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Veterinary Drugs Residues: Anthelmintics
VETERINARY DRUGS RESIDUES
Anthelmintics R Romero-Gonza´lez, A Garrido Frenich, and JL Martı´nez Vidal, University of Almeria, Almeria, Spain r 2014 Elsevier Inc. All rights reserved.
Glossary Anthelmintic Any drug that destroys or causes the expulsion of parasitic intestinal worms. Chromatography Physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) whereas the other (the mobile phase) moves in a definite direction. Helminth Parasitic worm found in the intestines of vertebrates, especially roundworms, tapeworms, and flukes. Liquid chromatography A separation technique in which the mobile phase is liquid. Mass spectrometry The art of measuring atoms and molecules to determine their molecular weight.
Introduction There are several types of veterinary drugs that can be applied to livestock, such as aminoglycosides, macrolides, tetracyclines, b-agonists, and anthelmintics. Among these, anthelmintics (also called parasiticides, endectocides, and nematocides), are usually used to treat parasitic worms infections, including flatworms (tapeworms and flukes) and roundworms (nematodes), which usually infect human, livestock, and crops, affecting food production. For instance, gastrointestinal nematodes are parasites that cause important economic losses to livestock worldwide due to reduced appetite, lower body weights, reduced egg production (poultry), and death. To combat helminthiasis, several approaches have been proposed including regulation of parasitic vectors in populations, zootechnical strategies, and breeding of resistant animals. However, treatment with anthelmintics is the option most widely used as vaccines have proven ineffective till date. Before 1940s, only natural compounds including arecoline, lead arsenate, nicotine, and carbon tetrachloride were applied, but they are also toxic to the host. In 1960s and 1970s, organophosphate anthelmintics were introduced, although some of them have been removed from the market due to their toxicity. In general, broad-spectrum anthelmintics are effective, although most of them are usually used for specific infections. In fact, the term anthelmintic refers to the spectrum of pharmaceutical activity and not to a common chemical substructure of these drugs. Furthermore, the efficacy of these
Encyclopedia of Food Safety, Volume 3
Maximum residue limit Maximum concentration of residue established by a standard-setting body in a food product obtained from an animal that has received a veterinary medicine or has been exposed to a biocidal product for use in animal husbandry. Metabolite Any intermediate or product resulting by metabolism or by a metabolic process. Nematode Unsegmented worms of the phylum Nematoda with elongated rounded body, pointed at both ends. Pharmacokinetics A branch of pharmacology dedicated to the study of the time course of drug absorption, distribution, metabolism, and excretion. Transformation products Chemical species resulting from environmental or metabolic processes.
products depends on the pharmacokinetics in the host, including the complex interaction between formulation and route of administration, physicochemical properties of the compound and the metabolites generated. New compounds have been developed to overcome drug resistance to conventional anthelmintics caused by direct exposure of helminths to the drug at therapeutic doses. Moreover, the presence of anthelmintic residues in livestock may have serious consequences on consumers, and international organizations and governments have established maximum residue limits (MRLs) of these compounds in several matrices, in order to assure food safety. The unauthorized or incorrect use of anthelmintics can result in the introduction of harmful residues into the food chain. As a consequence, the development of sensitive analytical methods that fulfill the requirements established by governments and international standard-setting bodies is needed. For that purpose, advanced analytical methods mainly based on liquid chromatography (LC) coupled to mass spectrometry (MS) have been widely used for the determination of anthelmintic residues in edible tissues, in order to assure food safety as well as to increase the knowledge of the pharmacokinetics of these compounds.
Types of Anthelmintics Anthelmintics are usually classified into several types on the basis of similar chemical structure and mode of action. Basically, three main families can be distinguished: benzimidazoles, nicotinic receptor agonists, and macrocyclic lactones.
doi:10.1016/B978-0-12-378612-8.00243-2
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Veterinary Drugs Residues: Anthelmintics
Most members within each group have similar effects, although they are small chemical differences.
Figure 1. Thus, they include imidazothiazoles, such as levamisole, and tetrahydropyrimidines, such as pyrantel and morantel. They cause spastic muscle paralysis of the worm due to prolonged activation of the excitatory nicotinic acetylcholine receptors on body wall muscle. This type of compound only affects adult and larval population worms, whereas benzimidazoles are also able to kill worm eggs.
Benzimidazoles They are considered as the first chemical class of modern anthelmintics. This group includes benzimidazole carbamates, thiabendazole analogs, triclabendazole and prodrug netobimin, and a phenylguanidine derivative which is rapidly converted into albendazole in vivo. Thiabendazole was first discovered in 1961, and subsequently other benzimidazoles were introduced as broad-spectrum anthelmintics such as albendazole, fenbendazole, flubendazole, mebendazole, oxfendazole, and triclabendazole. It can be observed in Figure 1, that they have a bicyclic ring system in their structures in which benzene is fused to the 4 and 5 positions of the heterocycle (imidazole). They are the largest chemical family to treat endoparasitic diseases in domestic animals and they are widely used for prevention and treatment of parasitic infections (i.e., nematodes) in aquaculture and livestock with an excellent nematocidal activity. Furthermore, some of them have been used as pre- or postharvest fungicides. In general they interfere with the worm’s energy metabolism at the cellular level. Thus, they bind to a specific building block, b-tubulin, preventing its incorporation into certain cellular structures.
Macrocyclic Lactones (Avermectins and Milbemycins) Macrocyclic lactones are considered as the last ‘traditional’ class of anthelmintics introduced consisting of two closely related chemical groups: avermectins, which include abamectin, doramectin, ivermectin, emamectin, eprinomectin, and selamectin; and milbemycins (also called nemodectins), with moxidectin being the most representative compound. The structure of these type of compounds are shown in Figure 2, and it can be observed that avermectins are complex macrocyclics containing a 16-membered ring, linked to a disaccharide, showing different polarity, whereas, milbemycins do not have saccharide substituents. Ivermectin was the first macrocyclic lactone introduced as a anthelmintic by Merck in the 1980s. It is a semisynthetic derivative of avermectin, which is a large macrocyclic lactone fermentation product of Streptomyces avermitilis. It was the first drug to kill migrating larval stages of worms as well as adults, and therefore, other companies developed analogs such as doramectin, selamectin, etc. In general, each drug has two homologs, with the major component comprising more than 80%, which is usually used
Nicotinic Receptor Agonists This class of anthelmintics is formed by several classes of compounds with different chemical structures as shown in Benzimidazoles H3C
O
S
O
S
N H
N H
N
OCH3 N H
Albendazole
O N H
O
N
N H
N H
O S
OCH3
OCH3
Flubendazole O
N N H
Mebendazole
N H
N OCH3
N H
N
O
N H
Thiabendazole
SCH3 Cl Cl Triclabendazole
Tetrahydropyrimidine
Imidazothiazole
N
N N
S
N S
N CH3
Levamisole
Pyrantel
Figure 1 Chemical structure of benzimidazoles and nicotinic receptor agonists.
N S
Oxfendazole Cl
N H
N H
F
Fenbendazole
O
O
N
N
N CH3
H3C S Morantel
OCH3
Avermectins
OH
O
O
O
CH3
CH3
OCH3
OCH3
CH3
O
H
CH3 O
CH3 H
O
H CH3
H3C O O OH O H
H3CO
H
H3CO
HO OH
H3C
H3C O
O
CH3 O
R=
CH3 CH3 B1b CH3
H
HN
O H
CH3
O
O
H3C
CH3 OH
O O O O CH3 CH3
HO
OO
CH3
H
CH3
CH3 O
Eprinomectin
O HO CH3
H3CO CH3
H
N CH3
O O
H3C
CH3 CH3 CH3
O OH
O
CH3 OH Moxidectin H
O H
OCH3
O H3C
O O OH
OH
H O H N HO
OCH3 Ivermectin
Milbemycins
CH3
CH3
Selamectin
Veterinary Drugs Residues: Anthelmintics
OCH3
O
CH3 O O
HO H
O
OH Emamectin
O
R
H3C
B1a
O
O OH OH H
O
OH Doramectin
O HO
H
O
O
O
CH3
H
O
CH3
H
H
O
OH
O
H3CO
O H
O O OH H
CH3
CH3
O
O H3CO
O
CH3
O H3C HN
O
O H H3C
Abamectin
H3C
CH3
O
O
H3C
CH3
Figure 2 Chemical structure of macrocyclic lactones.
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Veterinary Drugs Residues: Anthelmintics
as the marker compound to calculate total residues in edible tissues. Thus, abamectin is a mixture of avermectins containing more than 80% of avermectin B1a and less than 20% of avermectin B1b, being the a and b series sec-butyl and isopropyl homologs, respectively. By contrast, ivermectin can be obtained by the reduction of abamectin (cis-hydrogenated product at the 22,23 position) and has higher activity and broader spectrum. Abamectin, ivermectin, and doramectin only contain C, H, and O, and further modifications of the avermectin structure have been obtained by introducing other atoms, such as N, to achieve different insecticidal properties with reduced withdrawal times. Meanwhile, moxidectin was introduced in 1997. Isolated from Streptomyces cyanogrise and Streptomyces hygroscopicus, it is able to kill certain worms resistant to ivermectin. Finally it must be highlighted that macrocyclic lactones have been used against a wide range of nematode and arthropod parasites in livestock. Basically they interfere with gamma-aminobutyric acid-mediated neurotransmission, causing paralysis and death of the parasite. In fact, they are the most potent killer of worms and are more persistent in their effect. Although they have a wide margin of safety for livestock, and are effective against all stages of worms, they are ineffective against cestodes (tapeworms) and trematodes (liver flukes).
Other Anthelmintic Agents In addition to these major anthelmintic classes, there are some specific compounds that are more effective for treating certain infections. Thus, emodepside, nitazoxanide, piperazine, paraherquamide, and praziquantel can also be used as anthelmintics. For instance, emodepside is effective against parasites that are resistant to benzimidazoles, levamisole, and ivermectin. Furthermore, some parasitic worms are resistant to conventional classes of anthelmintics, and therefore, new classes of anthelmintics, with new modes of action, are being proposed. Thus, a new class of anthelmintic, named aminoacetonitrile derivative (AAD) has been developed. For instance, in 2009 Novartis introduced (Zolvixs), which is the AAD monepantel. It is effective against some nematodes resistant to other drugs, because its mode of action, which is based on a nematode-specific clade of acetylcholine receptor subunits, is different. This new class is well tolerated and has low toxicity to mammals. Another example is tribendimidine, with high activity against Ascaris lumbricoides and Necator americanus. No mutagenic and clastogenic effects were observed compared with other anthelmintic agents, and therefore it is considered safe with a broad range of activities.
Anthelmintic Toxicity and Legislation Although anthelmintics are effective against worms, they may also affect the host itself based on the same biochemical mechanism that operates against the parasite or by specific mechanism to the host. The widespread use of anthelmintics implies the possibility of the presence of residues in edible tissues, and toxic effects could be associated with chronic exposure to these compounds, such as teratogenicity, congenital
malformations, diarrhea, anemia, pulmonary edema, or necrotic lymphadenopathy. Among the different classes of anthelmintics discussed in the Introduction, it has been observed that avermectins and milbemycins are usually safe, considering they usually modify certain types of communication between two nerve cells. This type of communication is characteristic in nematodes and arthropods but in vertebrates it only exists in the central nervous system and these compounds do not penetrate the brain and spinal cord. Thus, ivermectin has been well tolerated in humans and no indication of associated central nervous system toxicity has been observed at doses up to 10 times the highest US Food and Drug Administration-approved dose of 200 mg kg1. In relation to benzimidazoles, several studies have been carried out to evaluate the toxicity of these compounds, considering that they have a wide range of toxicity including teratogenic effects, congenital malformations, and anemia effects. Furthermore, some of the transformation products of this class of compounds show higher toxicity than the parent compound. For instance, hydroxymebendazole has been found to be more embryotoxic than mebendazole. Furthermore, albendazole sulfoxide has teratogenic effects in several species including humans, but at different concentrations. However, several toxicological effects between the two isomers of this compound were noted. Thus, the ( þ ) enantiomer was the predominant enantiomer in the bovine, ovine, and caprine and in which teratogenicity and clinical development abnormalities have not been reported after conventional usage. Meanwhile, European Medicines Agency (EMA) has indicated that albendazole should be regarded as a potential mutagen and no level had been identified at which there would be ‘no mutagenic risk’ to consumers of meat containing albendazole residues. However, it has been observed that there is a threshold value below which aneuploidy genesis will not occur, ensuring that conventional therapeutic use of albendazole as well as other similar compounds will not pose a risk in humans. In fact, it is recognized that there is no indication of harmful effects of this compound in humans and animals despite its worldwide use. For instance, there are no reports of a teratogenic action in humans, not even in women dosed during pregnancy. In relation to other benzimidazoles it has been noted that mebendazole has low systemic toxic potential, but at high doses it can cause anemia and liver damage. Considering the toxic effect that this type of compound can provoke, considerable effort has been focused on the development of a new generation of benzimidazoles, which are more soluble and produce minimal acute damage to the animal – even at very high doses. By contrast, praziquantel is unlikely to be of toxicological concern to humans and the European Union (EU) has granted marketing authorization of this compound for use on several animals without the need to set an MRL. Finally, it must be emphasized that levamisole, pyrantel, and morantel are more toxic, considering that they affect transmission of neural impulses of the host, but at higher doses. In fact, levamisole has the narrowest margin of safety, although toxicity is usually due to excess dosage. Thus, EMA
Veterinary Drugs Residues: Anthelmintics suggest an MRL of 10 mg kg1 of this compound in muscle, fat, and kidney from bovine, ovine, porcine, and poultry. Although these compounds have toxic effects at high doses in laboratory animals, levels detected in food are generally below toxicity thresholds and pose no risk to consumers, though special attention should be paid to the presence of this type of residues in milk.
Legislation Although anthelmintics are more toxic to parasites than mammals, food produced from treated animals should not contain residues of such drugs which would pose a food safety risk. Therefore, national and international organizations establish MRLs of these drugs in foodstuffs. In this sense, only a limited number of products are licensed for treatment of animals during the lactating period and have a MRL set under European Commission Regulation 37/2010, in order to protect consumers from risks related to these compounds. An overview of the established MRLs are listed in Table 1. It can be observed that different MRLs have been established for the anthelmintic, depending on the matrix and different marker residue. Thus, the marker residue of most benzimidazoles in foodstuffs is defined as the sum of the parent and its persistent metabolites. For instance, the marker residue definition of albendazole includes the sum of albendazole sulfoxide, albendazole sulfone, and albendazole 2-amino-sulfone, whereas for oxfendazole, the marker residue is defined as the sum of extractable residues, which maybe oxidized to oxfendazole sulfone. Furthermore, it must be emphasized that flubendazole is the only benzimidazole registered for poultry in different countries, although other benzimidazoles such as albendazole are used to treat helminth infections in poultry because of cost reasons. In fact, MRLs have been established for this molecule in several target tissues of poultry, including eggs (400 mg kg1). Like the EU, other countries have established legislation and regulations regarding human health, food safety, and environmental protection. In the USA, MRLs or tolerances for veterinary drugs in foodstuffs can be found in the Code of Federal Regulations, namely Title 21 (Food and Drugs, 500–600). For instance, ivermectin has been approved in the USA for use in several animal species, including nonlactating cattle. Doramectin and moxidectin are approved for use in beef cattle and nonlactating dairy cows, whereas eprinomectin was recently approved for use in beef and dairy cattle. Furthermore, in 2006 the USA approved febendazole for the control of nematodes in turkeys, establishing a MRL of 6000 mg kg1 in liver and 2000 mg kg1 in muscle, fenbendazole sulfone being the marker residue. In Canada, the Department of Health is in charge of administering a variety of laws, and develops and enforces regulations. It has also established MRLs for monitoring of residues of veterinary drugs in food. For instance, in this country, abamectin, doramectin, eprinomectin, ivermectin, and moxidectin are used to treat food animals such as bison, cattle, deer, sheep, swine, and reindeer against nematodes and arthropods; emamectin is used to control sea lice in fish farms and selamectin is used for the treatment of pets against
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heart- and roundworms. Only eprinomectin and moxidectin are permitted for use in dairy cattle, with no milk withholding time, considering that eprinomectin was designed to exhibit a low milk/plasma ratio, and moxidectin is less toxic with a larger acceptable daily intake. China has also established legislation regarding the presence of anthelmintics in edible tissues. For instance, the MRLs for triclabendazole in edible ruminant tissues were set at 200, 300, and 300 mg kg1 in muscle, liver, and kidney tissues of bovines respectively, whereas in goats, the MRL was set at 100 mg kg1 in the same tissues. It must be highlighted that, depending on the country or government organization, the MRLs set for the same combination compound/matrix maybe different. For instance, eprinomectin has an MRL in milk of 20 mg kg1 in Canada and EU, whereas the MRL is lower (12 mg kg1) in the USA. Meanwhile, in China, MRLs are set at 100 mg kg1 for avermectin, ivermectin, and doramectin in bovine liver and 20 and 100 mg kg1 for avermectin and doramectin, respectively in bovine muscle, whereas in the EU, MRLs in bovine liver are 20, 100, 100, and 600 mg kg1 for avermectin, ivermectin, doramectin, and eprinomectin, respectively. In relation to benzimidazoles, for instance, China and EU have established MRLs for these compounds, which range from 50 to 400 mg kg1 and 10 to 5000 mg kg1 respectively, depending on the matrix and compound. Thus, albendazole and related products (albendazole sulfoxide, sulfone, and amino sulfone) have an MRL of 100 mg kg1 in milk, muscle, and fat, whereas it is set at 5000 mg kg1 for kidney and liver. However, for flubendazole, an MRL of 400 mg kg1 was set in eggs. Finally, it must be emphasized that although most residue surveillance programs test for the presence of anthelmintic residues in edible tissues, few positive results were found. Thus, the presence of anthelmintics in muscle in several countries of Europe were tested and of 1061 beef samples, only 2.45% contained detectable amounts of anthelmintic residues at concentrations ranging from 0.2 to 171 mg kg1. However, none of the positive results were above EU MRL or action level, indicating that the risk of exposure of the European consumer to this type of drugs is negligible. Furthermore, albendazole and metabolites have been analyzed in porcine muscle and livers, but only two metabolites, albendazole sulfone and albendazole 2-amino-sulfone were detected in some porcine livers but at concentrations lower than the MRLs established by the EU.
Pharmacokinetics of Anthelmintics After administration, anthelmintics are usually absorbed into the bloodstream and transported to different parts of the body, including the liver, where they maybe metabolized through oxidation and cleavage reactions, and excreted in the feces and urine. The characterization of the kinetic behavior, metabolic fate, excretion, and residue profile of anthelmintics in the target animal will contribute to its optimized use as well as the determination of the length of the withdrawal time. However, the administration of anthelmintics does not always result in the expected therapeutic success, because
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Veterinary Drugs Residues: Anthelmintics
Table 1
MRLs for some anthelmintic drug residues in several matrices under European Commission Regulation 37/2010
Compound
Marker residue
Animal species
MRL (mg kg1)
Target tissues
Albendazole
Sum of albendazole sulfoxide, albendazole sulfone, and albendazole 2-amino sulfone, expressed as albendazole
All ruminants
100 100 1000 500 100
Muscle Fat Liver Kidney Milk
Fenbendazole
Sum of extractable residues which maybe oxidized to oxfendazole sulfone
All ruminants, porcine, and Equidae
50 50 500 50 10
Muscle Fat Liver Kidney Milk
50 50 400 300 400
Muscle Skin and fat Liver Kidney Eggs
All ruminants Flubendazole
Sum of flubendazole and (2-amino 1H-benzimidazol-5yl)(4fluorophenyl)methanone
Poultry and porcine
Flubendazole
Poultry
Mebendazolea
Sum of mebendazole methyl (5-(1-hydroxy, 1-phenyl)methyl-1H-benzimidazol-2-yl)carbamate and (2-amino-1H-benzimidazol-5-yl)phenylmethanone, expressed as mebendazole equivalents
Ovine, caprine, and Equidae
60 60 400 60
Muscle Fat Liver Kidney
Oxfendazole
Sum of extractable residues which maybe oxidized to oxfendazole sulfone
All ruminants, porcine, and Equidae
50 50 500 50 10
Muscle Fat Liver Kidney Milk
All ruminants Thiabendazole
Sum of thiabendazole and 5-hydroxy thiabendazole
Bovine and caprine
100 100 100 100 100
Muscle Fat Liver Kidney Milk
Triclabendazolea
Sum of extractable residues which maybe oxidized to keto-triclabendazole
All ruminants
225 100 250 150
Muscle Fat Liver Kidney
Abamectina
Avermectin B1a
Bovine
10 20 20 50 25 20
Fat Liver Muscle Fat Liver Kidney
Ovine
Doramectin
Doramectin
All mammalian food producing species
40 150 100 60
Muscle Fat Liver Kidney
Emamectin
Emamectin B1a
Fin fish
100
Muscle and skin
Eprinomectin
Eprinomectin B1a
Bovine
50 250 1500 300 20
Ivermectina
22,23-dihydro-avermectin B1a
All mammalian food producing species
100 100 30
Muscle Fat Liver Kidney Milk Fat Liver Kidney (Continued )
Veterinary Drugs Residues: Anthelmintics
Table 1
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Continued
Compound
Marker residue
Animal species
Moxidectin
Moxidectin
Bovine, ovine, and Equidae
Bovine and ovine
MRL (mg kg1)
Target tissues
50 500 100 50 40
Muscle Fat Liver Kidney Milk
Levamisoleb
Levamisole
Bovine, ovine, porcine, and poultry
10 10 100 10
Muscle Fat Liver Kidney
Morantel
Sum of residues which may be hydrolyzed to N-methyl1,3-propanediamine and expressed as morantel equivalents
All ruminants
100 100 800 200 50
Muscle Fat Liver Kidney Milk
a
Not for use in animals from which milk is produced for human consumption. Not for use in animals from which milk or eggs is produced for human consumption. Abbreviation: MRLs, maximum residue limits. b
host-related factors can modify pharmacokinetic behavior and efficacy of the selected compound. Thus, the factors affecting drug pharmacokinetics can be distinguished between two groups: interindividual (species, sex, and genetics) and intraindividual (age, gestation, stress, medication, food, and environment) factors. Interindividual factors remain constant during the life of an organism, whereas intraindividual ones change during the life. Basically they are related to physiological and pathological state of an organism. For instance, it has been observed that the metabolic interconversion between fenbendazole sulfide and sulfoxide differs in horses compared to ruminants, and the bioavailability and residence time were lower and shorter in horses than in ruminants. Extensive work has been carried out to study the pharmacokinetic and metabolism of certain benzimidazoles. In general, benzimidazoles are limited in absorption from the gastrointestinal tract due to the poor solubility of these drugs and the absorption is generally fast (from 2–7 h with flubendazole to 6–30 h for other compounds). Mebendazole is absorbed from the gastrointestinal tract and is intensively metabolized in sheep through ketoreduction and decarbamylation followed by conjugation. Furthermore, it is known that the absorption and the efficacy are influenced by their administration with food. Thus, the bioavailability of benzimidazole sulfides is markedly reduced in ruminants which have had unrestricted access to food compared with those given restricted access prior to treatment. Sometimes in order to minimize the metabolic oxidation of benzimidazole sulfides and sulfoxides to sulfones, metabolic inhibitors are added. For instance, piperonyl butoxide has been added to modify the pharmacokinetic profile of febendazole. The pharmacokinetics of albendazole has been studied in several animal species, although few studies have been carried out in poultry. In general, it is rapidly metabolized in all species, and the plasma levels of oxidized metabolites (sulfoxide and sulfone) are much higher than the parent drug. In
fact, the sulfoxide transformation product is considered the active metabolite responsible for the therapeutic activity of albendazole, whereas albendazole sulfone is considered an inactive compound. Furthermore, it can be noted that the coadministration of other drugs such as cimetidine and praziquantel affects the kinetics of albendazole probably by the induction of the second sulfonation step. In poultry it has been observed that the maximum concentration was obtained at 1 h and it was only detected for 6 h after treatment. However, albendazole sulfoxide was detected for 25 h after treatment. Furthermore, these results were compared with those obtained in other species such as calf, dogs, goats, pigs, and sheep, and the maximum concentration (Cmax) and time to reach it (Tmax) are lower in chickens. Therefore, it was concluded that albendazole was absorbed to a greater extent and more quickly in poultry than in mammalian species. Finally, only albendazole metabolites can be measured in eggs if single oral dose was given. However, if the treatment is applied during 7 days, albendazole and its metabolites can be simultaneously detected in yolk, whereas albendazole cannot be detected in egg albumin. The absence of albendazole in albumin can be explained considering the relatively higher lipid solubility of albendazole compared with its metabolites. In the same way, it has been observed that the concentration of flubendazole in egg yolk was about five times higher than in the albumin. Other compounds that have been studied are mebendazole and triclabendazole. Thus, some metabolites such as hydroxymebendazole (reduced metabolite) and aminomebendazole (hydrolyzed metabolite) can be found in food from animals treated with mebendazole, with the highest concentrations in liver of the reduced metabolite. In fact, this metabolite can be detected in liver for 14 days after administration. In relation to triclabendazole, it is rapidly removed from blood by the liver, and it is oxidized to the sulfoxide and sulfone, which are the main metabolites detected in plasma.
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Veterinary Drugs Residues: Anthelmintics
For nicotinic receptor agonists, it has been shown that the absorption and excretion of levamisole is fast and it is not affected by the route of administration because of its high solubility. It has been observed that the levels of this drug in blood cattle are maximum in less than 1 h, and 90% of the total dose is excreted in urine in 24 h. However, pyrantel is poorly soluble in water and its metabolism is fast, with the metabolites excreted rapidly in the urine. Morantel is absorbed rapidly from the upper small intestine of sheep and metabolized rapidly in liver (17% of the initial dose is excreted in the urine as metabolites within 96 h after dosing). In general, macrocyclic lactones are hydrophobic and they are distributed throughout the body and some of them can be concentrated in adipose tissue. Depending on the species, they have an elimination half-life of 32–178 h. In this sense, a pharmacokinetic study of emamectin benzoate has been carried out in Atlantic salmon and it was shown that emamectin B1a was the major residue component (480% in edible tissues). Therefore, this compound has been used as marker residue in edible tissues of Atlantic salmon for monitoring emamectin benzoate. Further metabolic studies have been carried out with other anthelmintic drugs. Thus, the metabolism of praziquantel has been studied in several tissues of rainbow trout maintained at different temperatures (12 and 18 1C), observing that absorption was faster at higher temperatures, whereas the elimination of the drug was less dependent on the temperature. Thus, after 32 h, 67–96% of the drug has been eliminated from the tissues. In relation to the elimination of anthelmintics, biliary secretion is an important pathway for elimination of macrocyclic lactones. Thus, they are primarily excreted in the feces and the remainder (o10%) in the urine. However, more lipophilic compounds are also excreted in milk. Biliary route is the most important pathway for secretion and recycling of benzimidazoles to the gastrointestinal tract, as well as praziquantel, which is partly excreted by bile fluid and partly as water soluble metabolites through the kidneys. Furthermore, the intestinal clearance of albendazole sulfoxide exhibits a stereoselective elimination of the ( ) form. The excretion of anthelmintics in the feces of livestock has given rise to concern as avermectins have adverse effects in dipteran flies and coleopteran beetles. However, benzimidazoles are unlikely to affect dung-dwelling arthropods and the environmental impact is not limited to specific effect on scavenger insects. For instance, the fecal concentration of febendazole and its metabolites was determined in horses, noting that no drug could be detected at 12 h post dosing. The maximal concentration was monitored at 24 h, and no target compounds were detected after 72 h. In feces the highest concentration was observed for the parent compound.
Analytical Methodology Anthelmintic residues are mainly determined applying LC techniques coupled to several detectors, although some benzimidazoles have been determined by gas chromatography (GC). However, GC is not suitable for quantification because
of thermal decomposition in the chromatograph or nonreproducible ionization in the source of the MS. Benzimidazoles could be detected using ultraviolet (UV) or fluorescence detection, considering that most of them (albendazole, flubendazole, and thiabendazole) have natural fluorescence. Furthermore in relation to UV, the use of diode array is very useful, considering that individual analytes have characteristic spectra. Levamisole can also be detected by UV. Meanwhile, avermectins can be detected using fluorescence detection including a derivatization reaction using trifluoroacetic acid, and this methodology has been used for the determination of emamectin in Atlantic salmon. Although the limits of detection provided by fluorescence detectors are below the MRLs established by governments, there are some problems related to the derivatization step such as instability of fluorescent derivatives, slow and/or incomplete formation of derivatives, and the low reproducibility of the results. Furthermore, and according to current legislation in Europe, MS should be used for confirmation purposes in the determination of residues in foodstuffs. Therefore, conventional detection has been replaced by MS applying several detectors, such as single quadrupole, triple quadrupole, time of flight (TOF), and Orbitrap. In this sense, several ionization techniques, such as electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization, have been used. Moreover, the application of MS analyzers allows the identification and quantification of metabolites of anthelmintics in the samples. However, in the case of avermectins, the use of MS is not a straightforward strategy. These compounds are heavy (molecular weight ranging from 600 to 900 Da), but most of them only contain one nitrogen atom in their structure. In fact, ivermectin, doramectin, and abamectin only contain C, H, and O, which have negative effects on the ionization. This implies that several assays have been carried out to study the best ionization of these compounds. For instance, it has been indicated that for abamectin, the determination of ammonium adduct by ESI is the best option, although other studies have indicated that the use of APCI with negative ionization provided [M H] as the predominant ion for ivermectins. In general, it has been reported that avermectins, which are devoid of nitrogen atoms, produce significantly higher abundances for [M þ Na] þ than [M þ H] þ . Furthermore, another difficulty is that sodium adducts are difficult to break and therefore several authors have evaluated alternative ionization processes, observing that negative ESI or APCI could overcome many of these problems. In relation to the LC separation, C18 or C8 columns are mainly used. Moreover, the selection of the mobile phase is critical for the correct ionization of the target compounds. Acetonitrile and methanol are usually used for the determination of anthelmintics, although for avermectins, the use of methanol increases the sensitivity of the compounds in relation to acetonitrile. The pH of the mobile phase maybe critical in order to get suitable chromatographic separation, and ammonium formate or acetate could provide suitable results. Furthermore, in the past decade, ultra highperformance LC has been widely used in order to reduce analysis time, obtaining narrower peaks, and increasing sample
Veterinary Drugs Residues: Anthelmintics
throughput. Furthermore, the combination of LC with tandem MS (MS/MS) allows the selective determination of these compounds. Finally, it must be emphasized that sample preparation is the critical step for the simultaneous determination of several analytes in complex matrices. In relation to the extraction of these compounds from several matrices, different methodologies have been applied. Thus, liquid–liquid partition using acetonitrile has been used, allowing the deproteination of proteins. Solid phase extraction (SPE) has also been evaluated, utilizing C18, polymer sorbents or strong cation cartridges. However, they are laborious and time-consuming, and new approaches could be applied. Thus, immunoaffinity chromatography (IAC) could be used as a valuable tool for cleanup, allowing a fast and selective extraction of avermectins from extracts of bovine and swine liver. Furthermore, the combination of IAC with LC–MS provides sensitive and selective methods. However, matrix solid phase dispersion has been applied for the determination of 37 anthelmintic drugs and metabolites in muscle. In addition, pressurized liquid extraction has been applied for the determination of benzimidazoles in tissues of animals such as swine, cattle, sheep, and chickens. Finally, it must be noted that low temperature clean-up has been used for the determination of ivermectins and moxidectin in bovine muscle. Furthermore, benzimidazoles are usually extracted by relatively polar aqueous extraction solutions. However, some metabolites are covalently bound to matrix components (liver or kidney) and chemical or enzymatic hydrolysis should be included in the extraction procedure, although simplified methods using anhydrous acetonitrile could be used. Nowadays, generic extraction conditions can be applied for the simultaneous extraction of several types of compounds in a single analytical method. However, the inclusion of some anthelmintics such as avermectins is not easy considering the low sensitivity obtained by the use of LC–MS/MS, and special attention should be paid to these compounds, when they are included in multiresidue methods. In this sense, QuEChERS approach (acronym for Quick, Easy, Cheap, Effective, Rugged and Safe), which is based on acetonitrile extraction followed by a phase separation induced by the addition of salts and a cleanup step based on dispersive SPE, has been successfully applied for the extraction of this type of compounds from edible tissues.
Conclusions and Future Trends As described in this article, the use of anthelmintics to treat helminth parasites and the resulting residues in edible tissues is a problem of major concern. Furthermore, the development of resistance to the currently used anthelmintics is an issue of increasing importance. In this sense, the development of new compounds, such as AAD, are needed in order to improve the characteristics of conventional classes of anthelmintics such as lower toxicity, favorable pharmacokinetic properties, and broad-spectrum capacities. Furthermore, additional studies should be carried out to more thoroughly examine the pharmacokinetics and metabolism of these compounds, especially during pregnancy. The possibility of elimination of some nonpolar compounds by
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milk should be also evaluated in order to minimize the risk of the presence of anthelmintic residues in this matrix. To assure a sustainable inventory of effective anthelmintics, monitoring of drug resistance and research with combinations of current or future drugs will be necessary. Moreover and despite the evaluation of the metabolism of anthelmintics in vertebrates, much attention should be paid to the evaluation of metabolism of these products in parasitic helminths. In relation to analytical methodologies, current methods should include known metabolites in routine analyses in order to fulfill current legislation. However, anthelmintic metabolism is complex, and it is necessary to develop and validate new analytical methods that allow the accurate determination of new metabolites in foodstuffs. For that purpose, high resolution MS analyzers such as TOF or Orbitrap are very helpful, considering that they acquire full-scan spectra, and target, nontarget and unknown analysis that could be carried out in one single analysis. Finally and considering the different MRLs established by several countries or organizations for the same combination anthelmintic/matrix, international legislation should be harmonized, in order to avoid some problems during export.
Acknowledgments The authors gratefully acknowledge the Spanish Ministry of Economy and Competitiveness (MINECO-FEDER) for financial support (Project Ref. AGL2010-21370). RRG is also grateful for personal funding through the Ramo´n y Cajal Program (MINECO and European Social Fund).
See also: Analytical Methods: Overview of Methods of Analysis for Chemical Hazards. Disciplines Associated with Food Safety: Food Safety Toxicology. Food Safety Assurance Systems: Food Safety and Quality Management Systems. Institutions Involved in Food Safety: FAO/WHO Codex Alimentarius Commission (CAC); Food and Agriculture Organization of the United Nations (FAO); World Health Organization (WHO). Public Health Measures: Food Control and Public Health Laboratories; Fundamentals of Food Legislation. Risk Analysis: Risk Assessment: Chemical Hazards. Veterinary Drugs Residues: Antibacterials; Control of Helminths; Veterinary Drugs – General
Further Reading Bartram DJ, Leathwick DM, Taylor MA, Geurden T, and Maeder SJ (2012) The role of combination anthelmintic formulations in the sustainable control of sheep nematodes. Veterinary Parasitology 186: 151–158. Beech RN, Skuce DJ, Martin RJ, Prichard RK, and Gilleard JS (2011) Anthelmintic resistance: Markers for resistance, or susceptibility? Parasitology 138: 160–174. Besier B (2007) New anthelmintics for livestock: The time is right. Trends in Parasitology 23: 21–24. Danaher M, De Ruyck H, Crooks SRH, Dowling G, and O’Keeffe M (2007) Review of methodology for the determination of benzimidazole residues in biological matrices. Journal of Chromatography B 845: 1–37. Dayan AD (2003) Albendazole, mebendazole and praziquantel. Review of nonclinical toxicity and pharmacokinetics. Acta Tropica 86: 141–159.
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Garrrido Frenich A, Plaza-Bolan˜os P, Aguilera-Luiz MM, and Martı´nez-Vidal JL (2010) Veterinary Drugs and Growth-Promoting Agent Analyses. New York: Nova Publishers. Holden-Dye L and Walker RJ (2007) Anthelmintic drugs. WormBook: The Online Review of C. elegans Biology 1–13. Kaminsky R, Ducray P, Jung M, et al. (2008) A new class of anthelmintics effective against drug-resistance nematodes. Nature 452: 176–181. Kaufmann A, Butcher P, Maden K, Walker S, and Widmer M (2011) Quantification of anthelmintic drug residues in milk and muscle tissues by liquid chromatography coupled to Orbitrap and liquid chromatography coupled to tandem mass spectrometry. Talanta 85: 991–1000. Keiser J and Utzinger J (2010) The drugs we have and the drugs we need against major helminth infections. Advances in Parasitology 73: 197–230. Krizova-Forstova V, Lamka J, Cvilink V, Hanusova V, and Skalova L (2011) Factors affecting pharmacokinetics of benzimidazole anthelmintics in food-producing animals: The consequences and potential risk. Research in Veterinary Science 91: 333–341. Lanusse CE, Alvarez LI, Sallovitz JM, Mottier ML, and Sanchez Bruni SF (2009) Antinematodal drugs. In: Riviere JE and Papich MG (eds.) Veterinary Pharmacology and Therapeutics, 9th edn., pp. 1053–1094. New Jersey: Wiley-Blackwell. Lanusse CE, Lifschitz AL, and Imperiales FA (2009) Macrocyclic latctones: Endectocide compounds. In: Riviere JE and Papich MG (eds.) Veterinary Pharmacology and Therapeutics, 9th edn., pp. 1053–1094. New Jersey: WileyBlackwell. Sutherland IA and Leathwick DM (2011) Anthelmintic resistance in nematode parasites of cattle: A global issue?Trends in Parasitology 27: 176–181.
Waller PJ (2006) From discovery to development: Current industry perspectives for the development of novel methods of helminth control in livestock. Veterinary Parasitology 139: 1–14.
Relevant Websites http://www.hc-sc.gc.ca/dhp-mps/vet/legislation/guide-ld/vich/guide-ligne-eng.php Canadian Health Ministry. www.codexalimentarius.net/vetdrugs/data/ Codex Alimentarius. http://www.ema.europa.eu/ema/ European Medicines Agency. http://www.fao.org/food/food-safety-quality/scientific-advice/jecfa/jecfa-vetdrugs/en/ Food and Agricultural Organization of the United Nations. http://www.vichsec.org/pdf/Gl07_st7final þ corr.pdf International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products. http://www.merckvetmanual.com/mvm/index.jspcfile=htm/bc/toc_191500.htm Merck Veterinary Manual. http://www.mrldatabase.com United States Department of Agriculture. http://www.fda.gov/AnimalVeterinary/default.htm United States Food and Drug Administration.
Anthelmintics R Romero-Gonza´lez, A Garrido Frenich, and JL Martı´nez Vidal, University of Almeria, Almeria, Spain r 2014 Elsevier Inc. All rights reserved.
Glossary Anthelmintic Any drug that destroys or causes the expulsion of parasitic intestinal worms. Chromatography Physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) whereas the other (the mobile phase) moves in a definite direction. Helminth Parasitic worm found in the intestines of vertebrates, especially roundworms, tapeworms, and flukes. Liquid chromatography A separation technique in which the mobile phase is liquid. Mass spectrometry The art of measuring atoms and molecules to determine their molecular weight.
Introduction There are several types of veterinary drugs that can be applied to livestock, such as aminoglycosides, macrolides, tetracyclines, b-agonists, and anthelmintics. Among these, anthelmintics (also called parasiticides, endectocides, and nematocides), are usually used to treat parasitic worms infections, including flatworms (tapeworms and flukes) and roundworms (nematodes), which usually infect human, livestock, and crops, affecting food production. For instance, gastrointestinal nematodes are parasites that cause important economic losses to livestock worldwide due to reduced appetite, lower body weights, reduced egg production (poultry), and death. To combat helminthiasis, several approaches have been proposed including regulation of parasitic vectors in populations, zootechnical strategies, and breeding of resistant animals. However, treatment with anthelmintics is the option most widely used as vaccines have proven ineffective till date. Before 1940s, only natural compounds including arecoline, lead arsenate, nicotine, and carbon tetrachloride were applied, but they are also toxic to the host. In 1960s and 1970s, organophosphate anthelmintics were introduced, although some of them have been removed from the market due to their toxicity. In general, broad-spectrum anthelmintics are effective, although most of them are usually used for specific infections. In fact, the term anthelmintic refers to the spectrum of pharmaceutical activity and not to a common chemical substructure of these drugs. Furthermore, the efficacy of these
Encyclopedia of Food Safety, Volume 3
Maximum residue limit Maximum concentration of residue established by a standard-setting body in a food product obtained from an animal that has received a veterinary medicine or has been exposed to a biocidal product for use in animal husbandry. Metabolite Any intermediate or product resulting by metabolism or by a metabolic process. Nematode Unsegmented worms of the phylum Nematoda with elongated rounded body, pointed at both ends. Pharmacokinetics A branch of pharmacology dedicated to the study of the time course of drug absorption, distribution, metabolism, and excretion. Transformation products Chemical species resulting from environmental or metabolic processes.
products depends on the pharmacokinetics in the host, including the complex interaction between formulation and route of administration, physicochemical properties of the compound and the metabolites generated. New compounds have been developed to overcome drug resistance to conventional anthelmintics caused by direct exposure of helminths to the drug at therapeutic doses. Moreover, the presence of anthelmintic residues in livestock may have serious consequences on consumers, and international organizations and governments have established maximum residue limits (MRLs) of these compounds in several matrices, in order to assure food safety. The unauthorized or incorrect use of anthelmintics can result in the introduction of harmful residues into the food chain. As a consequence, the development of sensitive analytical methods that fulfill the requirements established by governments and international standard-setting bodies is needed. For that purpose, advanced analytical methods mainly based on liquid chromatography (LC) coupled to mass spectrometry (MS) have been widely used for the determination of anthelmintic residues in edible tissues, in order to assure food safety as well as to increase the knowledge of the pharmacokinetics of these compounds.
Types of Anthelmintics Anthelmintics are usually classified into several types on the basis of similar chemical structure and mode of action. Basically, three main families can be distinguished: benzimidazoles, nicotinic receptor agonists, and macrocyclic lactones.
doi:10.1016/B978-0-12-378612-8.00243-2
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Veterinary Drugs Residues: Anthelmintics
Most members within each group have similar effects, although they are small chemical differences.
Figure 1. Thus, they include imidazothiazoles, such as levamisole, and tetrahydropyrimidines, such as pyrantel and morantel. They cause spastic muscle paralysis of the worm due to prolonged activation of the excitatory nicotinic acetylcholine receptors on body wall muscle. This type of compound only affects adult and larval population worms, whereas benzimidazoles are also able to kill worm eggs.
Benzimidazoles They are considered as the first chemical class of modern anthelmintics. This group includes benzimidazole carbamates, thiabendazole analogs, triclabendazole and prodrug netobimin, and a phenylguanidine derivative which is rapidly converted into albendazole in vivo. Thiabendazole was first discovered in 1961, and subsequently other benzimidazoles were introduced as broad-spectrum anthelmintics such as albendazole, fenbendazole, flubendazole, mebendazole, oxfendazole, and triclabendazole. It can be observed in Figure 1, that they have a bicyclic ring system in their structures in which benzene is fused to the 4 and 5 positions of the heterocycle (imidazole). They are the largest chemical family to treat endoparasitic diseases in domestic animals and they are widely used for prevention and treatment of parasitic infections (i.e., nematodes) in aquaculture and livestock with an excellent nematocidal activity. Furthermore, some of them have been used as pre- or postharvest fungicides. In general they interfere with the worm’s energy metabolism at the cellular level. Thus, they bind to a specific building block, b-tubulin, preventing its incorporation into certain cellular structures.
Macrocyclic Lactones (Avermectins and Milbemycins) Macrocyclic lactones are considered as the last ‘traditional’ class of anthelmintics introduced consisting of two closely related chemical groups: avermectins, which include abamectin, doramectin, ivermectin, emamectin, eprinomectin, and selamectin; and milbemycins (also called nemodectins), with moxidectin being the most representative compound. The structure of these type of compounds are shown in Figure 2, and it can be observed that avermectins are complex macrocyclics containing a 16-membered ring, linked to a disaccharide, showing different polarity, whereas, milbemycins do not have saccharide substituents. Ivermectin was the first macrocyclic lactone introduced as a anthelmintic by Merck in the 1980s. It is a semisynthetic derivative of avermectin, which is a large macrocyclic lactone fermentation product of Streptomyces avermitilis. It was the first drug to kill migrating larval stages of worms as well as adults, and therefore, other companies developed analogs such as doramectin, selamectin, etc. In general, each drug has two homologs, with the major component comprising more than 80%, which is usually used
Nicotinic Receptor Agonists This class of anthelmintics is formed by several classes of compounds with different chemical structures as shown in Benzimidazoles H3C
O
S
O
S
N H
N H
N
OCH3 N H
Albendazole
O N H
O
N
N H
N H
O S
OCH3
OCH3
Flubendazole O
N N H
Mebendazole
N H
N OCH3
N H
N
O
N H
Thiabendazole
SCH3 Cl Cl Triclabendazole
Tetrahydropyrimidine
Imidazothiazole
N
N N
S
N S
N CH3
Levamisole
Pyrantel
Figure 1 Chemical structure of benzimidazoles and nicotinic receptor agonists.
N S
Oxfendazole Cl
N H
N H
F
Fenbendazole
O
O
N
N
N CH3
H3C S Morantel
OCH3
Avermectins
OH
O
O
O
CH3
CH3
OCH3
OCH3
CH3
O
H
CH3 O
CH3 H
O
H CH3
H3C O O OH O H
H3CO
H
H3CO
HO OH
H3C
H3C O
O
CH3 O
R=
CH3 CH3 B1b CH3
H
HN
O H
CH3
O
O
H3C
CH3 OH
O O O O CH3 CH3
HO
OO
CH3
H
CH3
CH3 O
Eprinomectin
O HO CH3
H3CO CH3
H
N CH3
O O
H3C
CH3 CH3 CH3
O OH
O
CH3 OH Moxidectin H
O H
OCH3
O H3C
O O OH
OH
H O H N HO
OCH3 Ivermectin
Milbemycins
CH3
CH3
Selamectin
Veterinary Drugs Residues: Anthelmintics
OCH3
O
CH3 O O
HO H
O
OH Emamectin
O
R
H3C
B1a
O
O OH OH H
O
OH Doramectin
O HO
H
O
O
O
CH3
H
O
CH3
H
H
O
OH
O
H3CO
O H
O O OH H
CH3
CH3
O
O H3CO
O
CH3
O H3C HN
O
O H H3C
Abamectin
H3C
CH3
O
O
H3C
CH3
Figure 2 Chemical structure of macrocyclic lactones.
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Veterinary Drugs Residues: Anthelmintics
as the marker compound to calculate total residues in edible tissues. Thus, abamectin is a mixture of avermectins containing more than 80% of avermectin B1a and less than 20% of avermectin B1b, being the a and b series sec-butyl and isopropyl homologs, respectively. By contrast, ivermectin can be obtained by the reduction of abamectin (cis-hydrogenated product at the 22,23 position) and has higher activity and broader spectrum. Abamectin, ivermectin, and doramectin only contain C, H, and O, and further modifications of the avermectin structure have been obtained by introducing other atoms, such as N, to achieve different insecticidal properties with reduced withdrawal times. Meanwhile, moxidectin was introduced in 1997. Isolated from Streptomyces cyanogrise and Streptomyces hygroscopicus, it is able to kill certain worms resistant to ivermectin. Finally it must be highlighted that macrocyclic lactones have been used against a wide range of nematode and arthropod parasites in livestock. Basically they interfere with gamma-aminobutyric acid-mediated neurotransmission, causing paralysis and death of the parasite. In fact, they are the most potent killer of worms and are more persistent in their effect. Although they have a wide margin of safety for livestock, and are effective against all stages of worms, they are ineffective against cestodes (tapeworms) and trematodes (liver flukes).
Other Anthelmintic Agents In addition to these major anthelmintic classes, there are some specific compounds that are more effective for treating certain infections. Thus, emodepside, nitazoxanide, piperazine, paraherquamide, and praziquantel can also be used as anthelmintics. For instance, emodepside is effective against parasites that are resistant to benzimidazoles, levamisole, and ivermectin. Furthermore, some parasitic worms are resistant to conventional classes of anthelmintics, and therefore, new classes of anthelmintics, with new modes of action, are being proposed. Thus, a new class of anthelmintic, named aminoacetonitrile derivative (AAD) has been developed. For instance, in 2009 Novartis introduced (Zolvixs), which is the AAD monepantel. It is effective against some nematodes resistant to other drugs, because its mode of action, which is based on a nematode-specific clade of acetylcholine receptor subunits, is different. This new class is well tolerated and has low toxicity to mammals. Another example is tribendimidine, with high activity against Ascaris lumbricoides and Necator americanus. No mutagenic and clastogenic effects were observed compared with other anthelmintic agents, and therefore it is considered safe with a broad range of activities.
Anthelmintic Toxicity and Legislation Although anthelmintics are effective against worms, they may also affect the host itself based on the same biochemical mechanism that operates against the parasite or by specific mechanism to the host. The widespread use of anthelmintics implies the possibility of the presence of residues in edible tissues, and toxic effects could be associated with chronic exposure to these compounds, such as teratogenicity, congenital
malformations, diarrhea, anemia, pulmonary edema, or necrotic lymphadenopathy. Among the different classes of anthelmintics discussed in the Introduction, it has been observed that avermectins and milbemycins are usually safe, considering they usually modify certain types of communication between two nerve cells. This type of communication is characteristic in nematodes and arthropods but in vertebrates it only exists in the central nervous system and these compounds do not penetrate the brain and spinal cord. Thus, ivermectin has been well tolerated in humans and no indication of associated central nervous system toxicity has been observed at doses up to 10 times the highest US Food and Drug Administration-approved dose of 200 mg kg1. In relation to benzimidazoles, several studies have been carried out to evaluate the toxicity of these compounds, considering that they have a wide range of toxicity including teratogenic effects, congenital malformations, and anemia effects. Furthermore, some of the transformation products of this class of compounds show higher toxicity than the parent compound. For instance, hydroxymebendazole has been found to be more embryotoxic than mebendazole. Furthermore, albendazole sulfoxide has teratogenic effects in several species including humans, but at different concentrations. However, several toxicological effects between the two isomers of this compound were noted. Thus, the ( þ ) enantiomer was the predominant enantiomer in the bovine, ovine, and caprine and in which teratogenicity and clinical development abnormalities have not been reported after conventional usage. Meanwhile, European Medicines Agency (EMA) has indicated that albendazole should be regarded as a potential mutagen and no level had been identified at which there would be ‘no mutagenic risk’ to consumers of meat containing albendazole residues. However, it has been observed that there is a threshold value below which aneuploidy genesis will not occur, ensuring that conventional therapeutic use of albendazole as well as other similar compounds will not pose a risk in humans. In fact, it is recognized that there is no indication of harmful effects of this compound in humans and animals despite its worldwide use. For instance, there are no reports of a teratogenic action in humans, not even in women dosed during pregnancy. In relation to other benzimidazoles it has been noted that mebendazole has low systemic toxic potential, but at high doses it can cause anemia and liver damage. Considering the toxic effect that this type of compound can provoke, considerable effort has been focused on the development of a new generation of benzimidazoles, which are more soluble and produce minimal acute damage to the animal – even at very high doses. By contrast, praziquantel is unlikely to be of toxicological concern to humans and the European Union (EU) has granted marketing authorization of this compound for use on several animals without the need to set an MRL. Finally, it must be emphasized that levamisole, pyrantel, and morantel are more toxic, considering that they affect transmission of neural impulses of the host, but at higher doses. In fact, levamisole has the narrowest margin of safety, although toxicity is usually due to excess dosage. Thus, EMA
Veterinary Drugs Residues: Anthelmintics suggest an MRL of 10 mg kg1 of this compound in muscle, fat, and kidney from bovine, ovine, porcine, and poultry. Although these compounds have toxic effects at high doses in laboratory animals, levels detected in food are generally below toxicity thresholds and pose no risk to consumers, though special attention should be paid to the presence of this type of residues in milk.
Legislation Although anthelmintics are more toxic to parasites than mammals, food produced from treated animals should not contain residues of such drugs which would pose a food safety risk. Therefore, national and international organizations establish MRLs of these drugs in foodstuffs. In this sense, only a limited number of products are licensed for treatment of animals during the lactating period and have a MRL set under European Commission Regulation 37/2010, in order to protect consumers from risks related to these compounds. An overview of the established MRLs are listed in Table 1. It can be observed that different MRLs have been established for the anthelmintic, depending on the matrix and different marker residue. Thus, the marker residue of most benzimidazoles in foodstuffs is defined as the sum of the parent and its persistent metabolites. For instance, the marker residue definition of albendazole includes the sum of albendazole sulfoxide, albendazole sulfone, and albendazole 2-amino-sulfone, whereas for oxfendazole, the marker residue is defined as the sum of extractable residues, which maybe oxidized to oxfendazole sulfone. Furthermore, it must be emphasized that flubendazole is the only benzimidazole registered for poultry in different countries, although other benzimidazoles such as albendazole are used to treat helminth infections in poultry because of cost reasons. In fact, MRLs have been established for this molecule in several target tissues of poultry, including eggs (400 mg kg1). Like the EU, other countries have established legislation and regulations regarding human health, food safety, and environmental protection. In the USA, MRLs or tolerances for veterinary drugs in foodstuffs can be found in the Code of Federal Regulations, namely Title 21 (Food and Drugs, 500–600). For instance, ivermectin has been approved in the USA for use in several animal species, including nonlactating cattle. Doramectin and moxidectin are approved for use in beef cattle and nonlactating dairy cows, whereas eprinomectin was recently approved for use in beef and dairy cattle. Furthermore, in 2006 the USA approved febendazole for the control of nematodes in turkeys, establishing a MRL of 6000 mg kg1 in liver and 2000 mg kg1 in muscle, fenbendazole sulfone being the marker residue. In Canada, the Department of Health is in charge of administering a variety of laws, and develops and enforces regulations. It has also established MRLs for monitoring of residues of veterinary drugs in food. For instance, in this country, abamectin, doramectin, eprinomectin, ivermectin, and moxidectin are used to treat food animals such as bison, cattle, deer, sheep, swine, and reindeer against nematodes and arthropods; emamectin is used to control sea lice in fish farms and selamectin is used for the treatment of pets against
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heart- and roundworms. Only eprinomectin and moxidectin are permitted for use in dairy cattle, with no milk withholding time, considering that eprinomectin was designed to exhibit a low milk/plasma ratio, and moxidectin is less toxic with a larger acceptable daily intake. China has also established legislation regarding the presence of anthelmintics in edible tissues. For instance, the MRLs for triclabendazole in edible ruminant tissues were set at 200, 300, and 300 mg kg1 in muscle, liver, and kidney tissues of bovines respectively, whereas in goats, the MRL was set at 100 mg kg1 in the same tissues. It must be highlighted that, depending on the country or government organization, the MRLs set for the same combination compound/matrix maybe different. For instance, eprinomectin has an MRL in milk of 20 mg kg1 in Canada and EU, whereas the MRL is lower (12 mg kg1) in the USA. Meanwhile, in China, MRLs are set at 100 mg kg1 for avermectin, ivermectin, and doramectin in bovine liver and 20 and 100 mg kg1 for avermectin and doramectin, respectively in bovine muscle, whereas in the EU, MRLs in bovine liver are 20, 100, 100, and 600 mg kg1 for avermectin, ivermectin, doramectin, and eprinomectin, respectively. In relation to benzimidazoles, for instance, China and EU have established MRLs for these compounds, which range from 50 to 400 mg kg1 and 10 to 5000 mg kg1 respectively, depending on the matrix and compound. Thus, albendazole and related products (albendazole sulfoxide, sulfone, and amino sulfone) have an MRL of 100 mg kg1 in milk, muscle, and fat, whereas it is set at 5000 mg kg1 for kidney and liver. However, for flubendazole, an MRL of 400 mg kg1 was set in eggs. Finally, it must be emphasized that although most residue surveillance programs test for the presence of anthelmintic residues in edible tissues, few positive results were found. Thus, the presence of anthelmintics in muscle in several countries of Europe were tested and of 1061 beef samples, only 2.45% contained detectable amounts of anthelmintic residues at concentrations ranging from 0.2 to 171 mg kg1. However, none of the positive results were above EU MRL or action level, indicating that the risk of exposure of the European consumer to this type of drugs is negligible. Furthermore, albendazole and metabolites have been analyzed in porcine muscle and livers, but only two metabolites, albendazole sulfone and albendazole 2-amino-sulfone were detected in some porcine livers but at concentrations lower than the MRLs established by the EU.
Pharmacokinetics of Anthelmintics After administration, anthelmintics are usually absorbed into the bloodstream and transported to different parts of the body, including the liver, where they maybe metabolized through oxidation and cleavage reactions, and excreted in the feces and urine. The characterization of the kinetic behavior, metabolic fate, excretion, and residue profile of anthelmintics in the target animal will contribute to its optimized use as well as the determination of the length of the withdrawal time. However, the administration of anthelmintics does not always result in the expected therapeutic success, because
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Veterinary Drugs Residues: Anthelmintics
Table 1
MRLs for some anthelmintic drug residues in several matrices under European Commission Regulation 37/2010
Compound
Marker residue
Animal species
MRL (mg kg1)
Target tissues
Albendazole
Sum of albendazole sulfoxide, albendazole sulfone, and albendazole 2-amino sulfone, expressed as albendazole
All ruminants
100 100 1000 500 100
Muscle Fat Liver Kidney Milk
Fenbendazole
Sum of extractable residues which maybe oxidized to oxfendazole sulfone
All ruminants, porcine, and Equidae
50 50 500 50 10
Muscle Fat Liver Kidney Milk
50 50 400 300 400
Muscle Skin and fat Liver Kidney Eggs
All ruminants Flubendazole
Sum of flubendazole and (2-amino 1H-benzimidazol-5yl)(4fluorophenyl)methanone
Poultry and porcine
Flubendazole
Poultry
Mebendazolea
Sum of mebendazole methyl (5-(1-hydroxy, 1-phenyl)methyl-1H-benzimidazol-2-yl)carbamate and (2-amino-1H-benzimidazol-5-yl)phenylmethanone, expressed as mebendazole equivalents
Ovine, caprine, and Equidae
60 60 400 60
Muscle Fat Liver Kidney
Oxfendazole
Sum of extractable residues which maybe oxidized to oxfendazole sulfone
All ruminants, porcine, and Equidae
50 50 500 50 10
Muscle Fat Liver Kidney Milk
All ruminants Thiabendazole
Sum of thiabendazole and 5-hydroxy thiabendazole
Bovine and caprine
100 100 100 100 100
Muscle Fat Liver Kidney Milk
Triclabendazolea
Sum of extractable residues which maybe oxidized to keto-triclabendazole
All ruminants
225 100 250 150
Muscle Fat Liver Kidney
Abamectina
Avermectin B1a
Bovine
10 20 20 50 25 20
Fat Liver Muscle Fat Liver Kidney
Ovine
Doramectin
Doramectin
All mammalian food producing species
40 150 100 60
Muscle Fat Liver Kidney
Emamectin
Emamectin B1a
Fin fish
100
Muscle and skin
Eprinomectin
Eprinomectin B1a
Bovine
50 250 1500 300 20
Ivermectina
22,23-dihydro-avermectin B1a
All mammalian food producing species
100 100 30
Muscle Fat Liver Kidney Milk Fat Liver Kidney (Continued )
Veterinary Drugs Residues: Anthelmintics
Table 1
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Continued
Compound
Marker residue
Animal species
Moxidectin
Moxidectin
Bovine, ovine, and Equidae
Bovine and ovine
MRL (mg kg1)
Target tissues
50 500 100 50 40
Muscle Fat Liver Kidney Milk
Levamisoleb
Levamisole
Bovine, ovine, porcine, and poultry
10 10 100 10
Muscle Fat Liver Kidney
Morantel
Sum of residues which may be hydrolyzed to N-methyl1,3-propanediamine and expressed as morantel equivalents
All ruminants
100 100 800 200 50
Muscle Fat Liver Kidney Milk
a
Not for use in animals from which milk is produced for human consumption. Not for use in animals from which milk or eggs is produced for human consumption. Abbreviation: MRLs, maximum residue limits. b
host-related factors can modify pharmacokinetic behavior and efficacy of the selected compound. Thus, the factors affecting drug pharmacokinetics can be distinguished between two groups: interindividual (species, sex, and genetics) and intraindividual (age, gestation, stress, medication, food, and environment) factors. Interindividual factors remain constant during the life of an organism, whereas intraindividual ones change during the life. Basically they are related to physiological and pathological state of an organism. For instance, it has been observed that the metabolic interconversion between fenbendazole sulfide and sulfoxide differs in horses compared to ruminants, and the bioavailability and residence time were lower and shorter in horses than in ruminants. Extensive work has been carried out to study the pharmacokinetic and metabolism of certain benzimidazoles. In general, benzimidazoles are limited in absorption from the gastrointestinal tract due to the poor solubility of these drugs and the absorption is generally fast (from 2–7 h with flubendazole to 6–30 h for other compounds). Mebendazole is absorbed from the gastrointestinal tract and is intensively metabolized in sheep through ketoreduction and decarbamylation followed by conjugation. Furthermore, it is known that the absorption and the efficacy are influenced by their administration with food. Thus, the bioavailability of benzimidazole sulfides is markedly reduced in ruminants which have had unrestricted access to food compared with those given restricted access prior to treatment. Sometimes in order to minimize the metabolic oxidation of benzimidazole sulfides and sulfoxides to sulfones, metabolic inhibitors are added. For instance, piperonyl butoxide has been added to modify the pharmacokinetic profile of febendazole. The pharmacokinetics of albendazole has been studied in several animal species, although few studies have been carried out in poultry. In general, it is rapidly metabolized in all species, and the plasma levels of oxidized metabolites (sulfoxide and sulfone) are much higher than the parent drug. In
fact, the sulfoxide transformation product is considered the active metabolite responsible for the therapeutic activity of albendazole, whereas albendazole sulfone is considered an inactive compound. Furthermore, it can be noted that the coadministration of other drugs such as cimetidine and praziquantel affects the kinetics of albendazole probably by the induction of the second sulfonation step. In poultry it has been observed that the maximum concentration was obtained at 1 h and it was only detected for 6 h after treatment. However, albendazole sulfoxide was detected for 25 h after treatment. Furthermore, these results were compared with those obtained in other species such as calf, dogs, goats, pigs, and sheep, and the maximum concentration (Cmax) and time to reach it (Tmax) are lower in chickens. Therefore, it was concluded that albendazole was absorbed to a greater extent and more quickly in poultry than in mammalian species. Finally, only albendazole metabolites can be measured in eggs if single oral dose was given. However, if the treatment is applied during 7 days, albendazole and its metabolites can be simultaneously detected in yolk, whereas albendazole cannot be detected in egg albumin. The absence of albendazole in albumin can be explained considering the relatively higher lipid solubility of albendazole compared with its metabolites. In the same way, it has been observed that the concentration of flubendazole in egg yolk was about five times higher than in the albumin. Other compounds that have been studied are mebendazole and triclabendazole. Thus, some metabolites such as hydroxymebendazole (reduced metabolite) and aminomebendazole (hydrolyzed metabolite) can be found in food from animals treated with mebendazole, with the highest concentrations in liver of the reduced metabolite. In fact, this metabolite can be detected in liver for 14 days after administration. In relation to triclabendazole, it is rapidly removed from blood by the liver, and it is oxidized to the sulfoxide and sulfone, which are the main metabolites detected in plasma.
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Veterinary Drugs Residues: Anthelmintics
For nicotinic receptor agonists, it has been shown that the absorption and excretion of levamisole is fast and it is not affected by the route of administration because of its high solubility. It has been observed that the levels of this drug in blood cattle are maximum in less than 1 h, and 90% of the total dose is excreted in urine in 24 h. However, pyrantel is poorly soluble in water and its metabolism is fast, with the metabolites excreted rapidly in the urine. Morantel is absorbed rapidly from the upper small intestine of sheep and metabolized rapidly in liver (17% of the initial dose is excreted in the urine as metabolites within 96 h after dosing). In general, macrocyclic lactones are hydrophobic and they are distributed throughout the body and some of them can be concentrated in adipose tissue. Depending on the species, they have an elimination half-life of 32–178 h. In this sense, a pharmacokinetic study of emamectin benzoate has been carried out in Atlantic salmon and it was shown that emamectin B1a was the major residue component (480% in edible tissues). Therefore, this compound has been used as marker residue in edible tissues of Atlantic salmon for monitoring emamectin benzoate. Further metabolic studies have been carried out with other anthelmintic drugs. Thus, the metabolism of praziquantel has been studied in several tissues of rainbow trout maintained at different temperatures (12 and 18 1C), observing that absorption was faster at higher temperatures, whereas the elimination of the drug was less dependent on the temperature. Thus, after 32 h, 67–96% of the drug has been eliminated from the tissues. In relation to the elimination of anthelmintics, biliary secretion is an important pathway for elimination of macrocyclic lactones. Thus, they are primarily excreted in the feces and the remainder (o10%) in the urine. However, more lipophilic compounds are also excreted in milk. Biliary route is the most important pathway for secretion and recycling of benzimidazoles to the gastrointestinal tract, as well as praziquantel, which is partly excreted by bile fluid and partly as water soluble metabolites through the kidneys. Furthermore, the intestinal clearance of albendazole sulfoxide exhibits a stereoselective elimination of the ( ) form. The excretion of anthelmintics in the feces of livestock has given rise to concern as avermectins have adverse effects in dipteran flies and coleopteran beetles. However, benzimidazoles are unlikely to affect dung-dwelling arthropods and the environmental impact is not limited to specific effect on scavenger insects. For instance, the fecal concentration of febendazole and its metabolites was determined in horses, noting that no drug could be detected at 12 h post dosing. The maximal concentration was monitored at 24 h, and no target compounds were detected after 72 h. In feces the highest concentration was observed for the parent compound.
Analytical Methodology Anthelmintic residues are mainly determined applying LC techniques coupled to several detectors, although some benzimidazoles have been determined by gas chromatography (GC). However, GC is not suitable for quantification because
of thermal decomposition in the chromatograph or nonreproducible ionization in the source of the MS. Benzimidazoles could be detected using ultraviolet (UV) or fluorescence detection, considering that most of them (albendazole, flubendazole, and thiabendazole) have natural fluorescence. Furthermore in relation to UV, the use of diode array is very useful, considering that individual analytes have characteristic spectra. Levamisole can also be detected by UV. Meanwhile, avermectins can be detected using fluorescence detection including a derivatization reaction using trifluoroacetic acid, and this methodology has been used for the determination of emamectin in Atlantic salmon. Although the limits of detection provided by fluorescence detectors are below the MRLs established by governments, there are some problems related to the derivatization step such as instability of fluorescent derivatives, slow and/or incomplete formation of derivatives, and the low reproducibility of the results. Furthermore, and according to current legislation in Europe, MS should be used for confirmation purposes in the determination of residues in foodstuffs. Therefore, conventional detection has been replaced by MS applying several detectors, such as single quadrupole, triple quadrupole, time of flight (TOF), and Orbitrap. In this sense, several ionization techniques, such as electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization, have been used. Moreover, the application of MS analyzers allows the identification and quantification of metabolites of anthelmintics in the samples. However, in the case of avermectins, the use of MS is not a straightforward strategy. These compounds are heavy (molecular weight ranging from 600 to 900 Da), but most of them only contain one nitrogen atom in their structure. In fact, ivermectin, doramectin, and abamectin only contain C, H, and O, which have negative effects on the ionization. This implies that several assays have been carried out to study the best ionization of these compounds. For instance, it has been indicated that for abamectin, the determination of ammonium adduct by ESI is the best option, although other studies have indicated that the use of APCI with negative ionization provided [M H] as the predominant ion for ivermectins. In general, it has been reported that avermectins, which are devoid of nitrogen atoms, produce significantly higher abundances for [M þ Na] þ than [M þ H] þ . Furthermore, another difficulty is that sodium adducts are difficult to break and therefore several authors have evaluated alternative ionization processes, observing that negative ESI or APCI could overcome many of these problems. In relation to the LC separation, C18 or C8 columns are mainly used. Moreover, the selection of the mobile phase is critical for the correct ionization of the target compounds. Acetonitrile and methanol are usually used for the determination of anthelmintics, although for avermectins, the use of methanol increases the sensitivity of the compounds in relation to acetonitrile. The pH of the mobile phase maybe critical in order to get suitable chromatographic separation, and ammonium formate or acetate could provide suitable results. Furthermore, in the past decade, ultra highperformance LC has been widely used in order to reduce analysis time, obtaining narrower peaks, and increasing sample
Veterinary Drugs Residues: Anthelmintics
throughput. Furthermore, the combination of LC with tandem MS (MS/MS) allows the selective determination of these compounds. Finally, it must be emphasized that sample preparation is the critical step for the simultaneous determination of several analytes in complex matrices. In relation to the extraction of these compounds from several matrices, different methodologies have been applied. Thus, liquid–liquid partition using acetonitrile has been used, allowing the deproteination of proteins. Solid phase extraction (SPE) has also been evaluated, utilizing C18, polymer sorbents or strong cation cartridges. However, they are laborious and time-consuming, and new approaches could be applied. Thus, immunoaffinity chromatography (IAC) could be used as a valuable tool for cleanup, allowing a fast and selective extraction of avermectins from extracts of bovine and swine liver. Furthermore, the combination of IAC with LC–MS provides sensitive and selective methods. However, matrix solid phase dispersion has been applied for the determination of 37 anthelmintic drugs and metabolites in muscle. In addition, pressurized liquid extraction has been applied for the determination of benzimidazoles in tissues of animals such as swine, cattle, sheep, and chickens. Finally, it must be noted that low temperature clean-up has been used for the determination of ivermectins and moxidectin in bovine muscle. Furthermore, benzimidazoles are usually extracted by relatively polar aqueous extraction solutions. However, some metabolites are covalently bound to matrix components (liver or kidney) and chemical or enzymatic hydrolysis should be included in the extraction procedure, although simplified methods using anhydrous acetonitrile could be used. Nowadays, generic extraction conditions can be applied for the simultaneous extraction of several types of compounds in a single analytical method. However, the inclusion of some anthelmintics such as avermectins is not easy considering the low sensitivity obtained by the use of LC–MS/MS, and special attention should be paid to these compounds, when they are included in multiresidue methods. In this sense, QuEChERS approach (acronym for Quick, Easy, Cheap, Effective, Rugged and Safe), which is based on acetonitrile extraction followed by a phase separation induced by the addition of salts and a cleanup step based on dispersive SPE, has been successfully applied for the extraction of this type of compounds from edible tissues.
Conclusions and Future Trends As described in this article, the use of anthelmintics to treat helminth parasites and the resulting residues in edible tissues is a problem of major concern. Furthermore, the development of resistance to the currently used anthelmintics is an issue of increasing importance. In this sense, the development of new compounds, such as AAD, are needed in order to improve the characteristics of conventional classes of anthelmintics such as lower toxicity, favorable pharmacokinetic properties, and broad-spectrum capacities. Furthermore, additional studies should be carried out to more thoroughly examine the pharmacokinetics and metabolism of these compounds, especially during pregnancy. The possibility of elimination of some nonpolar compounds by
53
milk should be also evaluated in order to minimize the risk of the presence of anthelmintic residues in this matrix. To assure a sustainable inventory of effective anthelmintics, monitoring of drug resistance and research with combinations of current or future drugs will be necessary. Moreover and despite the evaluation of the metabolism of anthelmintics in vertebrates, much attention should be paid to the evaluation of metabolism of these products in parasitic helminths. In relation to analytical methodologies, current methods should include known metabolites in routine analyses in order to fulfill current legislation. However, anthelmintic metabolism is complex, and it is necessary to develop and validate new analytical methods that allow the accurate determination of new metabolites in foodstuffs. For that purpose, high resolution MS analyzers such as TOF or Orbitrap are very helpful, considering that they acquire full-scan spectra, and target, nontarget and unknown analysis that could be carried out in one single analysis. Finally and considering the different MRLs established by several countries or organizations for the same combination anthelmintic/matrix, international legislation should be harmonized, in order to avoid some problems during export.
Acknowledgments The authors gratefully acknowledge the Spanish Ministry of Economy and Competitiveness (MINECO-FEDER) for financial support (Project Ref. AGL2010-21370). RRG is also grateful for personal funding through the Ramo´n y Cajal Program (MINECO and European Social Fund).
See also: Analytical Methods: Overview of Methods of Analysis for Chemical Hazards. Disciplines Associated with Food Safety: Food Safety Toxicology. Food Safety Assurance Systems: Food Safety and Quality Management Systems. Institutions Involved in Food Safety: FAO/WHO Codex Alimentarius Commission (CAC); Food and Agriculture Organization of the United Nations (FAO); World Health Organization (WHO). Public Health Measures: Food Control and Public Health Laboratories; Fundamentals of Food Legislation. Risk Analysis: Risk Assessment: Chemical Hazards. Veterinary Drugs Residues: Antibacterials; Control of Helminths; Veterinary Drugs – General
Further Reading Bartram DJ, Leathwick DM, Taylor MA, Geurden T, and Maeder SJ (2012) The role of combination anthelmintic formulations in the sustainable control of sheep nematodes. Veterinary Parasitology 186: 151–158. Beech RN, Skuce DJ, Martin RJ, Prichard RK, and Gilleard JS (2011) Anthelmintic resistance: Markers for resistance, or susceptibility? Parasitology 138: 160–174. Besier B (2007) New anthelmintics for livestock: The time is right. Trends in Parasitology 23: 21–24. Danaher M, De Ruyck H, Crooks SRH, Dowling G, and O’Keeffe M (2007) Review of methodology for the determination of benzimidazole residues in biological matrices. Journal of Chromatography B 845: 1–37. Dayan AD (2003) Albendazole, mebendazole and praziquantel. Review of nonclinical toxicity and pharmacokinetics. Acta Tropica 86: 141–159.
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Garrrido Frenich A, Plaza-Bolan˜os P, Aguilera-Luiz MM, and Martı´nez-Vidal JL (2010) Veterinary Drugs and Growth-Promoting Agent Analyses. New York: Nova Publishers. Holden-Dye L and Walker RJ (2007) Anthelmintic drugs. WormBook: The Online Review of C. elegans Biology 1–13. Kaminsky R, Ducray P, Jung M, et al. (2008) A new class of anthelmintics effective against drug-resistance nematodes. Nature 452: 176–181. Kaufmann A, Butcher P, Maden K, Walker S, and Widmer M (2011) Quantification of anthelmintic drug residues in milk and muscle tissues by liquid chromatography coupled to Orbitrap and liquid chromatography coupled to tandem mass spectrometry. Talanta 85: 991–1000. Keiser J and Utzinger J (2010) The drugs we have and the drugs we need against major helminth infections. Advances in Parasitology 73: 197–230. Krizova-Forstova V, Lamka J, Cvilink V, Hanusova V, and Skalova L (2011) Factors affecting pharmacokinetics of benzimidazole anthelmintics in food-producing animals: The consequences and potential risk. Research in Veterinary Science 91: 333–341. Lanusse CE, Alvarez LI, Sallovitz JM, Mottier ML, and Sanchez Bruni SF (2009) Antinematodal drugs. In: Riviere JE and Papich MG (eds.) Veterinary Pharmacology and Therapeutics, 9th edn., pp. 1053–1094. New Jersey: Wiley-Blackwell. Lanusse CE, Lifschitz AL, and Imperiales FA (2009) Macrocyclic latctones: Endectocide compounds. In: Riviere JE and Papich MG (eds.) Veterinary Pharmacology and Therapeutics, 9th edn., pp. 1053–1094. New Jersey: WileyBlackwell. Sutherland IA and Leathwick DM (2011) Anthelmintic resistance in nematode parasites of cattle: A global issue?Trends in Parasitology 27: 176–181.
Waller PJ (2006) From discovery to development: Current industry perspectives for the development of novel methods of helminth control in livestock. Veterinary Parasitology 139: 1–14.
Relevant Websites http://www.hc-sc.gc.ca/dhp-mps/vet/legislation/guide-ld/vich/guide-ligne-eng.php Canadian Health Ministry. www.codexalimentarius.net/vetdrugs/data/ Codex Alimentarius. http://www.ema.europa.eu/ema/ European Medicines Agency. http://www.fao.org/food/food-safety-quality/scientific-advice/jecfa/jecfa-vetdrugs/en/ Food and Agricultural Organization of the United Nations. http://www.vichsec.org/pdf/Gl07_st7final þ corr.pdf International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products. http://www.merckvetmanual.com/mvm/index.jspcfile=htm/bc/toc_191500.htm Merck Veterinary Manual. http://www.mrldatabase.com United States Department of Agriculture. http://www.fda.gov/AnimalVeterinary/default.htm United States Food and Drug Administration.