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Food safety studies for veterinary drugs used in food-producing animals
This guideline is based on the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products guideline (VICH GL48), one of a series of guidelines developed to facilitate the mutual acceptance of food-safety (residue) data for veterinary drugs used in food-producing animals. This guideline was 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 GL48 covers many of the Australian recommended considerations in terms of food-safety studies performed to determine the maximum residue limit and the withholding period, there are some additional considerations that are unique to Australia. These additional food-safety considerations are detailed in this document.
1. Objective of this guideline
As part of the approval process for veterinary chemical products in food-producing animals, food-safety (marker-residue depletion) data are used to establish maximum residue limits, demonstrate compliance with existing maximum residue limits, determine appropriate withholding periods and determine export slaughter intervals. The food-safety studies submitted to support registration or approval of a new veterinary medicine product in the intended species should:
- demonstrate the depletion of the marker residue upon cessation of drug treatment to the regulatory safe level (that is, the maximum residue limit) in edible tissues including meat, milk and eggs
- generate data suitable to determine appropriate withdrawal periods to address consumer safety concerns in edible tissues including meat, milk, eggs and honey.
This guideline provides study-design recommendations to help you generate food-safetydata to satisfy the data recommendations for establishing maximum residue limits and recommending appropriate withdrawal periods for a specific product.
2. Scope of this guideline
When you conduct food-safety studies according to the recommendations described in this guideline, you should generate adequate data to determine maximum residue limits and establish withholding periods. The guideline encompasses the most common food species, namely cattle, pig, sheep and poultry, as well as bees and fish. The principles in this guideline can also be applied to other less common or novel food-producing animals.
3. Food-safety studies
3.1. Test product
The test product used for the study should be representative of the commercial formulation. Test product manufactured in accordance with good manufacturing practice (pilot scale or commercial scale) is the preferred source of test article.
The test product should be formulated to the top of its specified range of active constituent content and should be applied according to the proposed label directions. If there is more than one application method, separate trials or valid scientific argument may be necessary.
3.2. Trial design
3.2.1. General guidance
You should conduct food-safety studies in conformity with the Organisation for Economic Co-operation and Development’s principles of good laboratory practice, and follow internationally-recognised and regularly updated test protocols. In addition, you should provide the following detailed information where applicable:
- the species, breeds, strains or source of the test animals used
- the number, age, sex, body weight and production status of the dosed animals, including growth rate and milk, egg or honey yield
- the animal husbandry conditions; namely the water and food consumption of individual animals (especially for veterinary chemical products administered via drinking water and/or feed)
- details of the formulation of the administered veterinary chemical product and method of dose preparation
- details of the mode of dose administration—dose (in milligrams per kilogram of body weight), frequency of dosing, and duration.
The food-safety study should be conducted with previously unmedicated animals that are representative of the proposed target populations. Animals of both sexes should be used if a product is intended for use in both female and male animals. After animals have been treated with the veterinary chemical, edible tissues and biological fluids should be collected at appropriate times for residue analysis. The groups of animals used should be large enough to allow statistical assessment of the residues data.
Treatment regimens can be broadly classed as:
- Single treatments, where a veterinary chemical product is given once to an animal for a specific therapeutic effect. In this case, a single dose of veterinary chemical product (at the maximum dose rate) is the appropriate exposure regimen during residues trials.
- Repeat treatments, such as endo- or ecto-parasiticide treatments, where a single treatment is repeated. If a repeat dosage regimen can be reasonably anticipated in practice, then repeat treatments should be considered.
- Short-term treatments, where animals are dosed for a number of days, on one or more occasions. In this case, test animals should be dosed for the maximum period permitted on the label. Where the re-treatment interval is short, or the veterinary chemical has a propensity to remain in edible tissues, residue trials may need to incorporate multiple treatment periods at the shortest proposed re-treatment interval.
- Long-term treatments, where animals are treated for prolonged periods, for example medicated feed or water treatments, and where a zero day withholding period is desired. In this case, animals should be treated at the maximum proposed rate for a period that is sufficient to enable the residue concentrations in edible commodities to reach a steady-state equilibrium (that is, for the residue concentrations in tissues to reach a plateau).
Food-safety studies should characterise enough of the decline profile to enable the necessary withholding periods to be recommended with confidence. Australia does not subscribe to the concept of a ‘practical zero withdrawal period’. So, if a zero-day withholding period is being sought, one of the sampling times should be at zero hours after withdrawal, and the residues data should demonstrate compliance with the relevant maximum residue limits at all times after treatment.
Residues data from a well-designed residues decline trial are also likely to be used to determine an export slaughter interval for the product. To establish the export slaughter interval, it may be necessary for you to monitor the residues decline profile until it reaches concentrations below the maximum residue limit or the limit of quantification.
3.2.2. Dairy animals
In any submission for registration, you should address three aspects unique to dairy animals:
- the possibility of a treated dry animal re-joining the milking herd prematurely, soon after dosing, and her milk entering the farm milk supply
- the occurrence of residues in young animals as a result of access to milk from treated animals, either from suckling during the first few days post-partum or being fed discarded milk
- the occurrence of in utero drug transfer resulting in tissue residues in newborn animals.
Food-safety studies should establish the minimum recommended treatment-to-birthing interval during which application or administration of a product is appropriate. Normal animal husbandry practices should be considered.
When milk is destined for human consumption or for feeding to young animals destined for human consumption, the study should also establish the maximum period post-partum when milk could contain residues greater than the maximum residue limit. As most young animals destined for human consumption are allowed to suckle their dams or consume milk taken from treated animals, tissue residues in young animals should be addressed by residues data or valid scientific argument.
When determining maximum residue limits in milk, the potential effects of antimicrobial substances on microorganisms used in the manufacture of dairy products are also considered. Therefore, you should consider the effect of antimicrobial substances on the activity of dairy starter cultures when performing trials for antimicrobial products for use in dairy cows. The determination of the maximum concentration without effect for an antimicrobial substance on a starter culture is based on the guidelines established by the European Agency for the Evaluation of Medicinal Products—Committee for Veterinary Medicinal Products (2000).
3.2.3. Sheep ectoparasiticides
Food-safety studies for sheep ectoparasiticides involve many variables, including application technique, wool length at the time of treatment, animal type and environmental conditions. However, the studies should result in tissue residues as high as could be expected when the product is used according to label directions (the maximum residue scenario). Residues in wool may also need to be considered.
For plunge and shower dips, you should:
- use fine- or medium-wool merinos in store condition to achieve a maximum residue scenario
- dip sheep four to six weeks after shearing
- thoroughly mix plunge dips and spray dip tanks prior to use
- make sure the trial sheep are the first animals to pass through the dip
- take samples of the dip wash from the dip tanks immediately before and after treatment of the trial animals; determine and report the pesticide concentration
- make sure the dip concentration used during the trial approximates the maximum concentration expected under any top-up or reinforcement system of dip solution replenishment
- achieve 100 per cent wetting of the sheep to the skin. Common dry spots include behind the ears, around the neck and along the backline for plunge dips and under the neck and along the flanks from the shoulder to the hip for shower dips. Report wetting at the common dry spots for 10 sheep from each treatment
- report dip design and dipping procedure.
For jetting trials, you should:
- use fine- or medium-wool merinos in store condition to achieve a maximum residue scenario
- aim to achieve complete wetting to the skin over the target area
- describe the equipment and procedure used
- only use hand jetting
- report wetting to skin level under the treated area for 10 sheep from each treatment.
For off-shears treatments, short-wool and long-wool pour-ons, backliners and spray-ons, you should:
- use both a combination of fine- to medium-wool merinos (in store condition) and second-cross lambs
- calibrate the application equipment just before use in the trial
- unless otherwise recommended by label directions, apply off-shears treatments within 24 hours of shearing
- perform trials using sheep with six months’ wool growth to demonstrate the worst case for residues.
For wound dressings, treatment of fly strike commonly involves the application of the product to open wounds, increasing the likelihood of systemic absorption. In this circumstance, dosing of healthy sheep is not appropriate and there are significant animal welfare issues associated with the use of fly-struck sheep. Consequently, if the product is also to be registered for use as a jetting or dipping solution, specific data relating to its use as a wound dressing may not be necessary. If use as a wound dressing is the only use proposed for the product, you should conduct residues trials with the proposed product.
Within Australia, poultry may be farmed using a cage system, a free range system, or a barn or deep-litter system. The management and husbandry practices employed in each system will influence the way chemicals and drugs are administered, and the way any resulting residues are managed. The design of food-safety studies for veterinary chemicals used in the production of poultry should consider the type of poultry farm, the intended function of the birds, the developmental stage of the birds at the time of treatment and the route of treatment or medication.
The question of whether poultry are to be used as broilers, breeders or layers is fundamental to the design of residue trials, as it will determine the samples collected for residues analysis. Current industry practices should also be considered. For instance, both tissue and residues data should be generated from treated breeder birds if birds or eggs that are culled from the breeding programs are to enter the food chain. Residues data from the first eggs collected for human consumption should be provided for products used to treat pre-lay replacement pullets after sexual maturity (more than 12 weeks of age). Similarly, residues-decline data in eggs should be provided for treatment of pullets at the point of lay (that is, around 17 weeks), or for treatment of adult layers.
The method of administration, formulation type and extent of activity of a product will determine the design for food-safety studies. However, studies should endeavour to address the worst-case residue scenario, that is to say the maximum consumption or dose of medication. The following paragraphs detail some factors to be considered for specific routes of treatment or medication.
- Medicated drinking water: Administration via the drinking water is often preferable because domestic poultry will drink when they will not eat. However, fluid intake by the birds may vary due to the weather, to the ease of access or hygiene of drinking water dispensers, or to the unpalatability of the medicated water. Consequently, you should report the quantity of medicated water consumed by birds during residue trials, to determine the actual dosage received. You should also give consideration to the composition of the watering system or containers and the quality of the actual water. Galvanised metal may result in chelation of the drug with metal ions. Other materials may lead to adsorption of the drug onto the container surface. Chelation of the drug may also occur when ‘hard’ water is used to prepare the medication, and the use of chlorinated or otherwise sanitised water may destroy the medication. Each of these factors may result in a reduction of the intended dose delivered to the birds. Therefore, when the drug is to be administered in the water, samples of the medicated water should be collected and analysed to confirm the drug concentration.
- Feed additives: Feed additives, incorporated into pellets, crumble or mash, are commonly used in the mass medication of poultry, particularly when the physicochemical properties of the drug make it insoluble in water. Variations in feed consumption are associated with hot or cold weather; housing changes; breed, type, strain and age of bird; body weight; rate of lay; energy and fibre content of feed, and particle size of feed ingredients. These factors may alter the level of feed consumption by 10–20 per cent, thereby altering the effective dose rate (in the feed) by an equivalent percentage. Therefore, it is important that the quantity of medicated feed consumed by birds during residue trials be reported, to enable determination of the actual dosage received. It should also be noted that absorption of the drug by treated birds may be unpredictable because it may bind to feed ingredients. Also, the milling process may affect the stability of the drug, as pelleted feeds may be subjected to high temperatures, which may cause breakdown of the active constituents. Therefore, it is important to conduct residue studies using the predominant feed form (pellet, crumble or mash), and to analyse samples of the medicated feed for their drug content after milling or processing.
When designing a food-safety study for determining residues in honey, one trial involving a representative floral type should be conducted under conditions of good honey flow.
You should collect pre-treatment samples of honey from all hives from the brood nest and the super, and store and analyse them individually to determine if the test chemical is present. You should conduct and report cleaning validation. For example, after use, the extractor, uncapping knife, honey tank, bucket and the pipe from the extractor should be thoroughly cleaned using a high-pressure water cleaner, then washed with hot water, and finally dried. The honey from eight frames of an untreated control hive should then be extracted and the honey that drains from the extractor collected and poured over the internal walls of the previously-cleaned holding tank. A sample of this honey should be collected for assay to confirm that the equipment is free of any residual chemicals.
3.2.6. Aquatic animals
Within the aquaculture industry, withholding periods are typically expressed in terms of ‘degree days’—for example, a withholding period of 500 degree days is equivalent to 50 days at 10 °C or 25 days at 20 °C. This approach is used because water temperature may affect the rates of growth and residue depletion in the treated species. Consequently, you should record water temperature throughout all residue-decline trials conducted with aquatic species.
3.3. Animals and animal husbandry
3.3.1. General guideline
Generally, one food-safety study should be performed in pigs, sheep and poultry. For cattle, a single study in beef cattle could be applied to dairy cattle (or vice versa). However, because of differences in ruminant and pre-ruminant physiology, separate studies are recommended when the target species encompasses both adult and pre-ruminating animals. You should also do a separate study to demonstrate the residue-depletion profile in milk of dairy animals or in eggs produced by laying hens.
The food-safety study should take into account all factors that may contribute to the variability of residue levels in animal commodities. Factors such as animal breeds and physical maturity should be considered within the pool of animals included in the study without warranting an increase in the number of animals.
Animals should be healthy, identified individually and should not have been previously medicated. However, it is recognised that animals might have received biological vaccinations or prior treatment; for example, with anthelmintics. In the latter case, an appropriate wash-out time should be observed for the animals prior to enrolment in the actual study. Study animals should be representative of the commercial breeds and representative of the target animal population that will be treated. You should report on the sources of the animals, their weights, breeds, health statuses, ages and sexes.
Animals should be selected such that their age and body condition will not cause underestimation of residues. For example, residues of a fat-soluble compound could be diluted by applying the product to fat animals, or unduly concentrated by application to very lean animals. Coat length and body surface area to body-mass ratios are other factors that may require consideration depending on the product. Store condition animals are preferred.
Animals should be allowed adequate time to acclimatise and normal husbandry practices should be applied where possible. The feed and water supplied to the animals should be free from other drugs or contaminants and you should ensure adequate environmental conditions that are consistent with animal welfare requirements.
3.3.2. Food-safety studies for dairy commodities
After treatment, all dairy animals should be kept together according to the normal husbandry practices on the farm, except where the test treatment is externally applied to only part of the herd. In this case, the treated animals should be physically separated from the remainder of the herd. This is to ensure that licking and other direct contact does not affect absorption and subsequent excretion into the milk, and to prevent contamination of the remainder of the herd.
For studies using intra-mammary products, all animals should have visibly healthy udders, free from the effects of chronic mastitis. For dry-cow studies, pregnant animals with a predicted parturition date should be introduced into the study facility well in advance of study enrolment.
3.3.3. Food-safety studies for poultry commodities
The birds used in food-safety studies should be healthy, and sourced from contemporary, commercial genotypes. No concomitant drug therapy should be used on any bird or pen during the study. Animal housing, feeding and care should follow recommendations for welfare, including vaccination according to industry practices. The housing conditions for birds (that is, pen areas, stocking densities) should reflect commercial practices in the geographical location of the study, and take into consideration seasonal variations in temperature and the weight of the birds. Likewise, the number of birds per feeder or waterer should reflect commercial practices.
3.3.4. Food-safety studies in bees
The colonies used in food-safety trials should be standardised in terms of adult bee populations and brood. The hives should be uniquely identified and allocated to a treatment group; untreated control hives are not necessary. The hives are typically arranged in a row–column configuration and should be located to mimic the conditions found at the time of the year when apiarists would normally treat hives with the particular chemical.
3.3.5. Food-safety studies in aquatic species
Animals should be representative of the commercial species and of the target animal population that will be treated. You should report on the sources of the animals, their weights, health statuses, and their ages or developmental stages. The body-weight ranges should be consistent with the product label and appropriate for the proposed use. For example, if the product is intended for market-sized fish then you should use market-sized fish.
The table below classifies fish into groups based on taxonomic order. An ‘all fish’ claim could be obtained by conducting one study in each of the groups.
|Salmoniformes*||Coho salmon (Oncorhynchus kisutch); Rainbow trout (Oncorhynchus mykiss)|
|Cypriniformes||Carp (Cyprinus carpio)|
|Perciformes||Yellowtail (Seriola quinqueradiata)|
|Scorpaeniformes||Mebaru or rockfish (Sebastes inermis, Sebastes cheni, Sebastes ventricosus)|
|Silurformes||Channel catfish (Ictalurus punctatus)|
|Osmeriformes||Ayu or sweet fish (Plecoglossus altivelis)|
|Anguilliformes||Eel (Anguilla japonica)|
|Pleuronectiformes||Bastard halibut (Paralichthys olivaceus); Summer flounder (Paralichthys dentatus)|
|Tetraodontiformes||Japanese pufferfish (Takifugu rubripes)|
* Salmoniformes provide the worst-case scenario, as rearing conditions are the minimum water temperature across this group of fish. Therefore, you should conduct one residue depletion study performed at the minimum water temperature in Salmoniformes to gain approval for all the fish orders in this group.
You should conduct studies in an environment that mimics commercial conditions such as water flow and exchange rates, water temperature, water quality and light. The water quality should be the same quality and quantity that is appropriate for the animal’s developmental stage, and the water temperature should be recorded daily. The housing should be representative of commercial growing conditions. If more than one housing condition is used commercially, you should select the housing condition that results in maximum tissue residues for the study. Animals should be fed both the quality and quantity of food appropriate for their stage of development and to ensure adequate nutrition and growth, as seen in commercial conditions.
3.4. Number of animals
3.4.1. General guidance
You should use a large enough number of animals to allow a meaningful assessment of the data. From a statistical point of view, residue data from a minimum of 16 animals, with four animals being sacrificed at four appropriately distributed time intervals, are recommended. Larger numbers of animals should be considered if the biological variability is anticipated to be substantial, as the increased numbers may result in a better-defined withdrawal period. Control (non-treated) animals are not necessary as part of the actual study; however, sufficient amounts of control matrices should be available to test related analytical methods. Generally, for food-safety studies, at least four animals evenly mixed as per sex, for each sacrifice time, are recommended. These animals should be representative of the animals to be treated. Typical bodyweight ranges are around 40 to 80 kilograms for pigs, around 40 to 60 kilograms for sheep and around 250 to 500 kilograms for beef cattle. Non-lactating dairy cows could also be used for food-safety (tissue) studies.
3.5. Food-safety studies for dairy commodities—milk
For lactating animal studies, at least 20 animals, randomly selected from a herd where all lactation stages are represented, are recommended. High-yielding animals at an early lactation stage and low-yielding animals at a late lactation stage should be included in the group. The trial should take into account relevant factors such as the fat content of the milk and the volume of production. Production levels should be representative of those reported for Australia. You should include details of the animals’ breeds, weights, ages, pregnancy statuses, stages of lactation, milk production, fat content of milk and dates of calving.
For dry-cow (pre-parturition) studies, a minimum of 20 animals is recommended. The study should include randomly selected cows representative of commercial dairy practices. The veterinary chemical should be administered after the last milking (dry-off) and at a time consistent with the proposed treatment-to-calving interval. To reduce variability in the residues data, the study should be designed so that animals calve within two thirds to one hundred percent of the proposed treatment-to-calving interval. For example, for a treatment-to-calving interval of 49 days, residues data should be collected from animals giving birth between 33–49 days after treatment. For a pre-calving treatment interval of 56 days, residues data should be collected for at least 20 cows calving between 37–56 days after treatment.
3.6. Food-safety studies for poultry commodities—edible tissues and eggs
For food-safety (tissue) studies, a sufficient number of birds should be used to obtain at least six tissue samples at each sampling time. The actual numbers of birds used in each trial will depend on the type of residue trial being undertaken and whether pooling tissues from a number of birds (to form composite samples) is necessary to obtain adequate material for analysis (due to the immaturity of the bird, or small organ size). Usually, birds are sacrificed at zero day withdrawal and at three or more later points in time.
For food-safety (egg) studies, a sufficient number of birds should be used to collect ten or more eggs at each sampling time. Eggs from birds within a dosage group may be pooled, if necessary, to form composite samples so that adequate sample weight is available for analysis and retained samples. For composite egg samples, ten groups of laying birds should be treated (each group should contain sufficient birds for at least three eggs per group per day). You should record the numbers and weights of eggs per group per day.
3.7. Food-safety studies for bee commodities—honey
It is recommended that a minimum of five hives are used for each treatment at each sampling time. This will facilitate the extraction of honey from an entire super at each sampling point, thereby overcoming difficulties that would otherwise be associated with the variation within a super of honey and chemical residues that may occur in the individual combs. Harvesting honey in this manner also mimics commercial practice.
3.8. Food-safety studies for commodities from aquatic animals
Residue data from a minimum of 10 aquatic animals per time point is recommended. Small fish, crustaceans or Mollusca may require a composite sample of multiple animals. In cases where a composite is necessary, sufficient numbers of animals should be collected in order to facilitate assessment of the marker residue. It is recommended that animals should be sacrificed at four appropriately distributed time intervals.
3.9. Dosing and route of administration
3.9.1. General guidance
You should administer the highest intended treatment dose for the maximum intended duration. It is advised that you consider repeat treatments at the highest application rate, with the shortest interval and maximum number of treatments. Separate studies are appropriate for each formulation type and for each mode of treatment.
If an extended drug administration period is intended, duration of treatment sufficient to reach a steady state in target tissue(s) can be used instead of the full length of the treatment. However, a steady state in all tissues should be confirmed. The time-to-steady-state data are often obtained as part of the total residue study. Where daily or continuous exposure are involved, sampling should show when residues plateau in tissues and milk. You should take samples at an adequate number of time points (3 or 4 generally) at the plateau concentration. This allows the maximum concentration in tissues and milk to be established prior to the commencement of depletion.
3.9.2. Injectable products
Animal treatment should be consistent with the maximum treatment regime specified on the product label, including the volume, location and injection method. For example, if the label specifies 4–6 treatments and a maximum injection volume of 10 millilitres, you should conduct the study using six treatments and administer the maximum volume at the injection site that will be sampled. If the label specifies a dosage range, you should conduct the study using the highest dosage level. For multiple treatments, you should give the injections alternately between left and right sides of the animal. As the maximum injection volume needs to be considered, trials designed specifically for determination of injection site residue depletion are advisable.
Where the withdrawal period will clearly be determined by residue depletion at the site of injection, you generally have the option of collecting data from two injection sites per animal (and using the data from both sites to determine the withdrawal time). This practice can have a positive impact on study design with respect to animal welfare by reducing animal numbers.
An example of this approach is as follows: For a product that utilises only a single injection, treatment can be given on the right side of the neck on day zero and then on the left side of the neck on day four. Euthanasia on day 7 following the final treatment would provide depletion data at seven days’ (left injection site) and 11 days’ (right injection site) withdrawal. In this case, however, collection and assay of the other tissues would not be warranted since the product was administered contrary to the label (two injections rather than one injection) and as a result residues could be excessively elevated. Such a dosing regimen is designed specifically to determine injection site residue depletion.
If the drug product is intended to be administered via more than one parenteral route, you should provide a separate marker-residue depletion study for each route of administration. If the withdrawal period is clearly defined by depletion of residues from the injection site following subcutaneous or intramuscular dosing, a separate intravenous residue study (at the same dose) is not recommended provided the same withdrawal period (for subcutaneous or intramuscular dosing) can be applied to the intravenous route.
3.9.3. Intramammary products
Drug products intended for intra-mammary administration either for lactating animals or for pre-parturition (that is, dry-cow treatment) studies should be given to all quarters (that is, normally four quarters in bovines). It is unlikely that all quarters will be treated with an intra-mammary product during commercial practice; for residue studies this study design represents a worst-case scenario.
For pre-parturition (that is, dry-cow treatment) studies, the test article should be administered after the last milking (dry-off) and should be consistent with the desired pre-calving interval.
3.9.4. Ectoparasiticide products intended for cattle
Animals should receive the highest exposure to the pesticide allowed by the proposed label. This means the longest exposure time (maximum time in the dip or spray, 'thorough coverage'); the maximum amount of material per animal (pour-ons, back-line treatments, dusts); or free access of animals to the material, plus correct placement and recharging (back-rubbers and dust-bags).
The dosing should consider:
- the shortest recommended interval between treatments
- the maximum number of re-treatments in the season per year
- the maximum label dose rate.
Products applied without dilution should have an active concentration at or near the top end of product specification. Concentrations in dips should be at the maximum permissible concentration in relation to directions for use on the label. This concentration should be maintained for the trial. The stirrer group should be retained for re-dipping, and animals should be analysed as part of the trial. Due account needs to be taken, on recharging dips and sprays, nozzle types, and pressure and delivery rate of sprays.
You should make an effort to ensure that climatic conditions (for example, rain periods) do not compromise the maximum treatment regime. Rainfall occurring throughout the trial should be reported, as it may confound trial results.
3.9.5. Ectoparasiticide products intended for sheep
Animals should be dosed at the maximum label dose rate, with the maximum number of treatments applied at the minimum treatment interval in accordance with label directions. If you use a table specifying doses for defined live-weight groupings, the highest label rate is usually the rate applicable to the animal with the lightest body weight identified in the table. In some instances, the highest rate may be the rate applicable to suckling lambs. Trial sheep should be dosed with the quantity of product required for the heaviest sheep in the group. For dose rates based on time since shearing, the highest dose rate allowed on the label (usually that for the longest wool) should be used.
Application of these treatments should accurately address label directions using the prescribed application equipment and should follow recommended industry practice. The application equipment should be calibrated just prior to use in the trial and, unless otherwise recommended by label directions, off-shears treatments should be applied within 24 hours of shearing.
3.9.6. Bee products
The residue trial design should address the maximum treatment regimen (that is, the maximum dose rate and the maximum number of treatments at the minimum re-treatment interval). The method of applying the chemical in the trial should be identical to the intended commercial use. Chemicals are typically applied to beehives in sugar syrup (wet treatment), caster sugar (dry treatment), or as pest strips suspended in the brood nest. You may need to apply the chemical prior to the commencement of the main honey flow.
3.9.7. Products intended for aquatic species
Studies should address all modes of application and dosing of the veterinary chemical. These may include adding a solution of chemical directly to the water or in feed. You should use the highest dose per application rate that is likely to be used commercially and that is to be listed on the product label. The animals should consume the medicated feed within a short period of feeding so that the product does not leach into the water. To minimise the possibility of under dosing, all the medicated feed should be eaten. Where several treatments are likely, you should use the highest number of applications, with the shortest interval between successive applications.
3.10. Animal sacrifice
Animals should be euthanised using commercially applicable procedures and observing appropriate exsanguination times. Chemical euthanasia can be used unless it will interfere with the analysis of the marker residue.
3.11.1. General considerations
Following euthanasia, tissue samples in sufficient amounts 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 bear the responsibility for demonstrating residue stability through the time of assay.
The normal tissues collected for analysis are muscle, fat, liver and kidney. If metabolism studies show that the chemical has a predisposition for another edible tissue, that tissue should also be sampled. Table 2 indicates the recommended samples for collection.
|Edible tissue||Sample description by species|
|Cattle or sheep||Pigs||Poultry|
|Injection site muscle||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 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|
|Skin or 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
For formulations that are able to leave local residues such as dermal pour-on products, samples of relevant tissues (for example, muscle, subcutaneous fat or skin or fat from the application site) should be collected for analysis in addition to those specified in Table 2.
One additional tissue from Table 3 may be selected for residue assay if the results from the metabolism studies indicate that this would be appropriate. This would typically be the additional tissue with the highest residues or the slowest depletion rate. Only one additional tissue is recommended. For example, if the total-residue study indicates that cattle heart has the slowest depletion rate, heart should be selected for assay in the marker-residue depletion study, but cattle small-intestine marker-residue data are not recommended.
|Edible tissue||Sample description by species|
|Cattle or sheep||Pigs||Poultry|
|Small intestine||Composite, rinsed of content||Composite, rinsed of content||N/A|
3.11.2. Injection sites
For parenteral preparations, residue-depletion data from the injection site(s) should be included. Injection site residues are local residues that may or may not remain localised at the site of administration. As such, it is important for you to develop appropriate quality-control sampling procedures that ensure the collected tissue actually encompasses the injection site. Any approach you take should be justified on a case-by-case basis, taking into account the data available and the formulation characteristics. The following methodologies may be considered (regardless of the option selected, the primary core sample should target 500 grams plus or minus 20 per cent):
- Collection of an additional ring sample (300 grams plus or minus 20 per cent) around the primary core sample (500 grams plus or minus 20 per cent). These tissue amounts would generally not apply to small animals that do not allow sampling of 500 grams. For these situations, the optimum sampling strategy should be defined on a case-by-case basis and should be justified. However, collection of two samples (a core and surrounding sample) remains appropriate.
- Collection of an elliptical (or other appropriate shape) sample along the injection track and/or the site of irritation. You should provide evidence that this method correctly targets the injection site residues, such as with accompanying photographs of the site(s) of sampling.
Samples should be collected from the last injection site (or sites), as appropriate. In the case of products requiring multiple injections, the study design should be such that the last injection site will occur on the side of the animal receiving the higher number of injections. When a circular core sample is indicated, collection of the injection site muscle tissue (from large animals) should be centered on the point of injection and consistent with the recommendations shown in Table 2. For injections placed into the base (bottom third) of the ear, collection of neck muscle on the side of the injection, plus cheek muscle and tongue, should be considered.
When sampling, all four udder quarters should be milked out, the whole sample mixed thoroughly, and a sub-sample taken. You should determine the fat content of all milk samples analysed to demonstrate the validity of sampling. The time points for sample collection can be suggested by metabolism data. The morning and afternoon samples taken on the same day should be handled and analysed separately (that is, not combined). Milk samples from different animals should not be pooled. Residue analysis should be conducted and reported on whole milk. Records of milk volume and butterfat should be submitted with trial data for the individual cows for the period before and during the collection period.
For multiple-dosed products used in dairy animals, samples should be taken after the last treatment. For products that might qualify for zero-day withdrawal, samples should also be collected during treatment. Duplicate milk samples (200 millilitres each for residue and fat determinations, respectively) should be collected from individual animals:
- at the morning or afternoon milking on the day before treatment, and
- at the morning and afternoon milking on sufficient days after treatment to show the peak and decline of the residues below the appropriate reference point (for example, maximum residue limit or limit of quantitation) as determined by the chemical properties of the drug product.
The partitioning of parasiticides and other veterinary drugs between the aqueous phase and milk fat of whole milk should be considered as the degree of partitioning will determine the importance of the residue concentration in processed milk products such as butter, ice cream and cheese. A partitioning study, involving a minimum of two samples, is best accomplished on samples where residues in milk are at a maximum.
Egg samples should be obtained from 10 or more laying hens at every laying time point during the medication period and after the final medication. Egg samples should be collected after the period necessary to complete egg yolk development, which is usually up to 12 days. Egg white and yolk should be combined for analysis.
Honey from different hives in the same treatment group should be bulked for analysis to reflect the fact that honey is a bulked commodity. All samples of honey should be at least 100 grams. When honey is removed from hives, supers of honey combs from each treatment should be kept together, extracted and bulked in one batch. A portion of the honey from each treatment group should be collected in a uniquely identified pail for future sampling and analysis.
Cross-contamination of honey samples from different treatment groups should not occur and will invalidate the trial results. To prevent contamination from occurring during the extraction process, the extraction plant can be modified to allow honey to be bucketed from the extractor outlet rather than have it run through the pumps and lines.
3.11.6. Aquatic species
Separate samples should be collected from individual animals. If the amount of sample collected from one animal is not sufficient for the assay of marker residue, composite samples from multiple animals may be acceptable.
|Aqualculture species||Edible tissue type|
|Fish||Muscle including skin in natural proportions (that is the entire fillet with the overlying skin, without scales, in natural proportions from one side of the fish). The entire sample should be collected, homogenised and subsamples collected from the homogenate.|
|Crustaceans||Muscle excluding shell. For species marketed as ‘soft shell’, the entire animal including the unhardened shell is considered as edible tissue. The edible tissue for shrimp includes the mid-intestinal gland.|
|Mollusca||Whole animal after removal of the shell|
3.12. Analytical method for assay of marker residue
You should submit a validated analytical method to determine the marker residue in samples generated from the residue-depletion studies in the edible tissues and where applicable, in milk and eggs. The method(s) should be capable of reliably determining concentrations of marker residue which encompass the appropriate reference point (that is, maximum residue limit) for the respective tissues or products.
For further information on method validation refer to the guideline Analytical methodology.
4. Reporting of data—outcomes of the food-safety studies assessment
Assessment will involve review of the residue-decline data in the target animal species. Where maximum residue limits need to be determined, these limits should to be set using the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives maximum-residue-limit-setting approach (as adopted by the APVMA on 1 July 2006).
4.1. Maximum residue limit determination
The residues-decline data for each edible tissue will be subjected to a regression analysis and the maximum residue limit is determined as a point on the curve describing the upper side 95 per cent confidence limit of the 95th percentile. However, the determination of the maximum residue limit is an iterative process. It involves calculation of the daily dietary exposure levels using the theoretical maximum daily intake except in cases where the use of the estimated daily intake has been shown to be the appropriate approach. This is then reconciled with the health standard (acceptable daily intake). Where the total amount of residues consumed exceeds the acceptable daily intake, further iterations are performed with lower maximum residue limit proposals.
4.2. Withholding period determination
The residue-decline data should enable an appropriate withholding period to be determined that will ensure that the concentration of residues will be below the recommended or established maximum residue limits. The APVMA endeavours to use evidenced-based statistical analyses to establish withhold periods wherever possible. Therefore, the withholding period is determined to be the time point when the residues observed in the food-safety study, using the upper side 95 per cent confidence limit of the 95th percentile, are below the established maximum residue limits in all edible tissues.