A series of articles has been planned in order to give veterinarians some insight into certain aspects of the responsible and prudent use of antibiotics and other veterinary medicinal products (VMP). The OIE – Terrestrial Animal Health Code – 30/07/2015 publication was quoted in the previous article regarding the various responsibilities of veterinarians and the allowance for the extra-label or off-label use of veterinary medicinal products (VMP) containing antimicrobial agents in appropriate circumstances.
Extra label use of veterinary products must meet the national statutory requirements including tissue residues, and withdrawal periods.
This article will cover some pharmacokinetical factors and maximum residue levels of VMP’s that may be considered by practioners in their decision making process.
Pharmacokinetics describes quantitative changes in drug concentration in the body over time as a function of administered dose. Pharmacokinetic studies generate data on the absorption, distribution, metabolism, and excretion of drugs.
Lees and Toutain state that plasma or blood concentration-time profiles together with identification and quantitation of major metabolites of the VMP are important because plasma concentration is the driving force controlling all tissue concentrations. Serum/plasma concentration-time data may be subjected to mathematical models to determine quantitative terms which describe absorption, distribution, metabolism, and excretion of the drug and its metabolites.
Pharmacological terms describing or making inferences about concentrations are shown below.
Cmax is the highest concentration reached in the plasma or a specific tissue.
The volume of distribution (Vd) may be used as an indication of the relationship between vascular and extravascular concentrations of a drug.
According to Merck the apparent volume of distribution is the theoretical volume of fluid into which the total drug administered would have to be diluted to produce the concentration in plasma. For example, if 1000 mg of a drug is given and the subsequent plasma concentration is 10 mg/L, that 1000 mg seems to be distributed in 100 L (dose/volume = concentration; 1000 mg/x L = 10 mg/L; therefore, x= 1000 mg/10 mg/L = 100 L). In this case the volume of distribution has nothing to do with the actual volume of the body or its fluid compartments but rather involves the distribution of the drug within the body. For a drug that is highly tissue-bound, very little drug remains in the circulation; thus, plasma concentration is low and volume of distribution is high. Drugs that remain in the circulation tend to have a low volume of distribution.
According to Apley the apparent volume of distribution is the volume of fluid (expressed as l/kg of body weight) necessary to contain the total amount of drug in the body if it were uniformly distributed and the concentration in this hypothetical fluid were equal to the plasma concentration. Thus, in the example above the 100L volume of distribution would be divided by the body weight.
Therefore, in this method of calculation drugs that tend to stay in the plasma have a Vd much less than 1, drugs that have wide distribution have a Vd near 1, and drugs with very wide distribution have a Vd much greater than 1.
Volume of distribution provides a reference for the plasma concentration expected for a given dose but provides little information about the unique distribution of any drug in the body. Some drugs distribute mostly into fat, others remain in extracellular fluid, and others are bound extensively to specific tissues
Area under the curve (AUC) is described by Apley as the total area under the plasma concentration curve. The AUC following an IV injection of a drug essentially represents “all of the drug”. Comparing this AUC to the AUC following IM, SC, or oral administration allows the calculation of bioavailability. Bioavailability is the percent of a drug available after administration by a specified route (other than IV) compared to IV administration of the same amount.
Terms describing rates
Tmax describes time to peak concentration in the plasma, but this may also be described for a tissue. In the plasma, it is at the time of injection of an intravenous bolus while, after an intramuscular injection it reflects the rate of absorption.
T½ is the time required for the plasma concentration to decrease by ½ during the distribution phase of the plasma concentration curve (distribution half-time), and estimates the rate of distribution to the tissues. This phase consists primarily of distribution to the tissues, but also includes some elimination processes. It is greatly confounded by absorption following an IM injection.
T½ is the time required for the plasma concentration to decrease by ½ during the elimination phase of the plasma concentration curve (elimination half-time). Elimination from the plasma and tissues predominates in this phase. Although the plasma T½ may give an indication of the tissue T½, they are not necessarily equal.
The area under the plasma drug concentration time curve (AUC) reflects the actual body exposure to drug after administration of a dose of the drug and is expressed in mg*h/L.
This area under the curve is dependent on the rate of elimination of the drug from the body, the dose administered and the bioavailability. The total amount of drug eliminated by the body may be assessed by adding up or integrating the amounts eliminated in each time interval, from time zero (time of the administration of the drug) to infinite time. This total amount corresponds to the fraction of the dose administered that reaches the systemic circulation.
The AUC is directly proportional to the dose when the drug follows linear kinetics. The AUC is inversely proportional to the clearance of the drug. That is, the higher the clearance, the less time the drug spends in the systemic circulation and the faster the decline in the plasma drug concentration. Therefore, in such situations, the body exposure to the drug and the area under the concentration time curve are smaller.
According to Rees and Toutain, for most drugs administered at recommended dose rates, clearance is a parameter following first order pharmacokinetics, so that the rate of decrease in plasma concentration over time is exponential and proportional to concentration. There will always be a relationship between drug concentration in plasma and in tissues, because plasma concentration is the driving force for diffusion into tissues.
Lees and Toutain explain that tissue concentrations of veterinary drugs depend on a range of (mainly physicochemical) properties such as lipid solubility and acidic/basic characteristics, which influence the passive diffusion of drugs across cell membranes. In addition, for a few drugs, active uptake by, or extrusion from, tissues occurs. High lipid solubility drugs (fluoroquinolones, macrolides, phenicols and triamilides) cross cell membranes readily by passive diffusion to penetrate into intra- as well as extracellular compartments of tissues. Moderate to high lipid solubility drugs (diaminopyrimidines and tetracyclines) similarly generally enter readily into all water compartments of tissues.
On the other hand, drugs of low lipophilicity (cephalosporins, penicillins, aminoglycosides, and polymyxins) generally do not readily enter cells, so that these drugs are located mainly or solely in the extracellular compartment of tissues.
An additional factor is acid/base characteristics of the molecule. As mean intracellular fluid pH is somewhat lower than extracellular fluid pH, 7.0 versus 7.4, weak bases penetrate readily into cells, by the classical Henderson-Hasselbalch mechanism of ion/diffusion trapping, whereas weak acids do not. However, within cells, drugs may be located predominantly within different subcellular compartments; for example, fluoroquinolones and betalactams in the cytosol and macrolides, lincosamides and pleuromutilins (weak organic bases) in phagolysosomes. The pH of the environment in phagolysosomes is very acid (pH = 4 to 6) compared to extracellular fluid, so that very high concentrations of weak bases can be achieved. An example is tulathromycin, which accumulates in lung tissue of calves and pigs in concentrations some 50- to 100-fold greater than in plasma. By far the greater proportion of the drug within the lung is located intracellularly. Conversely, for those drugs with low lipophilicity and for lipophilic weak organic acids, the average/overall tissue concentration is made up of a high extracellular and low intracellular concentration.
The uptake and release of drugs from tissues varies. This process may be passive (the normal case for most drugs), or active. Passive uptake involves passage from a high (plasma) concentration down a concentration gradient to a low (tissue) concentration. Subsequently, the gradients are reversed and drug is off-loaded from tissues into circulation. Whilst plasma is an extracellular fluid, tissues contain both intra- and extracellular compartments.
The classical example of high affinity of drugs for particular tissues is the tendency of aminoglycosides to accumulate and persist in renal tissue.
For aminoglycosides, the decrease in plasma concentration is the sum of three separate exponential phases, each with its own slope and half-life. Thus, a plot of plasma concentration versus time reveals the three exponential phases (interpreted as a tricompartmental model). These are a, b, and g phases. The a-phase is short and represents rapid distribution from plasma to other extracellular fluids. The b-phase is also short and represents the phase during which most of the drug is rapidly eliminated from the body, almost exclusively by excretion in urine in high concentrations. Important, in relation to residues of aminoglycosides, is the existence of a very late terminal phase. This final phase can be detected using a sensitive analytical technique. The plasma concentrations in this phase are less than those that are microbiologically effective and thus without therapeutic significance. This slow terminal phase reflects persistence of aminoglycoside residues in deep compartments. This terminal phase is controlled by the redistribution rate constant from tissue to plasma and accounts for the persistence of residues in renal tissue for weeks or even years.
It is of importance to recognize both the differences from healthy animals and the potentially very variable tissue depletion rates of drugs when they are used for disease prophylaxis, metaphalaxis, and therapeutics.
Merck Manual pharmacokinetics by Le also describes the events after a drug enters the systemic circulation and is distributed to the body’s tissues. Distribution is generally uneven because of differences in blood perfusion, tissue binding (eg, because of lipid content), regional pH, and permeability of cell membranes.
The entry rate of a drug into a tissue depends on the rate of blood flow to the tissue, tissue mass, and partition characteristics between blood and tissue. Distribution equilibrium (when entry and exit rates are the same) between blood and tissue is reached more rapidly in richly vascularized areas, unless diffusion across cell membranes is the rate-limiting step. After equilibrium, drug concentrations in tissues and in extracellular fluids are reflected by the plasma concentration.
Metabolism and excretion occur simultaneously with distribution, making the process dynamic and complex.
After a drug has entered tissues, drug distribution to the interstitial fluid is determined primarily by perfusion. For poorly perfused tissues (eg, muscle, fat), distribution is very slow, especially if the tissue has a high affinity for the drug.
It is important to note differences in the interpretation and use of total tissue concentration from pharmacological and therapeutic perspectives on the one hand and for drug and metabolite depletion rates on the other.
Maximum residue level (MRL) is a drug substance property, which may be independent of any pharmacokinetic characteristics of the product. The MRL is thus fixed by regulatory authorities as a ‘regulatory constant’, with a universal meaning.
Maximum residue levels
Baynes et al report that risk assessment and regulation of veterinary drug residues in animal-derived food commodities, such as muscle, liver, kidney, fat, milk, and eggs, follow similar principles throughout the world. In the United States of America (USA), the Food and Drug Administration (FDA) is the regulatory body that sets maximum permitted concentrations for veterinary drug residues, known as tolerances.
In the European Union (EU), the equivalent regulatory body is the European Medicines Agency (EMA), which publishes maximum residue limits (MRLs) that have been set by the Committee for Medicinal Products for Veterinary Use (CVMP). There are also independent risk assessment bodies, such as the Joint Food and Agricultural Organization/World Health Organization Expert Committee on Food Additives, (JECFA) which also recommends MRLs. JECFA advises the Codex Alimentarius Commission (CAC), which as risk manager, determines whether or not to establish international standards for residues of veterinary drugs in terms of MRLs. According to Baynes et al developed countries that are not part of the EU develop their own MRLs while most developing countries adopt EU or Codex MRLs.
Calculation of the tolerance or MRL requires algorithms and assessment of available toxicological, pharmacological, and microbiological data bases. This is a risk assessment process where a standard battery of safety studies in animals and/or humans as well as in vitro studies are used to determine the acceptable daily intake (ADI).
The FDA published a draft document on general principles for evaluating human food safety of new veterinary medical products used in food producing animals and this has been used to compile parts of the discussion below.
The ADI is the daily intake that may be considered safe when food at that level is consumed daily for a life-time without adverse effects or harm to the health of a consumer. ADI is usually expressed in micrograms or milligrams of the new animal drug per kilogram of body weight per day (μg/kg bw/day) or (mg/kg bw/day) and may be calculated differently according to the characterisation of the product from perspectives of toxicology, residue chemistry and antimicrobial resistance.
Traditionally toxicology studies are designed to determine if a VMP produces an adverse effect in a biological test system and to identify the highest dose that produces no observable effect, which may be a no-observed-effects dose (NOEL), a no-observed-adverse-effects dose (NOAEL) or a benchmark dose (BMD). The BMD is the dose associated with a specified low incidence of response, generally in the range of 1 to 10%, of a health effect, or a dose associated with a specified measure or change in a biological effect.
It may be difficult to establish whether or not an observed effect in animal toxicological studies is also observable in humans in which case the VMP’s NOAEL is may be considered equivalent to its NOEL.
When calculating the ADI, the toxicities of the VMP and of its principal metabolites are considered, and the ADI is based on the toxicological end-point of most concern. For most endpoints, the NOAEL/NOEL or the BMDL (lower one-sided confidence limit of the BMD) are taken from the most appropriate toxicological study.
The toxicological ADI is derived by dividing the toxicological end point by an appropriate safety factor, using the following formula:
Toxicological ADI = NOAEL/NOEL or BMDL/ Safety Factor
The safety factor reflects uncertainties associated with the extrapolation of data and information from toxicology studies to humans, including extrapolation of long-term, chronic effects from laboratory studies with shorter-term exposures, extrapolation of animal data to humans, and variability in sensitivity to the toxicity among humans.
Generally, the safety factor consists of multiples of 10, with each factor representing a specific uncertainty inherent in the available data. Historically a safety factor of 100 for an ADI based on the NOAEL/NOEL from a chronic toxicity study (10-fold for extrapolating animal data to humans and 10- fold for variability in sensitivity to the toxicity of the new animal drug among humans) has been used.
The Microbiological Acceptable Daily Intake (mADI) on the other hand aims to ensure that residues in the edible tissues of food producing animals are safe for humans to consume and may be the lessor of the level resulting in a disruption of the colonization barrier or an increase of the population(s) of resistant bacteria. The National Antimicrobial Resistance Monitoring System (NARMS) monitors four major foodborne bacteria for antibiotic resistance trends over time as described in the previous article.
The ADI may be assessed across a range of edible tissues when consumption of those tissues may contribute to the total human exposure to a VMP and/or its residues.
The ADI is then adjusted with food consumption values for various tissues (300 g for muscle, 100 g for liver, 50 g form kidney, 50 g for fat, and if a dairy approval,1500 g for milk) to obtain MRLs or tolerance for each tissue. This requires kinetic data for each tissue which is used to ensure that the total food basket of residues at each tissue MRL or tolerance results in less than the ADI. Different jurisdictions may use slight modifications such as allocations of ADIs in how MRLs and tolerances are calculated
Finally, the safe concentration may be calculated
The safe concentration is the amount of total residue of a VMP that can be consumed from each edible tissue every day for the lifetime of a human without exposing the human to residues in excess of the ADI. The calculation of the safe concentration for each edible tissue uses the ADI, the weight in kilograms (kg) of an average adult human body (approximated at 60 kg), and a conservative estimate of the amount of the edible tissue eaten per day in grams.
Safe Concentration (edible tissue) = ADI × Human Body Weight Food Consumption Value
For example, the safe concentration for milk equals the ADI reserved for milk (μg/kg bw/day) times 60 kg divided by 1500 mL/day; safe concentrations for other tissues are calculated in a similar manner.
The FDA states that regulatory authorities traditionally use 60 kg as body weight for all life stages ranging from a two-year-old toddler to the eldest consumer. The weight adjustment assumes an arbitrary human body weight for either sex of 60 kg. While making this assumption, authorities recognize that some humans weigh less than 60 kg; these consumers are considered to be accommodated by the built-in safety factors used to determine an ADI.
There is the potential for some VMP’s to cause acute toxicity to the human consumer following consumption of a single meal or consumption of food over a single day. In these cases, the ADI is not the appropriate safe intake value for quantifying the dose above which exposure from a single meal or over a single day can produce acute adverse effects; instead, determining an acute reference dose (ARfD) may be considered the most appropriate approach.
The ARfD is an estimate of the amount of residues, expressed on a body weight basis, which can be ingested in a period of 24 hours or less without adverse effects or harm to the health of the human consumer.
Human consumption of all or part of a VMP residue in a single meal is considered a rare event, but may be associated with consumption of an injection site from which the VMP did not distribute to other tissues in the animal. In order for the FDA to ensure human food safety for this possible acute exposure, the safe concentration of residues in the injection site tissue is calculated for injectable products. Typically, injection site residues should be less than 10x the muscle safe concentration, or the injection site safe concentration calculated using the ARfD.
- When no specific acute toxicological concern has been identified, the safe concentration for injection sites is estimated by multiplying the safe concentration for muscle by a factor of 10.
- When a specific acute toxicological concern has been identified for an injected VMP, the safe concentration for the injection site is calculated using the ARfD as follows:
Safe Concentration (Injection Sites) = ARfD x Human Body Weight
THE Centre for Veterinary Medicine (CVM) applies a muscle consumption value (300 g) to determine the safe concentration for injection sites. Thus, a conservative estimate of the amount of the injection site tissue consumed in a single meal is estimated to be 300 g/day. CVM may accept an alternative consumption value or approach for calculating the safe concentration of injection sites with appropriate scientific justification.
For injection site assessment the JECFA requires information regarding drug dose, formulation, time elapsed since injection and concentrations of residues observed under standardized conditions of sampling. The Committee has accepted a sampling procedure laid down by both the European Medicines Agency (EMEA) and the USFDA. According to FAO and WHO the EMEA has recently modified its sampling procedure, which now requires a second “surrounding” sample (tissue surrounding the core 500 g sample) to confirm the quality and correctness of the original sampling.
FDA will not approve a compound for use as a new animal drug in food-producing animals when the compound or any of its metabolites has been found to cause cancer in animals or humans, unless: 1) the compound will not adversely affect the animals for which it is intended, and 2) no residue of the compound will be found by approved regulatory methods in any edible tissues.
There are also non-traditional biotechnology-derived animal drugs, including some enzymes, fusion proteins, hormones, and antibodies that may need to be evaluated differently.
The FAO and WHO published Principles and methods for the risk assessment of chemicals in food Environmental Health Criteria 240 in 2009 includes an overview of current principles and practices from the Joint FAO/WHO Meeting on Pesticide Residues (JMPR) and Joint Expert Committee for Food Additives (JECFA) for residue evaluation.
JECFA develops recommendations for MRLs, similarly to the FDA, based on chronic intake estimates calculated from the median residue levels and a theoretical food basket (consisting of 300 g muscle, 100 g liver, 50 g kidney, 50 g fat, 1500 g milk, 100g eggs and 20 g honey), to estimate a conservative daily intake of residues, known as the estimated daily intake (EDI). This estimate is then compared with the type and amount of residue considered to be without toxicological, pharmacological or microbiological hazard for human health, as expressed by the ADI.
In addition to specific residue data, JECFA also considers other factors, such as good veterinary practice (GVP) and the availability of suitable analytical methods for determining residues in food animal tissues.
Lees and Toutain point out that, for regulatory purposes pharmacokinetic profiles are established in healthy animals, using small numbers of animals, usually of a single breed, similar age, and possibly of the same gender. On the other hand the objective of population pharmacokinetics is to determine both pharmacokinetic variables which enable an optimal dose to be determined for the clinical population and also to explain inter- and intra-animal variability in terms of age, sex, breed, and health/disease status. Thus they believe that population pharmacokinetics are of relevance to tissue residue depletion rates.
It would be impractical and very expensive for a whole range of depletion profiles to be conducted in a target species with each study matching a particular circumstance/factor, such as age, sex, breed, weight, and disease status thus regulatory authorities build conservative assumptions into MRL determination and selection of the withdrawal time, designed (in theory) to ensure the safety of food, despite the variability in drug pharmacokinetics in differing circumstances.
The JECFA would normally request detailed pharmacological, toxicological, drug metabolism and other related studies to characterize the specific molecules for toxicological evaluation. Generally, identified metabolites that contribute 10% or more of the total residues would be considered for toxicological evaluation. However, in some instances, metabolites consisting of less than 10% of the total residues may be considered.
Additional specific data requirements for the consideration of MRLs on the basis of the ADI include authorized mode of administration, dose and formulation, toxicodynamic, toxickinetic, metabolism and residue depletion studies.
The JECFA uses residue depletion studies with radiolabelled parent drug as well as additional studies with unlabelled parent drug in intended target animal species for recommending MRLs in raw commodities of animal origin. The derived MRLs are defined on the basis of a marker residue substance (a substance with a known quantitative relationship to the total residue of concern). If MRLs cannot be recommended for every commodity of interest, JECFA may attempt to include at least appropriate target tissues for regulatory residue analysis for products and products marketed both domestically and internationally. Dose treatments in such depletion studies are required to include the maximum approved dose, administered in the commercial formulation and under the approved conditions of use. Residues are generally determined in several edible tissues and products, as appropriate for the intended use (e.g. in muscle, liver, kidney and fat of slaughter animals as well as in milk and eggs). These studies also have to provide the necessary information on all types of residues formed, such as free, conjugated and bound residues. For substances with an ADI derived from a toxicological end-point, all residues are considered to have the same toxicological significance as the parent drug unless data is provided to permit JECFA to discard them from consideration. Thus, the default assumption is that there may be dose additivity. Similar considerations apply to substances with a microbiologically defined ADI.
JECFA may make full recommendations for MRLs of a veterinary drug in appropriate food animal species and tissues on the basis of a permanent ADI and adequate residue data.
Alternatively temporary MRLs may be recommended either when there is a full ADI but adequate residue and/or method performance data are lacking or when the ADI is temporary.
The Committee may also recommend ”not specified” or ”unnecessary” MRLs when there is a wide margin of safety of residues when compared with the ADI.
Finally, JECFA may determine that MRLs cannot be recommended because of significant deficiencies in either residue data or available analytical methods or when an ADI is not established. JECFA also does not recommend MRLs when the required conditions of use would not be compatible with the GVP established by national authorities or when estimated chronic dietary intake of residues would substantially exceed the ADI.
The different approaches by different authorities has been addressed in the Veterinary International Conference on Harmonization (VICH) Guidance Documents which are available on the WEB. VICH guidelines are to be incorporated in future assessments to ensure consistency and transparency in the determination of microbiological ADIs
Beyene states that VMPs and agricultural chemicals used according to label directions should not result in residues at slaughter. He lists, however, possible Risk Factors for the Development of Residue in Food producing Animals
- Not following recommended label directions or dosage (extra-label usage);
- Not adhering to recommended withdrawal times;
- Administering too large a volume at a single injection site;
- Use of drug-contaminated equipment, or failure to properly clean equipment used to mix or administer drugs;
- Dosing, measuring, or mixing errors;
- Allowing animals access to spilled chemicals or medicated feeds;
- Animal effects- age, pregnancy, congenital, illness, allergies;
- Chemical interactions between drugs;
- Variations in water temperature for fish species;
- Environmental contamination; and improper use of agricultural chemicals such as pesticides.
Pharmaceutical companies may be inclined not to register products for use in different species due to the time and cost associated with the registration of products. This may lead to the off label use of available products and veterinarians should consider very carefully the consequences of the medicines under their sphere of influence coming into the human food chain or environment.
- Lees P and Toutain P-L The role of pharmacokinetics in veterinary drug residues. Drug Test. Analysis 2012, 4 (Suppl. 1), 34–39.
- Le J. Drug Distribution to Tissues Clinical Pharmacology Merck Manuals Professional Edition. April 2016
- Apley M. Antimicrobial Clinical Pharmacology, Iowa State University. 2003
- Beyene T. Veterinary Drug Residues in Food-animal Products: Its Risk Factors and Potential Effects on Public Health. J Veterinar Sci Technol 2016, 7:1
- FAO and WHO Principles and methods for the risk assessment of chemicals in food Environmental Health Criteria 240.2009. ISBN: 978 92 4 157240 8.
- Baynes RE, DedonderK, Kissell L, Mzyk D, Marmulak T, Smith G, Tell R, Davis J, Riviere JE. Health concerns and management of select veterinary drug residues. Food and Chemical Toxicology 88 (2016) 112-122
- Guidance for Industry General Principles for Evaluating the Human Food Safety of New Animal Drugs Used In Food-Producing Animals DRAFT REVISED GUIDANCE U.S. Department of Health and Human Services Food and Drug Administration Center for Veterinary Medicine July 2016