsor should consult with FDA prior to dose selection.
The sponsor should try to conduct toxicological studies in the laboratory animals whose profile of metabolites most closely resembles that observed in target animals. However, other factors also need to be considered. For example, knowledge that certain laboratory animals are especially sensitive to the parent compound or its chemical class may dictate their choice, even though the metabolite profile of other laboratory animals may more closely model that of the target animals. FDA does require that the strain of test species chosen for the comparative metabolism work be the one used for the toxicological testing.
E. SELECTION OF METABOLITES FOR TOXICOLOGICAL TESTING
FDA may ask for separate toxicological studies on a metabolite if it is not tested through autoexposure and it is likely to have toxicological potency significantly greater than the parent compound. FDA will normally conclude that autoexposure provides an adequate test of the toxicity of the sponsored compound if laboratory animals produce the metabolites that collectively comprise over 90% of the residue that people will consume from tissues of treated target animals. Failing that, FDA will use the information obtained from target animals on the concentration, persistence, and chemical structure or characterization of that metabolite to determine whether separate toxicological testing is desirable.
F. IDENTIFICATION OF TARGET TISSUE AND MARKER RESIDUE
The target tissue is the edible tissue selected to monitor for the total residue in the target animal. The target tissue is usually, but not necessarily, the last tissue in which residues deplete to the permitted concentration. A marker residue is a residue whose concentration is in a known relationship to the concentration of the total residue in the last tissue to deplete to its permitted concentration. The marker residue can be the sponsored compound, any of its metabolites, or a combination of the residues for which a common assay can be developed. The target tissue and marker residue are selected so that the absence of marker residue above a designated concentration R(M) will confirm that each edible tissue has a concentration of total residue at or below its permitted concentration.
When a compound is to be used in milk- or egg-producing animals, milk or eggs may be a target tissue in addition to one tissue selected to monitor for residues in the edible carcass because milk or eggs enter the food supply independently. In these cases, it may be necessary to select a marker residue for milk or eggs that is different from the marker residue selected for the target tissue representing the edible carcass.
Application of the concepts of marker residue and target tissue requires an experimental determination of the quantitative relationships among the residues that might serve as the marker residue in each of the various edible tissues that might serve as the target tissue. Because these relationships may change with time, the sponsor should measure the depletion of potential marker residues in potential target tissues starting after the last treatment with the sponsored compound and continuing until the residue has reached the permitted concentration for that tissue.
The sponsor may use the results from the total residue depletion study and metabolism study to determine the marker residue, target tissue, and R(M). If a new depletion study is advisable, the desired experimental parameters are as outlined for the total residue depletion study in section B. (A radiotracer depletion study provides an initial estimate of the R(M). The final R(M) is determined with the regulatory assay.)
G. REFERENCES
Paulson, G. and C. Struble (1980), I. A unique deaminated metabolite of sulfamethazine [4-amino-N-(4,6-dimethyl-2-pyridinyl) benzensulfonamide] in swine, Life Sci. 27:1811-1817.
Bakke, J. E., V- J. Feil, C. E. Price, and R. G. Zaylskie (1976), Metabolism of Carbon-14 crufomate (4-t-butyl-2-chlorophenyl methyl methylphosphoramidate) by the sheep, Biomed. Mass. Spectrom. 3:299-315
Gallo-Torres, H. E. (1977), Methodology for the determination of bioavailability of labeled residues, J. Tox and Environ. Health 2:827-845
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