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The Human Food Safety Evaluation Of New Animal Drugs
CHEMICAL FOOD BORNE HAZARDS AND THEIR CONTROL
0749-0720/99 $8.00
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THE HUMAN FOOD SAFETY EVALUATION OF NEW ANIMAL DRUGS Lynn G. Friedlander, PhD, Steven D. Brynes, PhD, and Ana H. Fernandez, DVM
Animal drugs are substances intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease in animals, and substances (other than food) intended to affect the structure or any function of the body. Before a new animal drug can be marketed in the United States, the drug sponsor must file a New Animal Drug Application (NADA), which must be approved by the US Food and Drug Administration (FDA). To file a NADA, the drug sponsor, usually a pharmaceutical firm, must conduct studies that demonstrate that the drug is safe and effective in the target animal species, is safe for the environment, and can be manufactured to uniform standards of purity, strength, and identity. When the drug is intended for use in foodproducing animals, the sponsoring firm also must demonstrate that the food products derived from treated animals are safe for human consumption. To conduct investigational studies with new animal drugs prior to approval, the drug sponsor must request an Investigational New Animal Drug (INAD) exemption from the FDA. Under the INAD, the sponsoring firm can conduct the studies needed to support the approval of the drug product. These studies are reviewed by scientists at the FDA's Center for Veterinary Medicine (CVM). Where appropriate, the
From the Division of Human Food Safety, Office of New Animal Drug Evaluation, Center for Veterinary Medicine, United States Food and Drug Administration, Rockville, Maryland
VETERINARY CLINICS OF NORTH AMERICA: FOOD ANIMAL PRACTICE VOLUME 15 • NUMBER 1 • MARCH 1999
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FDA may authorize the use in human food of products derived from animals treated in investigational studies. HUMAN FOOD SAFETY REQUIREMENTS
Human food safety studies are conducted to assess the nature and quantity of residues in foods derived from animals treated with the proposed new animal drug. Residue is defined as any compound present in the edible tissues of the target animal that results from the use of the sponsored compound, including the sponsored compound itself, its metabolites, and any substance formed in or on food as a result of the use of the sponsored compound. Substance is broadly defined, and even includes endogenous compounds that are altered owing to drug use. In this article, we discuss the types of studies submitted in a NADA to establish the human food safety of the drug and its metabolites in animal-derived edible products resulting from the use of new animal drugs and feed additives. Our guidance document describing the types of human food safety data that can be used to support a new animal drug approval may be found on our CVM website at www.fda.gov / cvm/. 2 Background
Although food safety has always been a major concern for the FDA, the human food safety requirements for animal drug residues have changed over the years. Originally, animal drugs were approved on the basis of a no residue or zero residue tolerance policy. Although many believed that zero residues could be achieved, most drugs are eliminated by first-order kinetics, and some residues always remain in the animal. The zero tolerance value actually represented the limits of the available analytic technology, not a true zero value. As a result, as the analytic methods continued to improve, the zero tolerance value was continually lowered. A refinement of the zero tolerance policy was the concept of negligible tolerance. Under this policy, toxicologic data requirements were limited to subchronic studies in dogs and rats. The tolerance was generally assigned as 0.1 ppm in all edible tissues. As with the zero tolerance, the negligible tolerance was not representative of the potential hazard of the compound. To remedy this situation, the CVM adopted its current risk-assessment-based procedure. The human food safety of animal drugs is determined following a review of the toxicologic, metabolic, and residue data. Under the current risk-assessment model, risk from an animal drug residue equals the hazard associated with the drug multiplied by the exposure to drug residues: Risk = Hazard X Exposure
THE HUMAN FOOD SAFETY EVALUATION OF NEW ANIMAL DRUGS
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Public health risk is regulated by assessing the hazard posed by the drug substance and by controlling the exposure to meet a risk standard of "reasonable certainty of no harm." Current Requirements
The human food safety studies conducted to assess the safety of drug residues fall into two general categories: toxicity studies and residue chemistry studies. Because the requirements for the residue chemistry studies are dependent on the toxicity of the drug as determined in the toxicity tests, it is usually more practical for the drug sponsor to conduct the preliminary toxicity studies before the residue chemistry work is begun. TOXICOLOGY (HAZARD ASSESSMENT)
All of the toxicity tests are designed to evaluate the toxicity of the drug or its metabolites to humans, who may be exposed to it through the consumption of food derived from animals treated with the new animal drug product. Although the human exposure would be at a chronic low dose via food, the toxicity studies are conducted using high doses, with the toxicologic model species (usually rats or mice and dogs) serving as human surrogates. The toxicity tests attempt to predict the effects of drugs over a lifetime of oral exposure by extrapolating from the toxicologic model species to man and from short-term exposure to long-term exposure. For some drugs, studies assessing acute toxicity also may be required. The basic toxicology package contains tests that are appropriate for most new animal drugs. The tests include genetic toxicity studies, 90day feeding studies in a rodent and a nonrodent mammalian species to test the oral toxicity of the drug in the toxicologic species, and a twogeneration reproduction study with a teratology component to evaluate the cross-generational toxicity and teratogenic potential of the new animal drug. Additional toxicity tests may be conducted to clarify the results obtained in the basic test battery or to provide additional data. Specialized tests may need to be conducted to selectively target toxic endpoints of concern for the drug (e.g., reproductive organs for synthetic hormones). For antimicrobial drugs, the sponsoring firm might also need to perform studies evaluating the effect of the drug residues on the human intestinal microflora (e.g., development of resistance, alteration of the barrier effect, overgrowth of potentially pathogenic microorganisms). Chronic studies may be conducted to evaluate the toxic effects of the drug over the lifetime of the toxicologic model species. All of the testing in the toxicologic species is conducted via the oral route of administration, because the tests are designed to evaluate the toxicity of the new animal drug to humans following oral exposure in food.
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Noncarcinogens
For compounds that are not carcinogens, the toxicity testing permits the calculation of the Acceptable Daily Intake (ADI). The ADI represents an amount of drug that can be consumed daily over the lifetime of a human individual without harmful effect. The ADI is calculated by dividing the no-observed-effect level (NOEL) of the most sensitive effect as determined by the most appropriate toxicity study by a safety factor: ADI
=
NOEL Safety Factor
The NOEL is the highest tested concentration of drug that produced no toxic effect. The safety factor is determined by the type of study in which the toxic effect was observed. In general, the closer the toxicity study is to assessing the true toxicologic significance to humans, the smaller the applied safety factor will be. Generally, a 10-fold factor is applied to account for variability within the human population, a 10-fold factor is applied to account for interspecies extrapolation, and a 10-fold factor is applied to account for extrapolation from sub chronic to chronic exposure. 3 Because the safety factor appears in the denominator, a smaller safety factor results in a larger AD!. Although the ADI is an indication of the inherent toxicity of the drug, it also reflects the uncertainty of the toxicity assessment. It may be possible for the drug sponsor to conduct additional toxicity studies for which the safety factor is smaller (e.g., a long-term study), which will result in the calculation of a higher final AD!. The calculation of the ADI completes the hazard analysis component of the human food safety section of the NADA. The ADI is used to calculate the safe concentration of residues, the amount of total drug-related residues that can be present in each of the edible tissues of animals treated with the new animal drug. In calculating the safe concentration of residues in edible tissues, the ADI is adjusted for the anticipated dietary consumption of animal-derived food products. The safe concentration is calculated by multiplying the ADI by 60 kg (132 lb), the weight of the average human, and dividing by a tissue consumption value: SAFE CONCENTRATION =
ADI X 60 kg Tissue Consumption Value
The tissue consumption values are assigned to each of the edible products based on the portion of the diet they comprise (Table 1). The consumption values assume that a person will not eat a full complement of all edible tissues in a single day; that is, a person does not normally eat a full helping of liver for lunch followed by a full serving of lean meat (muscle) for dinner. The consumption value for milk is high enough to accommodate exposure in that segment of the population for which milk may be the major food product consumed and where body weight is less than 60 kg (i.e., young children). Since milk, eggs, and
THE HUMAN FOOD SAFETY EVALUATION OF NEW ANIMAL DRUGS
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Table 1. CONSUMPTION VALUES FOR VARIOUS EDIBLE PRODUCTS Edible Product Muscle Liver Kidney Fat Milk
Eggs
Grams Consumed/day 300 100
50 50 1500 mL
100
meat may be consumed in a single day, drug sponsors who seek approval for drugs for use in lactating dairy cattle or laying hens must partition the ADI for meat and nonmeat (i.e., milk and eggs). That is, the sponsor must specify what portion of the total ADI is to be used for meat and what portion is reserved for milk and eggs. The ADI that is reserved for milk or eggs is not used to calculate the safe concentration for edible tissues. The safe concentrations establish the amount of total residue that can be present in each of the edible products derived from animals treated with the new animal drug. The calculation of the safe concentration begins the exposure-assessment portion of the human food safety section of the NADA. Carcinogens
The Delaney Clause of the Federal Food, Drug, and Cosmetic Act prohibits the use of compounds "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 the food additive, to induce cancer in man or animal" (section 409(c)(3)(A)). An exception to the Delaney Clause exists for animal drugs when "it is determined by methods of examination prescribed or approved by the Secretary ... that no residue of that compound will be found in the food produced from those animals under conditions of use reasonably certain to be followed in practice" (21 CFR 500.80(a)). To implement this latter provision of the Delaney Clause, the FDA relies on quantitative risk-assessment procedures to determine when the amount of residue of carcinogenic concern in the edible tissue produces an incremental estimated cancer risk for a lifetime of 1 in 1 million. This level functionally defines no residue. Following a determination of the no residue level, residue and metabolism studies are conducted as described below. RESIDUE CHEMISTRY (EXPOSURE-ASSESSMENT AND MITIGATION) STUDIES
The residue chemistry exposure-assessment studies are designed to determine the concentration of drug residue actually appearing in the edible tissues of the target animal species as a result of treatment with
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the proposed animal drug. Because tissue residue concentrations are often high immediately following treatment, residue chemistry studies also are used to determine how much time must elapse following treatment (e.g., how long milk from treated animals must be discarded or how long treated animals themselves must be withheld from slaughter) to allow residues in edible products to deplete to acceptable levels. Additionally, residue chemistry studies are used to confirm that the residues produced by the target (food) species have been tested toxicologically. Residue chemistry studies submitted to the FDA normally include total residue and metabolism studies, comparative metabolism studies, and residue depletion studies. Total Residue and Metabolism Studies
The initial residue chemistry study conducted is the total residue and metabolism study. The term total is used because the study is conducted with radiolabeled drug and is capable of monitoring all drugderived residues resulting from the administration of the test material. The total residue and metabolism study is conducted in the target (food) animal species and is intended to show where the administered drug is deposited in the target species, how it is metabolized, and what its pattern of elimination is. Because the only source of radioactivity is the administered drug, any detected radioactivity is drug-related. Usually, total radioactivity in the edible tissues is measured by combusting the tissue samples to liberate radiolabeled carbon dioxide, which is then trapped and measured by liquid scintillation counting. The measured carbon dioxide is correlated directly to the amount of total drug-derived residue in the tissue sample. Metabolism of the proposed drug also is evaluated using the tissues from the total residue and metabolism study. Commonly available analytic technology (e.g., high performance liquid chromatography and gas chromatography) makes it possible to separate the total residue into its components and identify individual drug-derived compounds. All major metabolites are identified as part of the total residue and metabolism study. A major metabolite is any radiolabeled compound, including the parent drug, comprising at least 10% of the total residue or having a tissue concentration of at least 0.1 ppm. By measuring the concentrations of the major radiolabeled drug-related components in tissue samples collected at different times following the cessation of treatment, it is possible to identify those drug residues that persist for the longest time following treatment and to determine in which of the edible tissues drug elimination proceeds most slowly. Because radiolabeled residues (i.e., total residues) represent all of the drug in edible tissues, they can be compared directly to the safe concentration (i.e., total daily exposure) established in the toxicity testing. Drug residues determined shortly after the cessation of treatment are evaluated to determine if a withdrawal period is needed for the
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intended use of the product. A practical zero withdrawal is established at 6 hours after the final dosing in poultry and at 8 to 12 hours after the
final dosing in large animal species (Le., cattle, sheep, and swine). The practical zero withdrawal is intended to reflect the time required for animals to reach the abattoir. If the total residues in the edible tissues at practical zero withdrawal are less than their respective safe concentrations, no preslaughter withdrawal period is assigned (zero withdrawal). The target tissue is the edible tissue selected to monitor for total residue in the target animal. The target tissue is usually, but not necessarily, the last tissue in which total residues deplete to their permitted safe concentration. A marker residue is a residue whose concentration is in a known relationship to the concentration of total residue in the last tissue to deplete to the safe concentration. The marker residue can be the parent compound, any of its metabolites, or a combination of residues for which a common assay can be developed (Fig. 1). The target tissue and the marker residue are selected so that the absence of the marker residue above a designated concentration (the tolerance) will confirm that each edible tissue has a concentration of total residues at or below the safe concentration (Fig. 2). Once the relationship between the total residue and the marker residue in the target tissue is established, it is possible to monitor depletion of total residues to the safe concentration. When the marker residue in the target tissue has depleted to the tolerance, the total residue will have depleted to the safe concentration in all edible tissues. The tolerance is the legal enforcement level; it is the codified value in the Code of Federal Regulations (specifically, 21 CFR 556).
10.0
o 1 - - - - - " " ' 0 ; : - - - - - " " ' - - = - - - - - - Safe
3
Concentration
7 Time (days)
Figure 1. Typical depletion curves for potential marker residues in the target tissue. Open circle = total residue; open square = metabolite 8; solid square = parent; solid oval = metabolite A.
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10.0
a
,-..,
S s:::l. S (J)
1-------=:".-.:;:------=-,.::::----- Safe Concentration
~
~ .S
'00 ~
~~
0.1
1_ _ _ _ _ _ _ _~----Assigned
Tolerance
(J)
u
~
0
u
3
7
Time (days) Figure 2. Selection of the marker residue and assignment of the tolerance. Open circle total residue; solid oval = marker residue.
=
Analytic Methods
In order to monitor drug residues, robust analytic methods must be available. It is necessary for the sponsor to develop and validate regulatory analytic methods that will accurately measure the marker residue in the target tissue at the tolerance. Regulatory analytic methods must be capable of accurately measuring the marker residue and verifying the identity of the marker residue. Consequently, regulatory methods submitted to the FDA usually have two components: a determinative method for quantification and a confirmatory method for identification. Regulatory analytic methods, both determinative and confirmatory, undergo a series of evaluations to ensure that they will perform well for monitoring drug residues in edible tissues. Since the tolerance for the marker residue is determined using the determinative analytic method, the tolerance is directly tied to that method. When a method is modified, it is necessary to confirm the relationship between the original and the revised analytic methods. The United States Department of Agriculture (USDA) uses the official regulatory methods to monitor animal-derived foods for residues of the new animal drugs. Screening tests (e.g., ELISA) are rapid, low-cost, on-site procedures that provide detection of residues for a general class of compounds. For example, the screening test may detect a residue as a tetracycline drug but not differentiate between oxytetracycline and chlortetracycline. Although developing screening tests is not a requirement for the approval of a new animal drug, sponsors are routinely asked to evaluate how their new animal drug will react with screening methods that are already
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available, especially those screening tests used by the USDA for the rapid analysis of tissue residues. Comparative Metabolism Study
Comparative metabolism studies are conducted in the toxicologic species to confirm that the animals used in the toxicity studies were exposed to the same metabolites present in food products derived from treated animals. Metabolic profiles in the toxicologic and target species are compared to assess whether the same major metabolites are present in both species. If both species produce the same metabolites, we conclude that the toxicologic species was autoexposed to the same compounds to which humans will be exposed when eating food derived from treated animals. Failure to demonstrate autoexposure may necessitate additional toxicity testing. Residue Depletion Study and Assigning a Withdrawal Period
When a withdrawal time is needed to support a new animal drug approval, the sponsoring firm conducts a residue depletion study using the commercial product under field-use conditions. The residue depletion study monitors the marker residue as it depletes to the tolerance level using the determinative analytic method. For the residue depletion study, the drug product is administered at the highest label dose for the longest label duration to provide a worst-case scenario for residue depletion. The withdrawal time is calculated by using the data from the residue depletion study and determining the 99th percentile tolerance limit with 95% confidence. When animals treated with the new animal drug according to the label indications are withheld from slaughter for the assigned withdrawal period, there is only a 5% probability that, by chance, 1 animal in 100 will have residues of the marker compound in the target tissue that exceed the established tolerance level. For most animals, drug levels will be well below the tolerance level before the end of the withdrawal time. Even for that fraction of animals that falls outside the predictive model, only the target tissue would be expected to have residues above the tolerance, if the label indications were followed. Furthermore, because animals are slaughtered when they reach market weight, not at the end of the withdrawal period, virtually no violative residues will occur when labeled directions are followed. To ensure that there is only a 5% probability that, by chance, 1 animal in 100 will have residues of the marker compound in the target tissue that exceed the established tolerance level when the drug product is used in accordance with its labeling, the assigned withdrawal time is the time when the 99th percentile data cross the tolerance value (Fig. 3).
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: . - 99th Percentile with 95% Confidence Actual
Residu~
Tolerance
Values
2
3 4 5 Withdrawal Time (days)
6.75
7
Figure 3. Calculating the preslaughter (tissue) withdrawal period. A 6.7S-day withdrawal period is calculated and a 7-day withdrawal period is assigned.
The observed (actual measured) residue data in the depletion study always are less than the 99th percentile data. If the calculated withdrawal time is a fraction of a day, the assigned withdrawal period is rounded up to the next whole day (e.g., if the calculated withdrawal period is 162 hours (6.75 days), a withdrawal period of 7 days is assigned). The assigned preslaughter withdrawal period must be consistent with standard production practices (i.e., likely to be followed in practice). The residue depletion study in milk is similar to the residue depletion study in tissues. The marker residue and tolerance for milk residues are determined in a radiolabeled study conducted in lactating cattle. The tolerance and marker residue in milk are determined independently from the marker residue and tolerance in tissue to provide for potential differences in drug metabolism in the udder. The statistical treatment of the data is also different. Unlike the tissue depletion study, where each animal provides data for only one sampling time (Le., the slaughter time for that animal), in the commercial production of milk, treated animals are milked over a series of days. Milk discard times are calculated using a statistical algorithm based on repeated measurements. As with the preslaughter withdrawal calculation, the final milk withholding period is determined from the combined data, applying the 99th percentile/ 95% confidence limit approach. As with the preslaughter withdrawal period, the milk discard time is rounded up to the next complete milking (i.e., a discard time of 33 hours is rounded up to 36 hours). The assigned milk discard period must be consistent with standard production practices. For drugs intended for use in laying hens, the sponsor must reserve a portion of the ADI for eggs. Since there is no withdrawal or discard
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time for eggs, drugs used in laying hens must qualify for a zero withdrawal period for the eggs. Harmonization with Canada
On January 1, 1989, the Free Trade Agreement between the United States and Canada went into effect. To implement certain provisions of the agreement concerning technical regulations and standards that would remove barriers to trade in agricultural products, CVM and Canada's Bureau of Veterinary Drugs formed the Working Group on Veterinary Drug Tolerances. The primary charges to the working group were the following: (1) to harmonize tolerances for approved drugs, with the goal of having the same tolerances in each country, and (2) to harmonize the procedures used for evaluating new drugs, performing risk assessments, and calculating tolerances. Three years after the start of the free trade initiative, Canada published a list of 37 drugs for which Canada and the United States held identical tolerances. 1 Although the 37 drugs were somewhat less than half the total number of approved drugs, this initial effort at harmonization represented a significant step forward. The working group reached agreement on the procedures that the United States and Canada would follow for the evaluation of new veterinary drugs. Both countries agreed to require the same toxicologic data and to use identical methods for calculating safe concentrations and tolerances. Nevertheless, it should be recognized that harmonized standards do not automatically lead to harmonized tolerances. For example, some differences between United States and Canada tolerances for the same drug were the result of different, but equally valid, scientific conclusions drawn by different scientists about the same data set. References 1. Canada Gazette. 1991: 1478, SOR/91-255, Part II. 2. General Principles for Evaluating the Safety of Compounds Used in Food-Producing Animals (CVM Guideline #3, parts I-VIII), revised July 1994. 3. Lehman AJ, Fitzhugh OC: Quarterly Report to the Editor on Topics of Current Interest: 100-Fold Margin of Safety. Journal of the Association of Food and Drug Officials 18:33, 1954
Address reprint requests to Lynn G. Friedlander, PhD Residue Chemistry Team, HFV-151 Division of Human Food Safety Office of New Animal Drug Evaluation Center for Veterinary Medicine US Food and Drug Administration 7500 Standish Place Rockville, MD 20855-2764
0749-0720/99 $8.00
+ .00
THE HUMAN FOOD SAFETY EVALUATION OF NEW ANIMAL DRUGS Lynn G. Friedlander, PhD, Steven D. Brynes, PhD, and Ana H. Fernandez, DVM
Animal drugs are substances intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease in animals, and substances (other than food) intended to affect the structure or any function of the body. Before a new animal drug can be marketed in the United States, the drug sponsor must file a New Animal Drug Application (NADA), which must be approved by the US Food and Drug Administration (FDA). To file a NADA, the drug sponsor, usually a pharmaceutical firm, must conduct studies that demonstrate that the drug is safe and effective in the target animal species, is safe for the environment, and can be manufactured to uniform standards of purity, strength, and identity. When the drug is intended for use in foodproducing animals, the sponsoring firm also must demonstrate that the food products derived from treated animals are safe for human consumption. To conduct investigational studies with new animal drugs prior to approval, the drug sponsor must request an Investigational New Animal Drug (INAD) exemption from the FDA. Under the INAD, the sponsoring firm can conduct the studies needed to support the approval of the drug product. These studies are reviewed by scientists at the FDA's Center for Veterinary Medicine (CVM). Where appropriate, the
From the Division of Human Food Safety, Office of New Animal Drug Evaluation, Center for Veterinary Medicine, United States Food and Drug Administration, Rockville, Maryland
VETERINARY CLINICS OF NORTH AMERICA: FOOD ANIMAL PRACTICE VOLUME 15 • NUMBER 1 • MARCH 1999
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FDA may authorize the use in human food of products derived from animals treated in investigational studies. HUMAN FOOD SAFETY REQUIREMENTS
Human food safety studies are conducted to assess the nature and quantity of residues in foods derived from animals treated with the proposed new animal drug. Residue is defined as any compound present in the edible tissues of the target animal that results from the use of the sponsored compound, including the sponsored compound itself, its metabolites, and any substance formed in or on food as a result of the use of the sponsored compound. Substance is broadly defined, and even includes endogenous compounds that are altered owing to drug use. In this article, we discuss the types of studies submitted in a NADA to establish the human food safety of the drug and its metabolites in animal-derived edible products resulting from the use of new animal drugs and feed additives. Our guidance document describing the types of human food safety data that can be used to support a new animal drug approval may be found on our CVM website at www.fda.gov / cvm/. 2 Background
Although food safety has always been a major concern for the FDA, the human food safety requirements for animal drug residues have changed over the years. Originally, animal drugs were approved on the basis of a no residue or zero residue tolerance policy. Although many believed that zero residues could be achieved, most drugs are eliminated by first-order kinetics, and some residues always remain in the animal. The zero tolerance value actually represented the limits of the available analytic technology, not a true zero value. As a result, as the analytic methods continued to improve, the zero tolerance value was continually lowered. A refinement of the zero tolerance policy was the concept of negligible tolerance. Under this policy, toxicologic data requirements were limited to subchronic studies in dogs and rats. The tolerance was generally assigned as 0.1 ppm in all edible tissues. As with the zero tolerance, the negligible tolerance was not representative of the potential hazard of the compound. To remedy this situation, the CVM adopted its current risk-assessment-based procedure. The human food safety of animal drugs is determined following a review of the toxicologic, metabolic, and residue data. Under the current risk-assessment model, risk from an animal drug residue equals the hazard associated with the drug multiplied by the exposure to drug residues: Risk = Hazard X Exposure
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Public health risk is regulated by assessing the hazard posed by the drug substance and by controlling the exposure to meet a risk standard of "reasonable certainty of no harm." Current Requirements
The human food safety studies conducted to assess the safety of drug residues fall into two general categories: toxicity studies and residue chemistry studies. Because the requirements for the residue chemistry studies are dependent on the toxicity of the drug as determined in the toxicity tests, it is usually more practical for the drug sponsor to conduct the preliminary toxicity studies before the residue chemistry work is begun. TOXICOLOGY (HAZARD ASSESSMENT)
All of the toxicity tests are designed to evaluate the toxicity of the drug or its metabolites to humans, who may be exposed to it through the consumption of food derived from animals treated with the new animal drug product. Although the human exposure would be at a chronic low dose via food, the toxicity studies are conducted using high doses, with the toxicologic model species (usually rats or mice and dogs) serving as human surrogates. The toxicity tests attempt to predict the effects of drugs over a lifetime of oral exposure by extrapolating from the toxicologic model species to man and from short-term exposure to long-term exposure. For some drugs, studies assessing acute toxicity also may be required. The basic toxicology package contains tests that are appropriate for most new animal drugs. The tests include genetic toxicity studies, 90day feeding studies in a rodent and a nonrodent mammalian species to test the oral toxicity of the drug in the toxicologic species, and a twogeneration reproduction study with a teratology component to evaluate the cross-generational toxicity and teratogenic potential of the new animal drug. Additional toxicity tests may be conducted to clarify the results obtained in the basic test battery or to provide additional data. Specialized tests may need to be conducted to selectively target toxic endpoints of concern for the drug (e.g., reproductive organs for synthetic hormones). For antimicrobial drugs, the sponsoring firm might also need to perform studies evaluating the effect of the drug residues on the human intestinal microflora (e.g., development of resistance, alteration of the barrier effect, overgrowth of potentially pathogenic microorganisms). Chronic studies may be conducted to evaluate the toxic effects of the drug over the lifetime of the toxicologic model species. All of the testing in the toxicologic species is conducted via the oral route of administration, because the tests are designed to evaluate the toxicity of the new animal drug to humans following oral exposure in food.
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Noncarcinogens
For compounds that are not carcinogens, the toxicity testing permits the calculation of the Acceptable Daily Intake (ADI). The ADI represents an amount of drug that can be consumed daily over the lifetime of a human individual without harmful effect. The ADI is calculated by dividing the no-observed-effect level (NOEL) of the most sensitive effect as determined by the most appropriate toxicity study by a safety factor: ADI
=
NOEL Safety Factor
The NOEL is the highest tested concentration of drug that produced no toxic effect. The safety factor is determined by the type of study in which the toxic effect was observed. In general, the closer the toxicity study is to assessing the true toxicologic significance to humans, the smaller the applied safety factor will be. Generally, a 10-fold factor is applied to account for variability within the human population, a 10-fold factor is applied to account for interspecies extrapolation, and a 10-fold factor is applied to account for extrapolation from sub chronic to chronic exposure. 3 Because the safety factor appears in the denominator, a smaller safety factor results in a larger AD!. Although the ADI is an indication of the inherent toxicity of the drug, it also reflects the uncertainty of the toxicity assessment. It may be possible for the drug sponsor to conduct additional toxicity studies for which the safety factor is smaller (e.g., a long-term study), which will result in the calculation of a higher final AD!. The calculation of the ADI completes the hazard analysis component of the human food safety section of the NADA. The ADI is used to calculate the safe concentration of residues, the amount of total drug-related residues that can be present in each of the edible tissues of animals treated with the new animal drug. In calculating the safe concentration of residues in edible tissues, the ADI is adjusted for the anticipated dietary consumption of animal-derived food products. The safe concentration is calculated by multiplying the ADI by 60 kg (132 lb), the weight of the average human, and dividing by a tissue consumption value: SAFE CONCENTRATION =
ADI X 60 kg Tissue Consumption Value
The tissue consumption values are assigned to each of the edible products based on the portion of the diet they comprise (Table 1). The consumption values assume that a person will not eat a full complement of all edible tissues in a single day; that is, a person does not normally eat a full helping of liver for lunch followed by a full serving of lean meat (muscle) for dinner. The consumption value for milk is high enough to accommodate exposure in that segment of the population for which milk may be the major food product consumed and where body weight is less than 60 kg (i.e., young children). Since milk, eggs, and
THE HUMAN FOOD SAFETY EVALUATION OF NEW ANIMAL DRUGS
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Table 1. CONSUMPTION VALUES FOR VARIOUS EDIBLE PRODUCTS Edible Product Muscle Liver Kidney Fat Milk
Eggs
Grams Consumed/day 300 100
50 50 1500 mL
100
meat may be consumed in a single day, drug sponsors who seek approval for drugs for use in lactating dairy cattle or laying hens must partition the ADI for meat and nonmeat (i.e., milk and eggs). That is, the sponsor must specify what portion of the total ADI is to be used for meat and what portion is reserved for milk and eggs. The ADI that is reserved for milk or eggs is not used to calculate the safe concentration for edible tissues. The safe concentrations establish the amount of total residue that can be present in each of the edible products derived from animals treated with the new animal drug. The calculation of the safe concentration begins the exposure-assessment portion of the human food safety section of the NADA. Carcinogens
The Delaney Clause of the Federal Food, Drug, and Cosmetic Act prohibits the use of compounds "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 the food additive, to induce cancer in man or animal" (section 409(c)(3)(A)). An exception to the Delaney Clause exists for animal drugs when "it is determined by methods of examination prescribed or approved by the Secretary ... that no residue of that compound will be found in the food produced from those animals under conditions of use reasonably certain to be followed in practice" (21 CFR 500.80(a)). To implement this latter provision of the Delaney Clause, the FDA relies on quantitative risk-assessment procedures to determine when the amount of residue of carcinogenic concern in the edible tissue produces an incremental estimated cancer risk for a lifetime of 1 in 1 million. This level functionally defines no residue. Following a determination of the no residue level, residue and metabolism studies are conducted as described below. RESIDUE CHEMISTRY (EXPOSURE-ASSESSMENT AND MITIGATION) STUDIES
The residue chemistry exposure-assessment studies are designed to determine the concentration of drug residue actually appearing in the edible tissues of the target animal species as a result of treatment with
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the proposed animal drug. Because tissue residue concentrations are often high immediately following treatment, residue chemistry studies also are used to determine how much time must elapse following treatment (e.g., how long milk from treated animals must be discarded or how long treated animals themselves must be withheld from slaughter) to allow residues in edible products to deplete to acceptable levels. Additionally, residue chemistry studies are used to confirm that the residues produced by the target (food) species have been tested toxicologically. Residue chemistry studies submitted to the FDA normally include total residue and metabolism studies, comparative metabolism studies, and residue depletion studies. Total Residue and Metabolism Studies
The initial residue chemistry study conducted is the total residue and metabolism study. The term total is used because the study is conducted with radiolabeled drug and is capable of monitoring all drugderived residues resulting from the administration of the test material. The total residue and metabolism study is conducted in the target (food) animal species and is intended to show where the administered drug is deposited in the target species, how it is metabolized, and what its pattern of elimination is. Because the only source of radioactivity is the administered drug, any detected radioactivity is drug-related. Usually, total radioactivity in the edible tissues is measured by combusting the tissue samples to liberate radiolabeled carbon dioxide, which is then trapped and measured by liquid scintillation counting. The measured carbon dioxide is correlated directly to the amount of total drug-derived residue in the tissue sample. Metabolism of the proposed drug also is evaluated using the tissues from the total residue and metabolism study. Commonly available analytic technology (e.g., high performance liquid chromatography and gas chromatography) makes it possible to separate the total residue into its components and identify individual drug-derived compounds. All major metabolites are identified as part of the total residue and metabolism study. A major metabolite is any radiolabeled compound, including the parent drug, comprising at least 10% of the total residue or having a tissue concentration of at least 0.1 ppm. By measuring the concentrations of the major radiolabeled drug-related components in tissue samples collected at different times following the cessation of treatment, it is possible to identify those drug residues that persist for the longest time following treatment and to determine in which of the edible tissues drug elimination proceeds most slowly. Because radiolabeled residues (i.e., total residues) represent all of the drug in edible tissues, they can be compared directly to the safe concentration (i.e., total daily exposure) established in the toxicity testing. Drug residues determined shortly after the cessation of treatment are evaluated to determine if a withdrawal period is needed for the
THE HUMAN FOOD SAFETY EVALUATION OF NEW ANIMAL DRUGS
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intended use of the product. A practical zero withdrawal is established at 6 hours after the final dosing in poultry and at 8 to 12 hours after the
final dosing in large animal species (Le., cattle, sheep, and swine). The practical zero withdrawal is intended to reflect the time required for animals to reach the abattoir. If the total residues in the edible tissues at practical zero withdrawal are less than their respective safe concentrations, no preslaughter withdrawal period is assigned (zero withdrawal). The target tissue is the edible tissue selected to monitor for total residue in the target animal. The target tissue is usually, but not necessarily, the last tissue in which total residues deplete to their permitted safe concentration. A marker residue is a residue whose concentration is in a known relationship to the concentration of total residue in the last tissue to deplete to the safe concentration. The marker residue can be the parent compound, any of its metabolites, or a combination of residues for which a common assay can be developed (Fig. 1). The target tissue and the marker residue are selected so that the absence of the marker residue above a designated concentration (the tolerance) will confirm that each edible tissue has a concentration of total residues at or below the safe concentration (Fig. 2). Once the relationship between the total residue and the marker residue in the target tissue is established, it is possible to monitor depletion of total residues to the safe concentration. When the marker residue in the target tissue has depleted to the tolerance, the total residue will have depleted to the safe concentration in all edible tissues. The tolerance is the legal enforcement level; it is the codified value in the Code of Federal Regulations (specifically, 21 CFR 556).
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Figure 1. Typical depletion curves for potential marker residues in the target tissue. Open circle = total residue; open square = metabolite 8; solid square = parent; solid oval = metabolite A.
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Analytic Methods
In order to monitor drug residues, robust analytic methods must be available. It is necessary for the sponsor to develop and validate regulatory analytic methods that will accurately measure the marker residue in the target tissue at the tolerance. Regulatory analytic methods must be capable of accurately measuring the marker residue and verifying the identity of the marker residue. Consequently, regulatory methods submitted to the FDA usually have two components: a determinative method for quantification and a confirmatory method for identification. Regulatory analytic methods, both determinative and confirmatory, undergo a series of evaluations to ensure that they will perform well for monitoring drug residues in edible tissues. Since the tolerance for the marker residue is determined using the determinative analytic method, the tolerance is directly tied to that method. When a method is modified, it is necessary to confirm the relationship between the original and the revised analytic methods. The United States Department of Agriculture (USDA) uses the official regulatory methods to monitor animal-derived foods for residues of the new animal drugs. Screening tests (e.g., ELISA) are rapid, low-cost, on-site procedures that provide detection of residues for a general class of compounds. For example, the screening test may detect a residue as a tetracycline drug but not differentiate between oxytetracycline and chlortetracycline. Although developing screening tests is not a requirement for the approval of a new animal drug, sponsors are routinely asked to evaluate how their new animal drug will react with screening methods that are already
THE HUMAN FOOD SAFETY EVALUATION OF NEW ANIMAL DRUGS
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available, especially those screening tests used by the USDA for the rapid analysis of tissue residues. Comparative Metabolism Study
Comparative metabolism studies are conducted in the toxicologic species to confirm that the animals used in the toxicity studies were exposed to the same metabolites present in food products derived from treated animals. Metabolic profiles in the toxicologic and target species are compared to assess whether the same major metabolites are present in both species. If both species produce the same metabolites, we conclude that the toxicologic species was autoexposed to the same compounds to which humans will be exposed when eating food derived from treated animals. Failure to demonstrate autoexposure may necessitate additional toxicity testing. Residue Depletion Study and Assigning a Withdrawal Period
When a withdrawal time is needed to support a new animal drug approval, the sponsoring firm conducts a residue depletion study using the commercial product under field-use conditions. The residue depletion study monitors the marker residue as it depletes to the tolerance level using the determinative analytic method. For the residue depletion study, the drug product is administered at the highest label dose for the longest label duration to provide a worst-case scenario for residue depletion. The withdrawal time is calculated by using the data from the residue depletion study and determining the 99th percentile tolerance limit with 95% confidence. When animals treated with the new animal drug according to the label indications are withheld from slaughter for the assigned withdrawal period, there is only a 5% probability that, by chance, 1 animal in 100 will have residues of the marker compound in the target tissue that exceed the established tolerance level. For most animals, drug levels will be well below the tolerance level before the end of the withdrawal time. Even for that fraction of animals that falls outside the predictive model, only the target tissue would be expected to have residues above the tolerance, if the label indications were followed. Furthermore, because animals are slaughtered when they reach market weight, not at the end of the withdrawal period, virtually no violative residues will occur when labeled directions are followed. To ensure that there is only a 5% probability that, by chance, 1 animal in 100 will have residues of the marker compound in the target tissue that exceed the established tolerance level when the drug product is used in accordance with its labeling, the assigned withdrawal time is the time when the 99th percentile data cross the tolerance value (Fig. 3).
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: . - 99th Percentile with 95% Confidence Actual
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3 4 5 Withdrawal Time (days)
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Figure 3. Calculating the preslaughter (tissue) withdrawal period. A 6.7S-day withdrawal period is calculated and a 7-day withdrawal period is assigned.
The observed (actual measured) residue data in the depletion study always are less than the 99th percentile data. If the calculated withdrawal time is a fraction of a day, the assigned withdrawal period is rounded up to the next whole day (e.g., if the calculated withdrawal period is 162 hours (6.75 days), a withdrawal period of 7 days is assigned). The assigned preslaughter withdrawal period must be consistent with standard production practices (i.e., likely to be followed in practice). The residue depletion study in milk is similar to the residue depletion study in tissues. The marker residue and tolerance for milk residues are determined in a radiolabeled study conducted in lactating cattle. The tolerance and marker residue in milk are determined independently from the marker residue and tolerance in tissue to provide for potential differences in drug metabolism in the udder. The statistical treatment of the data is also different. Unlike the tissue depletion study, where each animal provides data for only one sampling time (Le., the slaughter time for that animal), in the commercial production of milk, treated animals are milked over a series of days. Milk discard times are calculated using a statistical algorithm based on repeated measurements. As with the preslaughter withdrawal calculation, the final milk withholding period is determined from the combined data, applying the 99th percentile/ 95% confidence limit approach. As with the preslaughter withdrawal period, the milk discard time is rounded up to the next complete milking (i.e., a discard time of 33 hours is rounded up to 36 hours). The assigned milk discard period must be consistent with standard production practices. For drugs intended for use in laying hens, the sponsor must reserve a portion of the ADI for eggs. Since there is no withdrawal or discard
THE HUMAN FOOD SAFETY EVALUATION OF NEW ANIMAL DRUGS
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time for eggs, drugs used in laying hens must qualify for a zero withdrawal period for the eggs. Harmonization with Canada
On January 1, 1989, the Free Trade Agreement between the United States and Canada went into effect. To implement certain provisions of the agreement concerning technical regulations and standards that would remove barriers to trade in agricultural products, CVM and Canada's Bureau of Veterinary Drugs formed the Working Group on Veterinary Drug Tolerances. The primary charges to the working group were the following: (1) to harmonize tolerances for approved drugs, with the goal of having the same tolerances in each country, and (2) to harmonize the procedures used for evaluating new drugs, performing risk assessments, and calculating tolerances. Three years after the start of the free trade initiative, Canada published a list of 37 drugs for which Canada and the United States held identical tolerances. 1 Although the 37 drugs were somewhat less than half the total number of approved drugs, this initial effort at harmonization represented a significant step forward. The working group reached agreement on the procedures that the United States and Canada would follow for the evaluation of new veterinary drugs. Both countries agreed to require the same toxicologic data and to use identical methods for calculating safe concentrations and tolerances. Nevertheless, it should be recognized that harmonized standards do not automatically lead to harmonized tolerances. For example, some differences between United States and Canada tolerances for the same drug were the result of different, but equally valid, scientific conclusions drawn by different scientists about the same data set. References 1. Canada Gazette. 1991: 1478, SOR/91-255, Part II. 2. General Principles for Evaluating the Safety of Compounds Used in Food-Producing Animals (CVM Guideline #3, parts I-VIII), revised July 1994. 3. Lehman AJ, Fitzhugh OC: Quarterly Report to the Editor on Topics of Current Interest: 100-Fold Margin of Safety. Journal of the Association of Food and Drug Officials 18:33, 1954
Address reprint requests to Lynn G. Friedlander, PhD Residue Chemistry Team, HFV-151 Division of Human Food Safety Office of New Animal Drug Evaluation Center for Veterinary Medicine US Food and Drug Administration 7500 Standish Place Rockville, MD 20855-2764