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Comparative metabolism studies, selection of marker residues and ratios of marker residues to total residues
- 1. Guidance on metabolism studies
- 2. Introduction
- 3. Types of metabolism studies
- 4. Metabolism studies in laboratory animals
- 5. Test materials
- 6. Analytical standards
- 7. In vitro test systems
- 8. In vivo test systems
- 9. Determination of total radioactivity
- 10. Separation and comparison of metabolites
- 11. Analytical methods
- 12. Extent of characterisation of major metabolites
- 13. Non-extractable metabolites
- 14. Reporting of data—outcomes of the metabolism assessment
This guideline is based on the International Co-operation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products guidelines (VICH GLs) 46 and 47. These were intended to provide a general description of the criteria that have been found by the European Union, Japan, the United States, Australia, New Zealand and Canada to be suitable to identify the metabolites of veterinary drugs produced by laboratory animals and target animals in comparative metabolism studies.
VICH GLs 46 and 47 are two of a series developed to facilitate the mutual acceptance by national and regional regulators of residue information for veterinary drugs used in food-producing animals. They were originally prepared after consideration of the current national and regional requirements and recommendations for evaluating veterinary drug residues in the European Union, Japan, the United States, Australia, New Zealand and Canada.
While VICH GLs 46 and 47 cover most of the Australian recommended considerations in terms of comparative metabolism data for laboratory species and target animal species, there are some additional considerations that are unique to Australia. These additional considerations are detailed in this document.
1. Guidance on metabolism studies
This guidance is intended to provide recommendations for procedures to identify the metabolites of veterinary drugs produced by laboratory animals. The purpose of the comparative metabolism studies is to compare the metabolites of the laboratory animals used for toxicological testing to the residues of the veterinary drugs in edible tissues of food-producing animals. The studies aim to determine if the laboratory animals used for toxicological testing have been exposed to the metabolites to which humans can be exposed (that is, as residues in products of food-producing-animal origin).
The pharmacokinetic profiles of the veterinary chemical and its metabolites in the target animal species should be qualitatively comparable with those of the laboratory animal species used to establish the health standards, to verify the relevance of the toxicological effects and the no-observed-adverse-effect levels and/or no-observable-effect levels, and thereby validate the dietary exposure assessments.
Metabolism studies are used to assess the fate of the chemical in target animals and to assess the nature and disposition of chemical residues in food-producing animals. The composition of a residue (parent and metabolites) and the target tissue(s) or food commodities in which it is present (for example, meat, offal, milk, eggs) should be known, so that residue depletion trials and analytical methods deal with the relevant residue components.
Metabolism and kinetics studies in both target animals and laboratory animals help assess:
The human food-safety evaluation of veterinary drug residues helps ensure that food derived from treated food-producing animals is safe for human consumption. As part of the data collection process, you should conduct studies to:
- characterise the metabolites to which laboratory animals are auto-exposed during the toxicological testing of the veterinary drug. The purpose of these studies is to determine whether the metabolites that people will consume from tissues of target food-producing animals are also produced by metabolism in the laboratory animals used for the safety testing. It is understood that, if the laboratory animals produce substantially similar metabolites to those produced by the food-producing animal, the laboratory animals will have been auto-exposed to the metabolites that humans will consume from tissues of treated food-producing animals. Auto-exposure of metabolites will ordinarily be taken as evidence that the safety of metabolites has been adequately assessed in the toxicology studies
- permit an assessment of the quantity and nature of residues in food derived from animals treated with a veterinary drug. These metabolism studies provide data on
- the depletion of residues of concern from edible tissues of treated animals at varying times after drug administration
- the individual components, or residues, that comprise the residues of concern in edible tissues
- the residue(s) that can serve as a marker for analytical methods intended for compliance purposes (that is, monitoring of appropriate drug use)
- the ratio of marker residue to total radioactive residues
- the identification of target tissue(s).
3. Types of metabolism studies
Demonstration of metabolites from the laboratory animal can be generally accomplished in one or more in vitro studies or in an in vivo study.
You can conduct one or more in vitro laboratory animal metabolism studies (for example, laboratory animal liver slice metabolism) for comparison to the metabolism in the food-producing animal to demonstrate that the relevant laboratory animal produces the metabolites that are found as residues in the edible tissues of the target food-producing animal. Conducting in vitro studies can avoid the use of in vivo laboratory animal studies, reduce the number of animals that are euthanased, and reduce the cost of comparative metabolism studies. If the in vitro or in vivo studies do not demonstrate the metabolites produced by the target food-producing animal, you should address by other means the relevance to consumer safety of the food-producing-animal metabolites.
Laboratory animal in vitro and in vivo metabolism studies are most often accomplished using radiolabelled drugs. These studies are capable of monitoring all of the drug-derived residues resulting from the administration of test material (note: generally only the major metabolites should be identified). This guidance, therefore, recommends procedures for metabolism studies conducted with radiolabelled drugs. However, alternative approaches (that is, when not using radiolabelled drugs) to characterise the metabolites in laboratory animals can be suitable when the metabolites produced by the target food-producing animal as residues in edible tissues are readily identified in urine or tissues of the laboratory animals by chemical means.
You should conduct metabolism studies in compliance with applicable good laboratory practice.
4. Metabolism studies in laboratory animals
Generally, auto-exposure has been adequately demonstrated if laboratory animals produce each of the major metabolites of the residue that people will consume from edible tissues of treated food-producing animals. You should report qualitative information on the metabolites in laboratory animals. Quantification of the metabolites found in urine, fluids or tissues of laboratory animals is not generally an objective of the comparative metabolism studies. Generally, only the major metabolites found as residues in the food-producing animal should be identified in the laboratory animals. Metabolites observed in laboratory animals that are not observed in the food-producing animal are not relevant to the objective of assuring that the laboratory animals are auto-exposed to the residue metabolites that humans will consume.
5. Test materials
The chemical identity (including, for example, the common name, chemical name, CAS-number, structure, stereochemistry and molecular weight) and purity of the drug substance should be described. The test drug should be representative of the active ingredient to be used in the commercial formulation.
5.2. Radiolabelled drug
5.2.1. Nature and site of label
Carbon-14 (14C) is the label of choice because intermolecular exchange is not an issue. Other isotopes, such as 3H, 32P, 15N or 35S, might be appropriate. Tritium (3H) might be considered suitable if a rigorous demonstration of the stability of the tritium label is provided; for example, the extent of exchange with water is assessed and found to be equal to or less than 5 per cent.
You should indicate the position(s) of the radiolabel. The drug should be radiolabelled in a site, or in multiple sites, to assure that the portions of the parent drug that are likely to be of concern are suitably labelled. The radiolabel should be placed in a metabolically stable position(s).
5.2.2. Purity of radiolabelled drug
Radiolabelled drugs should have a high level of purity, preferably of approximately 95 per cent, in order to minimize artifactual results. You should demonstrate radiochemical purity via appropriate analytical techniques (for example, by using two chromatographic systems).
5.2.3. Specific activity
You should state the specific activity of the synthesised radiolabelled drug in the study report. The specific activity should be high enough to permit tracking of the residue of concern in edible tissues. The sensitivity should be determined by the potency of the drug. You can adjust the specific activity by mixing the radiolabelled drug with an unlabelled drug. To facilitate analytical measurements and conserve radiolabelled drugs, you can dose animals to be euthanised at early withdrawal periods with a drug of a lower specific activity, while animals to be euthanised at later withdrawal periods can be dosed with a drug of a higher specific activity.
6. Analytical standards
Analytical standards should be available for the parent drug and, if possible, for metabolites known or expected to exist, for use in the chromatographic comparison of drug metabolites. The metabolites can be isolated from tissues generated in the target food-producing-animal metabolism study.
7. In vitro test systems
You can use single or multiple in vitro metabolism test studies as an alternative for the in vivo comparative metabolism studies.
The laboratory animal species used in the comparative metabolism study should preferably be the same species (and for rodents the same strain) as was used in the pivotal study for determining the toxicological acceptable daily intake of the veterinary drug. In case another species is used, you should justify the choice of species in terms of relevance. You should report on the sources of the animals, their weights, health statuses, ages and sexes.
Various test systems have been published and are widely used. In vitro systems for comparative metabolism studies include primary hepatocytes, liver microsomes, the S9 sub-cellular fraction, cytosol, liver slices and whole cell lines. Protocols for these in vitro studies have not yet been standardised (for example, by the Organization for Economic Co-operation and Development), therefore some strengths and weaknesses of each of these systems are discussed below:
- Primary (fresh or cryopreserved) hepatocytes: primary hepatocytes are liver cells that are useful in evaluating phase-I and phase-II metabolism and have the added advantage of taking membrane transport effects into account. These hepatocytes can be prepared in suspension, monolayer culture or sandwich cultures. The sandwich cultures have the advantage that they maintain enzyme activities for a longer duration of time. If the food-producing-animal residue metabolites are demonstrated in a primary hepatocytes system, then comparative metabolism has generally been demonstrated. Use of a primary hepatocytes-based system can be complementary to one or more of the other in vitro systems to demonstrate the metabolism for a laboratory animal species.
- Liver microsomes: liver microsomes include most of activities of cytochrome P450 and flavin-containing monooxygenase systems for evaluating phase-I metabolism, along with uridine diphosphate-glucuronosyl-transferase for phase-II glucuronidation. If the food-producing-animal residue metabolites are demonstrated in a liver microsome system, then comparative metabolism has generally been demonstrated. Use of a liver-microsome-based system can be complementary to one or more of the other in vitro systems to demonstrate the metabolism for a laboratory animal species.
- S9 sub-cellular fraction: The S9 sub-cellular fraction contains the same phase-I and phase-II enzymes present in liver microsomes, as well as additional systems such as sulfotranferases and N-acetyltransferases. The S9 sub-cellular fraction is suitable for evaluating phase-I and phase-II metabolism or phase-I metabolism followed by phase-II conjugation. If the food-producing-animal residue metabolites are demonstrated in a S9 sub-cellular fraction system, then comparative metabolism has generally been demonstrated. Use of a S9 sub-cellular fraction-based system can be complementary to one or more of the other in vitro systems to demonstrate the metabolism for a laboratory animal species.
- Cytosol: this represents the supernatant fraction that remains following microsomal centrifugation. It contains some of the phase-II conjugation systems, but otherwise represents a relatively incomplete matrix for metabolic work. In general, the use of cytosolic systems alone is unlikely to provide a complete comparative metabolism profile, but if the food-producing animal residue metabolites are demonstrated in a cytosol system, comparative metabolism has generally been demonstrated. Use of a cytosol-based system can be complementary to one or more of the other in vitro systems to demonstrate the metabolism for a laboratory animal species.
- Liver slices: the use of whole liver slices for metabolism research is possible; however, the liver cell viability and corresponding enzyme activities decrease rather rapidly compared with the other alternatives. You should not conduct comparative metabolism studies using liver-slice methodology unless you can demonstrate cell viability and enzyme activity. However, if the food-producing-animal residue metabolites are demonstrated in a liver-slice system, then comparative metabolism has generally been demonstrated. Use of a liver-slice-based system can be complementary to one or more of the other in vitro systems to demonstrate the metabolism for a laboratory animal species.
- Whole cell lines: use of whole cell lines is not currently recommended because the enzymatic activity is generally low. However, if the food-producing animal residue metabolites are demonstrated in a whole cell line system, then comparative metabolism has generally been demonstrated. Use of a whole-cell-line-based system can be complementary to one or more of the other in vitro systems to demonstrate the metabolism for a laboratory animal species.
- It is generally possible that only one of these specific in vitro options could be used for demonstration of comparative metabolism. However, if the target-species metabolic profile includes evidence of both phase-I and phase-II biotransformation, you should consider investigating multiple options (for example, microsomes and S9) to reproduce the complete metabolic profile.
Although many variations in test conditions have been reported in the literature, the following represents some general guidance for conduct of in vitro comparative metabolism studies:
- Test molecules are usually incubated in the in vitro system at 37 oC.
- The concentrations of target molecules are typically lower than 100 μM.
- The incubation time is dependent upon the rate of metabolism of the target molecules and should be adjusted accordingly.
Cofactors of phase-I and phase-II metabolism are scientifically necessary for incubation of liver microsomes and S9, such as NADPH (nicotinamide adenine dinucleotide phosphate, NADPH regeneration system) for phase-I metabolism, UDPGA (uridine diphosphate-glucuronosyl-transferase) for glucuronidation, and PAPS (3’-phosphoadenosine 5’-phosphosulfate) for sulfation.
When more standardized in vitro system metabolism study protocols become available, the general guidance above can be replaced according to the standardized protocols.
8. In vivo test systems
Laboratory animals: the laboratory animal species used in the comparative metabolism study should preferably be the same species (and for rodents the same strain) as was used in the pivotal study for determining the toxicological acceptable daily intake of the veterinary drug. In case another species is used, the choice of species should be justified in terms of relevance. You should provide the sources of the animals, their weights, health statuses, ages and sexes.
Target animals: there are some national or regional differences regarding the designation of major and minor species, particularly for turkeys and sheep. These differences can affect national or regional data collection requirements and recommendations. In Australia, sheep are considered to be a major species, while turkeys are minor.
In certain instances, the total residue and metabolism data for a drug’s use in a major species might be extrapolated to the minor species. When a national or regional authority calls for a total residue and metabolism study for a minor species, or for a species considered to be major in one region but not another, the study design outlined in this guidance should be acceptable.
Animals used in the metabolism study should be representative of commercial breeds and of the target population. You should provide the source of the animals, their weights, health statuses, ages and sexes.
Ordinarily, a single study can be performed in pigs (around 40 to 80 kg), sheep (around 40 to 60 kg) and poultry. For cattle, a single study in beef cattle (around 250 to 500 kg) could apply to dairy cattle, and vice versa. Generally, the results of a metabolism study in adult cattle and sheep can be extrapolated to calves and lambs, respectively. However, a second study might be appropriate for pre-ruminating animals if there is sufficient reason to believe the pre-ruminating animal will have significantly different metabolism from the adults. You should perform a separate study to demonstrate the total residue in milk of dairy cows.
8.2. Animal handling
Animals should be allowed adequate time to acclimatise. You should apply normal laboratory animal caretaking or husbandry practices. It is recognized that these studies might call for metabolism cages, a departure from ‘normal’ practices; therefore, you should only use metabolism cages if the study is intended to collect urine and excreta, or other specifications.
Animals should be healthy and, preferably, should not have been previously medicated. However, it is recognized that animals might have received biological vaccinations or prior treatment, for example with anthelmintics. An appropriate wash-out time should be observed for the animals prior to enrolment in the actual trial. Animals should have a known history of medication.
The feed and water supplied to the animals should be free from other drugs and/or contaminants and you should ensure that environmental conditions are adequate, consistent with animal welfare, and in accordance with applicable national and regional regulations.
Animal caretaking practices and disposal of animals and tissues from animals should be in compliance with all applicable national and regional laws and regulations.
8.3. Numbers of animals
Laboratory animals: enough animals should be treated with the drug in the comparative metabolism study to provide enough composited tissue or excreta for analysis. The samples of like material from different animals can be composited for a single analysis. There is no minimum number of animals for a comparative metabolism study; however, four animals of each sex are often used (but less can be used) to assure there is enough sample material. Demonstration of comparative metabolism is not generally conducted in animals of each sex; therefore, the samples of like material can be pooled (without regard to sex) to increase the likelihood of demonstrating the metabolites of interest when sex differences in metabolic ratios might exist.
Target animal species: at least four groups of animals, evenly-mixed as to sex if the drug is intended for use in both males and females, should be euthanised at appropriately spaced time points. The following numbers of animals are recommended:
- large animals (cattle, pigs, sheep)—three or more per euthanasia time
- poultry—three or more per euthanasia time
- fish—10 or more per euthanasia time
- lactating cattle for milk collection—eight or more for representative multiparous cattle of high and low milk production
- laying birds for egg collection—sufficient to collect 10 or more eggs per day
- honey—five honey samples, each from a separate hive.
A sufficient amount of control tissues should be available to permit a determination of background concentrations and combustion efficiency, and to provide tissue for testing of related analytical methods.
8.4. Drug formulation
You should describe the drug formulation, the method of dose preparation, and the stability of the drug in the formulation during the treatment period in the study report. For studies in laboratory animals, it is not critical that the formulation used in the comparative metabolism studies is the same as the commercial product. However, in contrast, target animal studies should involve treatment with the intended final formulation whenever possible. Given that metabolism studies can be conducted well in advance of definitive formulation decisions, treatment with representative or prototype formulations can also be considered appropriate.
8.5. Route of administration
Laboratory animals: in studies with laboratory animals, you should administer the drug orally. Gavage or bolus dosing can be used to ensure that animals receive the complete dose and to minimise environmental concerns.
Target animal species: for metabolism studies with the target animal species, you should administer the drug via the intended route of administration (for example, orally, dermally, intramuscularly, subcutaneously). For drugs that are intended for oral administration, especially via feed or drinking water, you can use gavage or bolus dosing to ensure that animals receive the complete dose and to minimise environmental concerns.
For drugs that are intended for oral and parenteral administrations, you should usually conduct separate metabolism studies. Ordinarily, a single study with a parenteral route will be applicable to cover all parenteral routes including intramuscular, intramammary, subcutaneous and topical. Similarly, a single study with an oral route will ordinarily be applicable to all potential oral formulations (for example, drinking water, in-feed and quick-release tablets).
Laboratory animals: the dose should be high enough to result in concentrations of metabolites in excreta or tissues for comparison. You should administer the dose daily, for enough time that the drug undergoes all relevant metabolic events, including those associated with enzyme induction. Normally, administration for five days is used unless there are data to show that a longer time of administration can better demonstrate the formation of the metabolites of interest. You can use doses near the minimum toxic dose to generate high concentrations of the metabolites of interest in tissues and urine, but you can also use lower doses.
Target animal species: the dose should be the highest intended treatment concentration, and you should administer it for the maximum intended duration or for the time required for steady state to be achieved in edible tissues. Pre-dosing of animals with an unlabelled drug, followed by administration of a radiolabelled drug, is not recommended.
For continuously administered drugs, a separate study to determine the time for residues to reach steady state in edible tissues might be appropriate. When a drug administered in a single dose is intended to have zero withdrawal, you should demonstrate that the absorption phase has been completed.
When gavage dosing for the feed and water routes, you should divide the dose and give it in the morning and afternoon to better approximate actual-use conditions.
8.7. Animal euthanasia
Laboratory animals: you should humanely euthanase all animals. Chemical euthanasia can be used unless it will interfere with analysis of the metabolites of interest.
Animals should be euthanised for metabolite analysis at a single time point, usually 2–4 hours after the last dose of the test substance. Multiple days of dosing provides the presence of metabolites resulting from sequential metabolism of the parent drug over time, and therefore additional euthanasia time points are not called for.
Target animal species: you should euthanase animals using commercially applicable procedures, making certain to observe appropriate exsanguination times. Chemical euthanasia can be used unless it will interfere with analysis of metabolites of interest.
8.8. Sample collection
Laboratory animals: before euthanasia, urine, faeces, and blood can be collected for analysis. The samples should be analysed immediately or stored frozen (unless freezing causes a stability problem for the metabolites of interest) until analysis. Freezing of the samples is to reduce microbial and host metabolism from altering the metabolic profile. If the samples are stored after collection, the sponsor should ensure that the radiolabelled compound remains intact throughout the storage period.
Comparative metabolism can be demonstrated with one or more excreta or tissues. Samples that are typically taken for qualitative metabolite analysis can include blood or blood fractions, excreta, liver, bile, kidney, fat or other tissues. Enough tissue of each type should be taken from each animal for analysis.
Target animal species: following euthanasia, samples of sufficient amounts of edible tissues should be collected, trimmed of extraneous tissue, weighed, and divided into aliquots. If the analysis cannot be completed immediately, the samples should be stored under frozen conditions pending analysis. If samples are stored after collection, you should ensure that the radiolabelled compound remains intact throughout the storage period.
Table 1 lists how the recommended samples should be taken from the target animal in the metabolism study.
|Edible tissue type||Sample description by species|
|Cattle or sheep||Pigs||Poultry|
|Injection site tissue||Core of muscle tissue ~500 g 10 cm diameter × 6 cm deep for intramuscular 15 cm diameter × 2.5 cm deep for subcutaneous||Core of muscle tissue ~500 g 10 cm diameter × 6 cm deep for intramuscular 15 cm diameter × 2.5 cm deep for subcutaneous||Collect sample from entire site of injection, for example chicken: whole neck, whole breast or whole leg. Larger birds: not to exceed 500 g|
|Liver||Cross-section of lobes||Cross-section of lobes||Entire|
|Kidney||Composite from combined kidneys||Composite from combined kidneys||Composite from combined kidneys|
|Fat||Peri-renal and subcutaneous back fat||N/A||N/A|
|Skin/fat||N/A||Skin with fat in natural proportions||Skin with fat in natural proportions|
|Eggs||N/A||N/A||Composite from combined white and yolk|
N/A: not applicable
You should analyse the tissues shown in Table 1. Additional tissues should be collected and analysed to provide information on the one additional tissue to be analysed in the marker residue depletion study. As appropriate to species, the additional tissues you might analyse include heart (cattle, pigs, sheep, poultry), small intestines (cattle and pigs) and gizzard (poultry). Furthermore, it might be appropriate to collect and analyse other edible offal from the various species if it is deemed important for a safety assessment (for example, offal with expected high residue concentrations or with residues having slow depletion rates).
Excreta and blood are not always collected during metabolism studies with target animal species. However, analyses of these samples can be useful from several perspectives:
- analyses of the excreta and blood allow an estimate of the mass balance, a valuable tool in assessing the quality of the study
- the samples of excreta can be a good source of metabolites
- the samples can be of use in conducting an environmental risk assessment.
If you decide to collect such data, we recommend that you collect urine and excreta from selected animals on a daily basis.
Blood samples can be taken from selected animals at various time points and at euthanasia. Data on the total residue in blood can provide valuable pharmacokinetic information.
9. Determination of total radioactivity
Laboratory animals: determination of total radioactivity in samples and accounting for the mass balance of the radioactivity are not normally conducted for the in vivo metabolism studies conducted with laboratory animals. When total radioactivity is to be determined, you should follow the procedures presented below (for target animal species).
Target animal species: you should determine the total radioactivity in samples using established procedures, which might include, for example, combustion followed by liquid scintillation counting, solubilisation and counting, or direct counting, depending on the nature of the sample. You should completely describe the details of the radioassays, including the preparation of analytical samples, instrumentation, and data from standards, control tissues, fortified tissues, and incurred tissues. You should also demonstrate the ability of the procedure to recover radioactivity added to control tissues.
The results of analyses of samples for radioactivity should be reported on a wet weight basis and on a weight/weight basis, with micrograms per kilogram (μg/kg) as the preferred units. You should describe the sample calculations showing conversion from counts per minute/weight (cpm/weight) or disintegrations per minute/weight (dpm/weight) to the weight/weight basis in the study report.
10. Separation and comparison of metabolites
Commonly available analytical technology, including, for example, high-performance liquid chromatography, high-performance thin layer chromatography, gas chromatography, and mass spectrometry, enable separation of the total residue into its components and identification of the drug-derived residues.
11. Analytical methods
You should provide a complete description of the procedures used for chromatographic and chemical characterization of the drug residues components in the report. The description should include the preparation of standards, reagents, solutions, analytical samples; the extraction, fractionation, separation and isolation of the residues; the instrumentation; and the data derived from standards, control tissues, fortified tissues and incurred tissues. The analytical method should be validated at least to demonstrate the recovery, the limit of detection and the variability. You should also demonstrate the repeatability of the retention times for the analytical method.
12. Extent of characterisation of major metabolites
Laboratory animals: characterisation and structural identification of the metabolites and demonstration of the tissue extraction efficiency during the comparative metabolism study are not normally conducted when the comparison of the chromatographic retention time(s) demonstrates the presence of the metabolites of interest in the laboratory animal.
Target animal species: the degree of characterisation and structural identification depends on several factors, which include the amount of residue present, the concern for the compound or for the class of compounds to which the residue belongs, and the suspected significance of the residue based on prior knowledge or experience.
In general, characterisation and structural identification of major metabolites should be accomplished using a combination of techniques and might include chromatographic comparison to standards or mass spectrometry. As a point of reference, major metabolites are those comprising 100 micrograms per kilogram or more, or 10 per cent or more of the total residue in a sample collected at the earliest euthanasia interval (or following attainment of steady state, or at or near the end of treatment for continuous-use drug products). In some cases, chemical characterisation rather than unequivocal structural identification for a major metabolite will be appropriate (for example, when a conjugate is present or if mass spectrometry information indicates the likely biotransformation pathway, such as M+16 for hydroxylation). Ordinarily, no differentiation of the radioactivity below these levels (that is, of the minor metabolites) would be recommended unless there are toxicological concerns over residues occurring at the lower levels.
13. Non-extractable metabolites
Laboratory animals: characterisation of non-extractable metabolites in comparative metabolism studies in laboratory animals is normally not performed. You should only do a characterisation of the covalently bound metabolites of a veterinary drug in laboratory animals when the non-extractable residue contains a metabolite of interest that is not present in enough quantity for characterisation in the easily extractable portion. In that case, you should follow the procedures identified for target animal species below.
Target animal species: the use of veterinary drugs in food-producing animals can result in residues that are neither extractable from tissues using mild aqueous or organic extraction conditions nor easily characterized. These residues arise from:
- incorporation of residues of the drug into endogenous compounds
- chemical reaction of the parent drug or its metabolites with macromolecules (bound residues), or
- physical encapsulation or integration of radioactive residues into tissue matrices.
Those non-extractable residues shown to result from incorporation of small fragments of the drug (usually one or two carbon units) into naturally occurring molecules are usually not of significance.
Characterisation of the bound residues of a veterinary drug is usually prompted when the bound residue comprises a significant portion of the total residue or when the concentration of bound residue is so high as to preclude the assignment of a practicable withdrawal period for the drug (that is, the total residue does not deplete below the residue of concern because of the amount of bound residue). The extent of data you should collect on the bound residue depends on a number of factors, including the amount of bound residue, the nature of the bound residue and the potency of the parent drug or metabolite on which the acceptable daily intake is based. You may need to investigate the nature of bound residues in certain situations. The information obtained from such an investigation might warrant the discount of some of the residues from the total residue of concern.
Characterisation of the bound residue: the characterisation of bound residues is usually difficult, involving vigorous extraction conditions or enzymic preparations that can lead to residue destruction or artefact formation.
However, the biological significance of residues of veterinary drugs in foods usually depends on the degree to which those residues are absorbed when the food is ingested. Therefore, the determination of the bioavailable residues that result when tissue containing bound residue is fed to test animals can be a useful characterisation tool.
14. Reporting of data—outcomes of the metabolism assessment
14.1. Comparative metabolism studies
Assessment will involve review of the absorption, distribution, metabolism and excretion of the drug in laboratory animals and target animal species.
Absorption: the primary purpose of absorption studies is to assess the bioavailability of the veterinary chemical, which relates to the rate and extent of absorption. The same considerations of absorption apply, regardless of the route of administration.
In the case of veterinary chemicals where it is proven that systemic absorption is negligible (that is, where metabolism studies demonstrate that the levels of total radioactive residues in edible commodities are below the limit of quantification of the analytical method used for monitoring and surveillance purposes), further residue studies are not required. However, if there is significant systemic absorption and total radioactive residues in any of the edible commodities are above the method limit of quantification, you should provide full residues studies.
Absorption involves estimation of:
- the rate of absorption (such as plasma Tmax reached within time of treatment)
- the extent of absorption
- the absolute bioavailability of the drug.
Distribution: following absorption, the veterinary chemicals are distributed throughout various tissues and organs of the target animal species. The results of distribution studies are useful in identifying the target tissue(s).
Distribution involves consideration of:
- where in the body the drug residues partition into the relative rank order of total radioactive residue(s) distribution in standard tissues from different species
- the extent of protein binding that occurs.
Metabolism: different biotransformation products may possess different toxic potentials. Therefore, you should provide information on the chemical nature, concentration, and persistence of the total residues. The purpose of the metabolism study in the target animal species is to provide the necessary information on the metabolic fate of the veterinary chemical in the edible tissues, and to enable the establishment of the marker residue(s).
These studies are also necessary to establish whether the metabolite(s) found in target animals are the same as those found in the laboratory animals used for toxicity testing. This is referred to as comparative metabolism, and determines whether the metabolites that people are exposed to when consuming tissues from treated animals are also produced in the laboratory animals used to establish the health standards. To validate the approach to assessing dietary exposure and the potential risk to human health, this information should be provided.
Metabolism involves consideration of:
- the profiles of total radioactive residues in excreta (urine, faeces), and tissues (muscle, liver, kidney, fat, skin/fat1, or injection sites) from treated animals
- the characterisation of total radioactive residues, and identification of the main residues components
- the postulation of the routes of metabolism of the drug in animals.
Ideally, the outcome of the comparative metabolism assessment is that the biotransformation pathways for a veterinary drug are qualitatively (if not quantitatively) similar in laboratory animals and target animal species. It is understood that, if the laboratory animals produce substantially similar metabolites as those produced by the food-producing animal, the laboratory animals will have been auto-exposed to the metabolites that humans will consume from tissues of treated food-producing animals. Auto-exposure of metabolites will ordinarily be taken as evidence that the safety of metabolites has been adequately assessed in the toxicology studies.
If the conclusion of the comparative metabolism studies is that laboratory animals are not exposed to all significant metabolites, you should provide bridging toxicology studies where laboratory animals are dosed with the significant metabolite that is not auto-exposed in the laboratory species.
Excretion: Information on the route(s) of excretion reflects the pathways by which the veterinary chemical is metabolised. It is recommended that you provide data on excretion in target animal species, including renal and faecal excretion. You should also consider other routes of excretion when appropriate (for example, milk).
Excretion involves examination of:
- the extent of recovery of administered dose in excreta (over a certain time frame)
- the estimation of the terminal plasma half-life, used as an indicator of residues persistence
- the comparison across the different species.
1 skin/fat means a sample of skin with associated fat in natural proportions, and refers to samples collected from pigs and poultry
14.2. Selection of marker residue(s)
You should report the components of the total residues in each tissue for each collection time point, for comparison to the total residue concentrations. The components of the total residues (parent drug plus metabolite[s]) should be examined to select the marker residue. The marker residue might be the parent compound. However, the marker residue might also be defined as a combination of parent compound plus a metabolite(s) or as a sum of residues that can be chemically converted to a single derivative or fragment molecule.
After consideration of the metabolism data, it should be possible to select the marker residue. An appropriate marker residue has the following properties:
- there is a known relationship established between the marker residue and the total residue concentration in the tissue of interest
- the marker residue should be appropriate to test for the presence of residues at the time point of interest; that is, adherence to the withdrawal period
- there should be a practicable analytical method to measure the marker residue at the level of the maximum residue limit at a level that is acceptable to Australia’s major trading partners.
14.3. Selection of a target tissue
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 tissue with the slowest depletion rate of the residues.
14.4. Ratios of marker residues to total residues
You should report the total residue concentration for each tissue for each collection time point. Also provide the amounts of total residue radioactivity extracted (percentage extractable) using various treatments (enzyme, acid). You should present the data so that it is possible to determine the ratios of marker residue to total residue ratio in each tissue, without extrapolation. These ratios are an integral part of the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives approach to establishing maximum residue limits for veterinary drugs.