MARKER RESIDUE DEPLETION STUDIES TO ESTABLISH VETERINARY DRUG WITHDRAWAL PERIODS
Revision: April 2013
TECHNICAL GUIDELINE TO CONDUCT METABOLISM AND RESIDUE KINETIC STUDIES OF VETERINARY PHARMACOLOGICAL AGENTS IN FOOD-PRODUCING ANIMALS
MARKER RESIDUE DEPLETION STUDIES TO ESTABLISH VETERINARY DRUG WITHDRAWAL PERIODS
Table of contents
1. Introduction 6
1.1 Objective of guideline 6
1.2. Background 6
2. Scope 6
3. Glossary 7
4. Marker residue depletion studies 7
4.1. Investigational veterinary drug 9
4.2. Animals and animal husbandry 9
4.2.1. Intramammary studies 10
4.2.2. Other parameters 10
4.3. Number of animals for the study 10
4.3.1. Ruminants, pigs and horses for tissue residue studies 10
4.3.2. Dairy animals for milk residue studies 10
4.3.3. Poultry 11
4.4. Route of administration 11
4.4.1. General guidance 11
4.4.2. Considerations for veterinary drugs intended for multiple routes of administration 11
4.4.3. Recommendations for the application of a spray veterinary drug 11
4.5. Animal euthanasia 12
4.6. Sampling 12
4.6.1. General considerations 12
4.6.2. Injection sites 14
4.6.3. Other considerations 15
4.6.4. Milk sampling 16
4.6.5. Egg sampling 16
5. Recommendations for veterinary drugs proposed for 0-day withdrawal periods 16
6. Withdrawal period confirmation studies 17
6.1. Criteria to determine the need of a residue study or a withdrawal period confirmation study. 17
6.2. Interpretation of results in withdrawal period confirmation studies 17
6.3. Study design 18
7. Analytical method for marker residue assays 18
As part of the approval process for veterinary medicinal products in food-producing animals, regulatory authorities require data from marker residue depletion studies in order to establish appropriate withdrawal periods in edible products, including meat, milk, eggs and honey. The objective of this guidance is to provide study design recommendations which will facilitate the universal acceptance of the generated residue depletion data to fulfill this requirement.
General recommendations here established apply for most situations in which a period of use restriction must be determined. Nevertheless, it should be born in mind that there may be situations in which these recommendations may result inadequate or not applicable. In those cases, the health authority will evaluate the specific proposed designs as long as they are appropriately justified.
1.1. Objective of the guidance
This document, based on recognized previous works, aims at proposing protocols for the conduction of residue depletion studies to establish withdrawal periods of veterinary drugs. These protocols have been adapted to meet the characteristics of the reality of the country members of CAMEVET and could be useful for other countries.
VICH GL 48 (November 2009) – Marker Residue Depletion Studies.
EMEA/CVMP/036/95-Final (January 1997). Approach Towards Harmonization of Withdrawal Periods.
In order to register a veterinary drug intended for food-producing animals, marker residue depletion studies are recommended in the target species to:
• Demonstrate the depletion of the marker residue upon cessation of drug treatment to the regulatory safe level (e.g. maximum residues limit [MRL] or tolerance).
• Generate suitable data for the elaboration of appropriate withdrawal periods to warranty food safety.
The idea is that one residue depletion study (per species), conducted within any global region, be sufficient to satisfy the data requirements for the determination of appropriate withdrawal periods for a specific product in food-producing animals. The guideline encompasses the most common species, namely cattle, pig, sheep and chicken; however, the principles of this guideline allow sufficient flexibility for application to related species not mentioned in this core group (e.g. cattle vs. all ruminants, chickens vs. all poultry).
This guideline does not include considerations regarding aquiculture nor apiculture products.
Studies should be conducted in conformity with the applicable principles of Good Laboratory Practice (GLP) and Good Clinical Practices (GCP).
Standard food basket: It is an estimate of the total amount of food of animal origin consumed daily by an adult weighting 60 kg. The basic food basket employs arbitrary figures of consumption, based on percentiles higher than the daily intake of products of animal origin.
The figures of daily consumption of food of animal origin are:
300 g. of muscle, 50 g. of fat or fat and skin, 100 g. of liver and 50 g. of kidney.
300 g. of muscle, 90 g. of fat or fat and skin, 100 g. of liver and 10 g. of kidney.
300 g. of muscle and skin in natural proportions.
Also an intake of 1.5 l. of milk, 100 g. of eggs and 20 g. of honey are considered.
The estimate of the risk of consumption of present residues in a food basket is calculated taking into account the ADI.
Active Pharmaceutical Ingredient –Drug substance (API): Any substance intended to cure, mitigate, treat, prevent or diagnose diseases in humans or animals.
Acceptable Daily Intake (ADI): It is the estimate of the residue, expressed in terms of weight per kilogram of body weight (kg/bw) that can be daily consumed during consumer´s whole life without noticeable risks to his health.
Confidence Interval: Range of values in which the value of a population parameter is expected to be found with certain degree of certainty.
Maximum Residue Limit (MRL): Maximum concentration of residues founded in an animal product or by product that not introduce any risk for the consumer safety, based on known facts at the time of its publication (CAMEVET definition).
Limit of Quantitation (LOQ): It is the smallest concentration of an analyte that can be quantified with a specific degree of accuracy and precision, within statistically defined limits.
Limit of Detection (LOD): It is the smallest concentration of an analyte from which it is possible to deduce the presence of the analyte in the test sample but it is not possible to quantify it, within statistically defined limits.
Tolerance limits: The extreme values of a series of values (interval) within which a determined percentage of the individuals of a determined population is expected to be found with certain degree of certainty.
Veterinary drug: Any chemical, biological, biotechnological substance or manufactured (elaborated) preparation administered either individually or collectively, directly or mixed with food, aimed at the prevention, cure or treatment of animal diseases.
Safety Period – Use restriction- Withdrawal Period – Elimination Period: Minimum period of time that must happen between the last application of a veterinary medicinal product to an animal, in the normal conditions of use, and the extraction of food products from that animal, to warranty that such food products do not contain residues in quantities that exceed the established maximum limits.
Veterinary Product (as defined by CAMEVET): A veterinary product is any chemical, biological, biotechnological substance or manufactured preparation administered either individually or collectively, directly or mixed with food or water, aimed at the prevention, cure or treatment of animal diseases, including additives, supplements, promoters, animal production enhancers, antiseptics, disinfectants for environmental use or for devices and ectoparasiticides and any other product that, used in animals and their habitat, protects, restores or modifies the organic and biological functions. It also encompasses products destined to enhancing animals’ beauty.
Marker Residue: It is an analyte that is reliable to determine the presence of residues of a specific drug in the tissue. The marker residue can be a mother cell or any of its metabolites, degradation products or a combination of any of them. The marker residue can also be a chemical derivate of one or various components of the residue. The relationship between the marker residue and the concentration of the residues of interest in edible tissues must be known (marker residue/ residue of interest). The MRL reflects the maximum allowed concentration of the marker residue in the edible tissues.
Total Residue: The total residue of a drug in animal derived food consists of the parent drug together with all the metabolites and drug based products that remain in the food after administration of the drug to food producing animals.
The total sum of the residues normally includes all parent residues of the pharmacological agent (parent drug and its metabolites), and in most cases it is identical to the sum of residues determined by radiometric tissue depletion studies.
Residues of toxicological relevance: For the estimate of an exposure based on a toxicological ADI, the residue of relevance is the residue of toxicological relevance. This normally includes all the parent compounds and the molecule (parent drug and the metabolites) and in most cases it is identical to the sum of residues determined by radiometric tissue depletion studies. However, if a component of the residue or a fraction of the total residues is demonstrated to be toxicologically inactive, it is possible to deduct it from the total residue or any other fraction of residues that is not bioavailable by oral route or the metabolites known to be toxicologically inactive.
Residues of pharmacological relevance: For the estimate of an exposure based on a pharmacological ADI, the residue of relevance is the residue of pharmacological relevance. In general, the mother cell plus any other of its residues are considered. If there is lack of data on pharmacological activity of the total residue components, the total residues are presumed to present the same pharmacological activity as the parent drug.
Residues of microbiological relevance: For the estimate of an exposure based on a microbiological ADI, the residue of relevance is the residue of microbiological relevance. In most cases, it is identical to the residues determined in microbiological assays. In case of lack of such data, the total residue can be used, or alternatively the sum of the individual components known to present microbiological activity. Therefore, the microbiological activity of the total residue or of the metabolites and/or products of degradation is the same as the parent drug.
Injection site: It is the area of tissue where the veterinary medicinal product has been injected. The samples of tissue obtained from the injection sites for the conduction of residue studies should be representative of the edible tissue that is feasible of being selected in the slaughter procedures. The tissue sample should include muscular tissue, connective tissue and subcutaneous fat in natural proportions (cutting off the samples to eliminate the connective tissue and the fat attached to muscle are considered artificial procedures that differ from the real situation). Injection site should not include the skin portion that covers it, since it is not required for the residues analysis.
Tissue: Any edible animal tissue, including muscles and sub-products (Definitions established and adopted by the Joint FAO/WHO Expert Committee on Food Additives – JECFA).
4. Marker residue depletion studies
4.1. Investigational veterinary drug
The investigational veterinary drug used must be representative of the commercial formulation. Preferably, it should come from material elaborated with the use of good manufacturing practices (GMP) (at pilot and commercial scale); however, duly documented preparations at laboratory scale are also acceptable.
4.2. Animals and animal husbandry
In general, a single marker residue depletion study (in tissues) can be conducted in pigs, horses and poultry. In the case of ruminants, a single study can be applied for meat and milk-producing animals. However, due to differences in the physiology of ruminants and pre-ruminants, separate studies are recommended when target species include both adults and pre-ruminants. A separate study should be performed to demonstrate the residue depletion profile in milk of dairy animals or in eggs produced by laying hen.
Animals should be healthy and, preferably, should not have been previously medicated. However, it is recognized that animals might have received vaccinations or prior treatment, for example with anthelmintics. In the latter case, an appropriate wash-out period should be observed for the animals prior to enrollment in the actual trial. Study animals should be representative of the commercial breeds and of the target animal population that will be treated. The source of the animals, their weights, health status, ages and sex should be informed.
Animals should be allowed adequate time to acclimatize to the trial conditions, and good clinical practices should be applied. The feed and water supplied to the animals should be free from other drugs and/or contaminants and adequate environmental conditions should be ensured, in accordance with animal welfare practices.
4.2.1. Intramammary studies
For studies with veterinary products of intramammary application, all animals should have healthy udders, free from the effects of mastitis. For pre-parturition studies, pregnant animals with a predicted parturition date should be introduced in study facilities in advance of study enrollment.
4.2.2. Other parameters
All factors that may contribute to the variability of the residue levels in the animal products should be taken into account in the planning and conduct of trials. The intent is that these factors (e.g. animal breeds, physical maturity) be considered without increasing the number of animals recommended in 4.3. For example, if a milk residue depletion study recommends 20 animals, any factor determining variability should be represented within the 20 initially selected animals (20 more animals representing “other factors” should not be added).
4.3. Number of animals for the study
The number of animals used should be large enough to allow a statistically significant assessment of data. From a statistical point of view, for meat residue studies, data from a minimum of 16 animals should be collected: 4 animals euthanized at 4 appropriately distributed time intervals. A larger number of animals can be considered if the biological variability is anticipated to be substantial, as an increase in the number can contribute to determine more precisely the withdrawal period. Control (non-treated) animals are not necessarily called for as part of the actual marker residue depletion study; however, sufficient amounts of target tissue should be available for the preparation of matrices in the assessment of related analytical methods. The following section provides general recommendations on the number of animals to be included in the study design.
4.3.1. Ruminants, pigs and horses for tissue residue studies
At least 4 (evenly mixed as per sex) per each slaughter time are recommended. The body weight should be consistent with the class for which the veterinary drug is indicated.
Según lo expuesto en la sección 4.2, las vacas lecheras también pueden ser utilizadas para estos estudios de residuos en tejidos.Following section 4.2., milking cows can also be used for these tissue residue studies.
4.3.2. Dairy animals for milk residue studies
For lactating studies, at least 20 animals, randomly selected from a herd where all lactating stages are represented, are recommended. High yielding animals at an early lactation stage and low yielding animals at a late lactating stage should be included.
For pre-parturition and dry cow therapy studies, a minimum of 20 animals is recommended. The study should include randomly selected cows, representative of commercial dairy practices.
A sufficient number of birds should be used to obtain at least 6 samples at each slaughter time for tissue residue studies.
For egg residue studies, a sufficient number of birds should be used to collect 10 or more eggs at each interval time point.
4.4. Route of administration
4.4.1. General guidance
Animal treatment should be consistent with the dosage and indications intended for the investigational veterinary product and must include, for injectable products the location and injection method. For multiple treatments, injections should be given alternatively between left and right sides of the animal.
The highest intended treatment dose should be administered for the maximum intended duration. For extended treatments including various doses, if there is available data indicating that the API concentration reaches a steady stage (moment when the API concentration in the target tissue neither increases nor decreases because the absorption speed is the same as the depletion speed) before the end of treatment, sampling could start at that time. Veterinary products intended for intra-mammary administration should be given to all four quarters of each cow.
For dry cow and pre-parturition studies the test article should be administered after the last milking (dry-off) and observing the interval until calf birth (usually 60 days).
4.4.2. Considerations for veterinary drugs intended for multiple routes of administration
If the veterinary drug is intended to be administered via more than one parenteral route (intramuscular, subcutaneous or intravenous), a separate marker residue depletion study for each route of administration should be provided. Note: If the withdrawal period is clearly defined by depletion of residues from the injection site following subcutaneous (SC) or intramuscular (IM) dosing, a separate intravenous residue study (at the same dose) is not needed, provided the same withdrawal period than the one applied for IM or SC route can be applied for IV route.
4.4.3. Recommendations for the application of a spray veterinary drug:
Spraying veterinary products are widely used in many member countries, indicated for ectoparasites treatment. To establish the standard dose is a critical requirement and it has to be done before the beginning of a residue trial. The following protocol is recommended.
a- The spraying equipment should be filled with a previously measured amount of the veterinary drug, prepared and ready for application.
b- Once the backpack sprayer or spraying equipment is filled the system should be cleaned.
c- Start spraying the animal´s dorsal part, from head to tail and then back to cranial through the contiguous lower part, covering thus a route that ensures the correct drenching of the animal. This should be done in both sides of the animal, up to the dripping point and always avoiding product application at the animal`s eyes. The dripping point indicates that the animal has been correctly drenched, and that from that point on, all products applied will not stay on the animal, but it will drain to the floor. This is the parameter usually employed to treat animals in field conditions, and one of the most appropriate ones to reduce dosage variables in this kind of application.
d- If feasible, once the application is done, the remaining product should be collected from the sprayer in a measuring glass to calculate the applied volume. This information should be registered in the "treatment record”.
Loaded volume - Remaining volume = Applied volume
The applied volume should be correlated to the animal weight in order to accurately know the applied dose per animal in terms of total milligrams and milligrams per kilograms.
4.5. Animal euthanasia
Animals should be euthanized following OIE regulations on animal health, using when possible commercially applicable procedures, making certain to observe appropriate exsanguination times. Chemical euthanasia should be avoided.
4.6.1. General considerations
Following euthanasia, edible tissue samples in sufficient amounts should be collected, trimmed of extraneous tissue, weighed and divided into aliquots. If the analysis cannot be completed immediately, samples should be adequately stored pending analysis. If samples are stored after collection, residue stability should be demonstrated through the assay time.
Table 1 indicates samples recommended being collected during euthanasia.
Table 1. Sample collection from animals used in a marker residue depletion study (All regions).
Edible tissue type
Species/ Sample Description
Cattle/ Sheep/ Pigs / Horses
Muscle from the lumbar region
Core of muscle tissue ~0,5 kg.
10 cm. diameter x 6 cm. deep for IM
15 cm. diameter x 2,5 cm. deep for SC
Cross-Section of lobes
Composite from combined kidneys
Composite from combined kidneys
Omental and/or peri-renal
(pigs and poultry)
Skin with fat in natural proportions
Muscle with skin in natural proportions
(sheep and cattle)
--- Clean shell, break the egg, white and yolk can be combined.
However, VICH guideline to conduct “Studies to evaluate the metabolism and determine the quantity and identify the nature of residues” recommends the collection of additional tissues to quantify total residues in order to address specific regional concerns. The additional tissues suggested for samples are included in Table 2.
Table 2 Additional tissues that can be collected to answer regional concerns in the marker residue depletion study.
Edible tissue type
Species/ Sample Description
Cattle/ Sheep/ Pigs / Horses
Composite, rinsed of content
For the purposes of this guidance, if necessary, one of the additional tissues (per species) will be selected to evaluate the marker residue and address regional concerns. The selected additional tissue is based on the results of the total residue (TTR) study and it would typically be the additional tissue with the highest residue concentration or with the slowest residue depletion rate. For instance, if the TTR study indicates that cattle heart presents the slowest depletion rate, that additional tissue should be selected for assay in the marker residue study, but cattle small intestine marker residue data will not be necessary. Likewise, if poultry gizzard presents the highest residue concentration, assays of poultry heart are not recommended.
4.6.2. Injection sites
For parenteral preparations (IM or SC), residue depletion data from the injection site(s) should be included. Samples should be collected from the last injection site. In case of products requiring multiple injections, the study design should be such that the last injection site will occur on the side where the animal received the higher number of injections. A minimum 10-centimeters distance between injection sites is recommended in order to obtain a better sample quality. 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 1.
During the conduction of residue studies, injection site should be permanently marked, so that it can be easily and correctly identified at slaughter time. The veterinary drug should be administered at the center of the subjacent tissue and the injection site should be extracted immediately after euthanasia.
Sample collection technique should include, whenever possible: The needle track, the site where drug was released and any site presenting tissue irritation.
With the aim of ensuring that the described sampling procedures are adequate to represent residues concentration, it is recommended, whenever possible, to take from every injection site a ring control sample of around 300 g. to the primary sample.
The collection of an additional ring sample or a circular sample surrounding the injection site during the conduction of tissue residue depletion studies is required for the EU, but generally not for other regions.
This second sample may not be always obtained, especially in injections applied in neck sites; therefore the sample size of peripheral tissue to the primary sample may be reduced to the possible extent. However, this sample needs to present an adequate size to allow analytical processing.
For injectable products in which the applied dose exceeds the recommended volume for the application site, injection should be applied in more than one site (e.g.: IM or SC 1 mL/10 kg dose for an animal of 360 kg. when maximum recommended volume is 20 mL). In those cases, the injection site sample should be taken from the injection site receiving the highest volume (the site injected with 20 mL. and not the second site injected with 16 mL).
In real practice, it is difficult to obtain samples of the exact recommended weight, so it is accepted for the real weight to vary within certain limits with respect to the proposed theoretical weight. The core injection site samples presenting a weight fluctuating between 400 and 500 g. (500 g ± 20%) are acceptable.
Following tissue removal, the injection site samples collected (core and peripheral) should be appropriately homogenized before the final sampling (300 g.) for the determination of residues, thus avoiding analytical processing of a potentially non homogenous material.
Proposed dimensions and weights of injection site tissue samples cannot be applied in small animals, whose size and anatomy do not allow samples of 500 g.
A general strategy cannot be applied here, instead a case-by-base strategy should be designed and the sampling technique chosen and the weight of the tissue to be analyzed should be conveniently justified. In this case, a sample of the injection site peripheral tissue should be taken, as long as possible, to corroborate the reliability of the analytical method used.
It should be born in mind that residues concentration determined on the basis of samples taken from small animals or samples of tissues with a weight or size smaller than the one proposed in this guidance should be used without any type of correction or dilution for the calculation of withdrawal time.
4.6.3. Other considerations
• For formulations that are able to leave local residues, such as dermal pour-on products, samples of relevant tissues (e.g., muscle, subcutaneous fat or skin/fat from the application site) should be harvested for analysis (in addition to those specified in Table 1).
• For clarity, if two or more of the tissues are assayed as composite tissues such as skin plus fat in natural proportions (pig and poultry), it is not recommended to assay separate samples of skin and fat.
• Muscle samples can be obtained from skeletal muscles that include intramuscular fat in natural proportion.
• Collection of only one type of fat sample (omental for ruminants and horses) or skin with fat (pigs and poultry).
4.6.4. Milk sampling
Milk samples should be taken from all animals included in the assay. Collection should be done at every milking time, at evenly spaced intervals (12 hours approximately). Samples of each animal will be milk from four quarters.
For multiple dose treatments, samples should be taken after the last treatment, except when the product might qualify for 0-day withdrawal periods, in which case samples should also be collected during treatment.
Although beyond the scope of this guidance, sponsor may be asked to analyze the tissue residues in calves fed by milk (including colostrum) from treated adults (e.g. mothers), if these animals are intended for human consumption.
4.6.5. Egg sampling
Egg samples should be obtained from 10 or more laying hen at every laying time point during medication period and after the final medication. Samples should be collected after the period necessary to complete egg yolk development, which is usually up to12 days. Egg white and yolk can be combined for analysis.
5. Recommendations for veterinary drugs proposed for 0-day withdrawal periods (Single Time-Point Studies)
For veterinary drugs administered as one or several treatment (e.g. daily for 3-5 days) or for continuous use products in which residues have reached steady state, a single time point study should be sufficient to qualify for 0-day withdrawal period, provided the total residue depletion characteristics of the drug have been adequately described. If such data are available, then a single time point study conducted with the specified number of animals is recommended to demonstrate acceptability of 0-day withdrawal period.
• Poultry: 12 birds
• Ruminants, horses and pigs: 6 animals
• Milk 10 animals
The time chosen for euthanasia for this study should be consistent with the peak concentrations observed during the total residue depletion study, a minimum transit time (e.g., not less than 3 hr.) and a maximum time that would still qualify for 0-day withdrawal period (e.g., ? 12 h).
The increased number from that recommended in Section 4.3. is appropriate for the single time point. However, for females producing milk for consumption or manufacturing, a minimum of 10 animals is recommended, since that amount is enough to determine milk concentration in a single time point (0 days). The concentrations of active principle remaining below the appropriate reference point (e.g. MRL, tolerance) will be considered to determine 0-day withdrawal time.
While a 0-day withdrawal designation is possible based on a single time point (i.e. 12 hours) sampling protocol, it is recommended that additional samples (e.g. 1-4 milking) be collected for full assessment of the residue profile, in case of lack of these data. As milk studies do not call for terminal euthanasia for sample collection, this recommendation is straightforward.
In case of egg producing hens, a 0-day withdrawal period could be determined based on consecutive samples below the reference point (maximum residue limit or tolerance) during the 12-days egg collection period for analysis, given the physiological conditions of the oogenesis process.
6. Withdrawal period confirmation studies.
The single confirmation point assay is a special test whose technical validity is restricted to the comparison of results of residue depletion of a veterinary drug widely known and used in the veterinary practice, a drug whose similar formulations have been previously registered for the country where the product is presented. In this study, fewer animals are used and the result only indicates if the same use restriction can be applied. It only serves one purpose: it determines if residues are the same or below the allowed MRL at the (time) point selected for the test. In practice, it does not calculate the withdrawal period, but it does confirm a specific withdrawal period compared to the already known veterinary drug.
6.1. Criteria to determine the need of a residue study or a withdrawal period confirmation study.
The criteria to determine which veterinary drugs should be submitted to a residue study will depend on the type of product (generic or similar), the present active principle(s) and the application criterion defined by each regulatory authority.
6.2. Interpretation of results in withdrawal period confirmation studies
- If an assay demonstrates that the residues of a veterinary drug submitted for trial are below the MRL, confirmation is accepted.
- If the study demonstrates the contrary, that is to say that residues exceed the MRL, then the trial is not accepted, and the period established for the reference product cannot be used and a complete residue depletion study should be conducted.
6.3. Study design
The species, age, breed, genetic line, sex and source of the animals should be informed. The assay should be conducted with the minimum number of animals informed in the following table:
Animal species/ product
Lactation/ dry-off/pre partum
The trial should follow the indications stated at points 4.2; 4.4 and 4.5 of this guideline. The sampling (point 4.6) and the residue analysis have to be performed only using target tissue (the tissue that was used in previous residue trials to define the withdrawal).
7. Analytical method for marker residue assays
The sponsor is responsible for submitting validated analytical methods for the determination of the marker residue in samples generated from the residue depletion studies in edible tissues and where applicable in milk, eggs and honey. The method(s) should be capable of reliably determining the concentrations of marker residue which encompass the appropriate reference point (i.e. MRL/tolerance) for the respective tissues or products. (See: Guideline of recommendation for the validation of analytical methods).
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