Toxicity Studies

Before any drug can be approved by a regulatory authority for use in a food‐producing animal, it undergoes an extensive toxicological evaluation of the drug and its residues. The toxicology studies characterize the toxicity of the antimicrobial in various in vitro and in vivo animal models as well as available human data to predict the potential toxicity of residues of the drug in food. In the US, a battery of four toxicological tests is required to satisfy human food safety requirements for any new animal drug intended for use in a food‐producing animal species.

• Metabolism studies for identification of residues for toxicological testing. This includes metabolite identification in the target species and metabolite identification in a laboratory animal species.
  
• Toxicological testing in laboratory animals. This includes genetic toxicity tests, acute toxicity tests, subchronic (90‐day) toxicity tests, and a 2–3‐generation reproduction study with a teratology component in rats. Lifetime carcinogenicity studies in two rodent species are required only if genetic toxicity tests indicated that the drug or metabolites are potentially carcinogenic (the decision by the FDA to require lifetime carcinogenicity studies is based on a decision tree process referred to as threshold assessment). Other specific toxicity tests are carried out as needed.
  
• Residue depletion studies in the target species.
  
• Regulatory analytical methodology for identification and quantitation of marker residues in animal tissues, milk, or eggs.

Residues of veterinary drugs in food are primarily evaluated for toxic effects following chronic exposures, because people may be exposed daily to antimicrobial residues through regular consumption of the same food (e.g., meat or milk), and because chronic exposures often have a lower threshold for toxicity than infrequent or acute exposures. Typically, multiple doses of the antimicrobial are administered to test animals to identify a dose that results in a no observable effect level (NOEL). Based on the NOEL, regulatory agencies establish an acceptable daily intake (ADI) for the veterinary drugs. The ADI represents a level of daily intake of a drug (and/or its metabolites) which, during an entire lifetime, appears to be without appreciable risk to the health of the consumer. It is calculated by multiplying the NOEL by a safety factor (SF). The SF usually has a value of 100 or higher based on factors that allow for interspecies differences and human variability.

The ADI is used to determine the maximum concentration of a marker residue in edible tissues, honey, milk, or eggs that is legally permitted or recognized as acceptable. In the US, these acceptable concentrations are termed tolerances while other jurisdictions use the term maximum residue limit (MRL). The MRL is calculated such that daily intake of food with residues at the MRL will result in a total daily consumption of residues in quantities at or below the ADI. The ADIs are based on the total residue of a drug present in food (parent compound and all metabolites) whereas MRLs are based on a single, measurable marker residue, which may be the parent compound or any of its metabolites.

In establishing MRLs, consumption estimates for different foods are taken into account, so that foods consumed infrequently or in small amounts are allowed greater MRL values than those foods likely to be consumed daily or which represent a major component of a usual diet. A typical “food basket” is used to determine the quantity of residues potentially ingested by the “average” 60 kg consumer (Reuss, 2013) (Table 26.1). The US FDA assumes that: (i) an individual eats more edible muscle than organ tissue, (ii) an individual does not eat a full portion of meat product from another species, and (iii) milk and egg products are consumed in addition to edible muscle or tissue (Baynes et al., 1999). This approach has a number of limitations: it is not based on actual food consumptions in various regions of the world; it does not consider acute toxicities (e.g., ingestion of an injection site from an injectable antimicrobial); and there can be a special occasion/event where people may consume a large portion of food derived from an edible tissue where the veterinary drug residues are concentrated. Despite these limitations, it is a comprehensive and carefully developed approach to reducing risk from residues.

The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has developed models for estimating chronic and acute dietary exposure to veterinary drugs: the Global Estimate of Chronic Dietary Exposure (GECDE) for chronic exposure assessment and the Global Estimate of Acute Dietary Exposure (GEADE) for acute exposure assessment (Boobis et al., 2017). The GECDE uses food consumption data from consumer surveys to provide more realistic and accurate data of a population’s actual consumption patterns. For acute exposures to veterinary drug residues, the GEADE establishes the acute reference dose (ARfD), the acceptable acute intake based on short‐term studies, that can be considered the upper limit of occasional fluctuations above the ADI. The ARfD is “an estimate of the amount of a substance in food and/or drinking‐water, normally expressed on a body‐weight basis, that can be ingested in a period of 24 hours or less, without appreciable health risk to the consumer, on the basis of all the known facts at the time of the evaluation” (Moretto, 2000).

Because of differences in consumption factors, MRLs and label withdrawal times may differ between countries, even though ADIs are equivalent (Fitzpatrick et al., 1995, 1996). The US tolerances and Canadian, EU, and Australian MRLs can be found online. China issued MRLs for veterinary drugs that took effect in 2020 (USDA, 2019). Although the World Trade Organization and the Codex Alimentarius have attempted to harmonize MRLs worldwide, they still vary from one geographical location to another. The MRLs in a particular animal product may differ from one country to another depending on the local food safety regulatory agencies and drug usage patterns, and most developing countries have yet to develop their own MRLs.

In many jurisdictions, drug approval agencies now consider the microbiological effects of antimicrobial residues in new drug submissions (Cerniglia et al., 2016). A microbiological ADI is established considering disruption of the intestinal colonization barrier and an increase in the selection and emergence of antimicrobial‐resistant bacteria. In 2003, the FDA released guidance document #152: Evaluating the Safety of Antimicrobial New Animal Drugs with Regard to their Microbiological Effects on Bacteria of Human Health Concern. Other jurisdictions use the guidelines of the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH GL36): Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food: General Approach to Establish a Microbiological ADI (revised 2019). These documents are not regulations, but science‐based processes that drug sponsors may use when they seek official approval of an antimicrobial for use in food‐producing animals. These guidance documents recommend studies to assess microbiological endpoints, including: (i) changes in the colonization resistance properties of the microflora; (ii) changes in the metabolic activity of the intestinal microflora; (iii) changes in antimicrobial resistance patterns of the microflora; and (iv) changes in the number and composition of the microorganisms that constitute the intestinal microflora. The “microbiological ADI” is then compared with the “toxicological ADI” and the lower of the two ADIs for the drug is used to establish the overall ADI (“the ADI”) to ensure safety to the consumer.

In the absence of approvals and defined ADI and MRLs (e.g., chloramphenicol), the concentration of residues allowed in food is considered to be zero. In practical terms, this is defined by the limit of detection of the analytical method. The ALARA (as low as reasonably achievable) approach recognizes that absolute zero is unattainable, and considers what is technically achievable, the resources needed to achieve that technical goal, and the benefit gained.

Withdrawal Times

Much of the human health risk from antimicrobials used in food animals is mitigated by established withdrawal times (WDT). The WDT is defined as the time interval from the last administration of a product to when the animal can be slaughtered for human food, or when milk, eggs or honey can be safely consumed. If the WDT is followed, food animal products should not contain antimicrobial residues that exceed the MRL.

In order to establish the WDT, the drug sponsor carries out residue depletion studies: (i) with nonradiolabeled drug; (ii) in the product formulation proposed for marketing; and (iii) administered at the highest dose rate, shortest dose interval, and longest duration specified in the product literature. Typically, a minimum of four animals of the target species at each of at least four slaughter times is used to define the depletion profile. The animals should be typical of target animals in clinical use, for example, young calves or lactating adult cattle. Both EU and FDA authorities assume log‐linear decline of residue concentrations and apply least‐squares regression to derive the fitted depletion line. Then the one‐sided upper tolerance limit (95% in EU and 99% in USA) with a 95% confidence level is computed. The WDT is the time when this upper one‐sided 95% tolerance limit for the residue is below the MRL with 95% confidence.

Direct Antimicrobial Residue Toxicity

Reports of acute adverse reactions in humans from ingestion of antimicrobial drug residues are rare. Allergic reactions are manifested in many ways, from life‐threatening anaphylactic reactions to lesser reactions such as rashes. Although animal drug residues do not cause primary sensitization of individuals because exposures are too low and for short duration, violative residues of animal drug residues in food can potentially cause allergic reactions in sensitive individuals.

Of the few reports that document adverse reactions in people consuming residue‐contaminated foods, the overwhelming majority are allergic reactions to penicillin. In reference to these allergic reactions, Burgat‐Sacaze et al. (1981) stress the following: “1) Involvement of residues constitutes a very low number of cases (a small percentage) of food allergies. The major allergens involved are natural food constituents or human food additives; 2) The clinical observations report rashes the most frequently, but never, to our knowledge, has anaphylactic shock been noted; 3) In most cases, residues are implicated without sufficient diagnostic evidence.” Most cases follow this pattern: hypersensitivity reaction observed following food intake, tests demonstrate that the individual is not allergic to the food eaten but is to some drugs, and the possibility of the presence of residues of these drugs in the food is promoted without documenting the presence of residues.

Nearly all reports of acute adverse reactions from food‐borne residues implicate penicillin as the offending agent, and the source of penicillin residues is most often thought to be milk or dairy products. Although a substantial number of farm milk samples have been found to contain small amounts of penicillin, there have been relatively few published reports of adverse reactions from milk residues (Dayan, 1993). In all instances, the victims reported a history of penicillin allergy or skin disease unrelated to penicillin allergy. Symptoms varied in intensity from mild skin rashes to exfoliative dermatitis. Many drugs other than penicillin, including other beta‐lactams, streptomycin (and other aminoglycosides), sulfonamides, and to a lesser extent, novobiocin and the tetracyclines, are known to cause allergic reactions in sensitive persons (Bacanlı and Başaran, 2019); however, there is only a single report of a reaction to meat suspected of containing streptomycin residues (Tinkelman and Bock, 1984).

Although there are no reports of direct human toxicity from residues in food, there are a number of antimicrobials that are globally banned for use in food animals because of their toxicity risk even though they may be directly prescribed to humans. Chloramphenicol is of concern because of its association with potentially fatal, dose‐independent aplastic anemia in humans. However, it is still used systemically and in ophthalmological formulations in human medicine. While use in most food animals has been eliminated, chloramphenicol residues in shrimp and fish have been an issue for importing countries (Hanekamp and Bast, 2015). The nitrofurans (furazolidone, nitrofurazone, furaltadone, nitrofurantoin) are known to be carcinogenic and mutagenic, causing mammary and ovarian tumors in animals, yet are still used for the treatment of urinary tract infections in people (Hiraku et al., 2004). According to the International Agency for Research on Cancer, there is strong evidence that metronidazole is carcinogenic in animals but not in humans, and it is frequently prescribed for people (Bendesky et al., 2002).

Potential Effects of Antimicrobial Residues on Human Gastrointestinal Microflora

The commensal bacteria that populate the gastrointestinal tract of humans provide a barrier to infectious agents, metabolize toxins and carcinogens, produce vitamins, and aid in food digestion. Ingested antimicrobial residues from food animal products have the theoretical potential for: (i) selection of resistant bacteria; (ii) domination of microbiota by pathogenic bacteria; (iii) loss of bacterial diversity; (iv) decrease or even loss of certain bacterial species; (v) increase in susceptibility to infections; and (vi) risk of new infection and/or recurrence of disease (Pilmis et al., 2020). Commensal anaerobic bacteria restrict colonization of the gut by enteric pathogens and/or the overgrowth of these pathogens after implantation. Indirectly, the commensal flora interacts with the host through antimicrobial peptide production, epithelial barrier maintenance, bile acid metabolism, and in other ways.