Cross-contamination of non-target feedingstuffs by diclazuril authorised for use as a feed additive 1

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1 The EFSA Journal (2008) 716, 1-31 Cross-contamination of non-target feedingstuffs by diclazuril authorised for use as a feed additive 1 Scientific opinion of the Panel on Contaminants in the Food Chain Question N EFSA-Q J Adopted on 30 May 2008 PANEL MEMBERS Jan Alexander, Diane Benford, Andrew Cockburn, Jean-Pierre Cravedi, Eugenia Dogliotti, Alessandro Di Domenico, Maria Luisa Férnandez-Cruz, Peter Fürst, Johanna Fink-Gremmels, Corrado Lodovico Galli, Philippe Grandjean, Jadwiga Gzyl, Gerhard Heinemeyer, Niklas Johansson, Antonio Mutti, Josef Schlatter, Rolaf van Leeuwen, Carlos Van Peteghem, Philippe Verger. SUMMARY Diclazuril is a non-ionophoric synthetic compound that is authorised as a coccidiostat for use at a maximum concentration of 1 mg/kg complete feed in chickens and turkeys for fattening (maximum 12 and 16 weeks, respectively) with a withdrawal period of 5 days and chickens reared for laying (maximum 16 weeks) (Commission Regulation (EC) No 1800/ ). Recently, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) has evaluated the use of diclazuril in rabbits for fattening and breeding and demonstrated its 1 For citation purposes: Opinion of the Scientific Panel on Contaminants in the Food Chain on a request from the European Commission on cross-contamination of non-target feedingstuffs by diclazuril authorised for use as a feed additive, The EFSA Journal (2008) 716, OJ L 317, , p. 37 European Food Safety Authority, 2008

2 safety of use at 1 mg/kg feed. However, the legislation authorising the use of diclazuril in rabbits is not yet in place. Despite the requirements set for feed business operators in Regulation No (EC) 183/2005, it is generally acknowledged that under practical conditions during the production of mixed feeds, a certain percentage of a feed batch remains in the production circuit and these residual amounts can contaminate the subsequent feed batches. This cross-contamination may result in the exposure of non-target animal species, and hence the potential health risks for non-target animal species as well as the potential residue deposition in foods derived from these non-target animal species have been evaluated. A number of tolerance studies performed by industry on non-target animal species proved a lack of toxicity of diclazuril in ducks, quails, guinea fowl, pigs and ruminants exposed to the maximum authorised feed level for target animal species (1 mg/kg feed). At a level of cross-contamination of 10% of the maximum authorised concentration in feed, the intake of diclazuril would be well below the overall no observed effect level (NOEL) of 2.9 mg/kg per day derived by the FEEDAP Panel based on a 2 year oral toxicity/carcinogenicity study in male mice. Hence, the Panel on Contaminants in the Food Chain (CONTAM Panel) concluded that adverse effects are unlikely to occur in non-target animal species exposed to feed that is cross-contaminated with diclazuril up to 10% of the maximum authorised level for target animal species. No kinetic or occurrence data were available to estimate the amount of diclazuril residues in milk, meat or offal from non-target animal species. Consumer exposure was estimated using kinetic data after one-day withdrawal time from rabbits that were given the maximum level assessed by the FEEDAP Panel for authorisation: 1 mg diclazuril/kg feed. These data were extrapolated to a concentration of 0.1 mg/kg feed to correspond to feed cross-contaminated with diclazuril at 10% of the maximum authorised level for target animal species. Aggregate consumption of rabbit (100 g of liver, 300 g muscle, 50 g skin/fat and 50 g kidney) and chicken eggs (100 g eggs) could give an intake of approximately 0.40 µg/kg b.w. per day, which represents approximately 1.5% of the acceptable daily intake (ADI) of 29 µg/kg b.w. established by the FEEDAP Panel. Therefore, even though kinetics and tissue deposition may differ between rabbits and non-target animal species, consumers are unlikely to exceed the ADI. The CONTAM Panel concluded that the limited dataset provides no indication of an appreciable risk to consumers health from the ingestion of diclazuril residues in products from animals exposed to feed cross-contaminated up to a hypothetical level of 10% of the maximum authorised level for diclazuril in target animal species. KEYWORDS: diclazuril, cross-contamination, carry-over, coccidiostat, anticoccidial, feed additive, occurrence, exposure, animal health, intoxication, human health. The EFSA Journal (2008) 716, 2-31

3 TABLE OF CONTENTS BACKGROUND AS PROVIDED BY THE REQUESTOR Cross-contamination Legal provisions as regards minimisation of cross-contamination Legal provisions as regards the authorisation of coccidiostats (and histomonostats) for use as feed additive Unavoidable cross-contamination (under practical conditions) Tolerances...6 TERMS OF REFERENCE AS PROVIDED BY THE REQUESTOR...7 ACKNOWLEDGEMENTS...8 GLOSSARY OF TERMS USED BY THE PANEL IN ITS OPINIONS ON COCCIDIOSTATS...8 ASSESSMENT Introduction Biological activities of diclazuril Previous evaluations of the toxicity and safety of diclazuril Cross-contamination of feed batches Specific data for diclazuril-based feed additive products Methods of analysis for diclazuril Analysis of diclazuril in premixes and animal feeds Analysis of diclazuril residues in animal products Occurrence of diclazuril Occurrence of diclazuril residues in feed materials for non-target animal species Occurrence of diclazuril residues in animal products derived from non-target animal species Toxicity of diclazuril Mechanisms of toxicity Toxicity of diclazuril in target animal species Toxicity of diclazuril in non-target animal species Kinetics and tissue disposition Kinetics of diclazuril in the target animal species Kinetics of diclazuril in the non-target animal species Risk characterization Animal health risks in non-target animal species associated with the accidental consumption of feed materials designated for target animal species Adverse health effects in non-target animal species as a consequence of crosscontamination of feed batches Residues of diclazuril in foods derived from non-target animal species Human health risk associated with residues in foods derived from non-target animal species following exposure of these animals to contaminated feed batches...27 CONCLUSIONS...28 RECOMMENDATIONS...28 REFERENCES...29 DOCUMENTATION PROVIDED TO EFSA...31 The EFSA Journal (2008) 716, 3-31

4 BACKGROUND AS PROVIDED BY THE REQUESTOR 1. Cross-contamination A feed manufacturing company produces a broad range of compound feedingstuffs. Therefore, in the same production line, different compound feedingstuffs have to be manufactured after each other. At the switch-over from one product to the subsequent one, it is unavoidable that traces of the first product remain in the production line and end up in the beginning of the production of the following product. The transfer from one production batch to the following batch is called carry-over or cross-contamination. Cross-contamination in purchased premixtures Purchased premixtures can contain traces of contamination of other substances due to crosscontamination during the production. Product-related cross-contamination The following properties of the feed additives and premixes also have an important influence on the cross-contamination behaviour: - adhesive strength-adhesion to walls - particle size and density (carrier, substance) - electrostatic properties. The cross-contamination decreases according to the product being less adhesive and electrostatic. Establishment related cross-contamination The design of the dosage, grinding and mixing equipment has an important influence on the level of cross-contamination. Also the transport and storage facilities and conditions are an important factor for cross-contamination 2. Legal provisions as regards minimisation of cross-contamination Directive No (EC) 95/69 3 Council Directive No (EC) 95/69 of 22 December 1995, laying down the conditions and arrangements for approving and registering certain establishments and operating in the animal feed sector, provides in Article 2 and 3, that establishments manufacturing coccidiostats, manufacturing premixtures prepared from coccidiostats, or manufacturing compound feedingstuffs containing premixtures prepared from coccidiostats have to receive approval for 3 OJ L 332, , p. 15. As last amended by Regulation No (EC) 806/2003 of 14 April 2003 (OJ L 122, , p. 1) The EFSA Journal (2008) 716, 4-31

5 these activities. Also intermediaries putting these products into circulation must be approved. The approval is subject to compliance with the minimum conditions laid down in the Annex. One of these conditions concerning the facilities and the equipment provides that the lay-out, design and operation of the facilities and equipment must be as such to minimize the risk of error and permit effective cleaning and maintenance in order to avoid contamination, crosscontamination and any adverse effects generally on the quality of the products. Regulation No (EC) 183/ Regulation No (EC) 183/2005 of the European Parliament and of the Council of 12 January 2005 laying down requirements for feed hygiene was applicable from 1 January 2006 onwards and has replaced Council Directive No (EC) 95/69. Article 10 of Regulation No (EC) 183/2005 provides that feed business operators shall ensure that establishments under their control are approved by the competent authority in case these establishments are manufacturing and/or placing on the market coccidiostats and histomonostats, manufacturing and/or placing on the market premixtures prepared using coccidiostats and histomonostats, manufacturing for placing on the market or producing for the exclusive requirements of their holdings, compound feedingstuffs using coccidiostats and histomonostats or premixtures containing coccidiostats and histomonostats. Annex II to Regulation No (EC) 183/2005 contains requirements for the feed businesses mentioned in previous paragraph. As regards facilities and requirements it is provided under point 2 of Annex II that The lay-out, design and construction and size of the facilities and equipment shall: (a) permit adequate cleaning and/or disinfection; (b) be such as to minimize the risk of error and to avoid contamination, cross-contamination and any adverse effects generally on the safety and quality of the products. Machinery coming into contact with feed shall be dried following any wet cleaning process. 3. Legal provisions as regards the authorisation of coccidiostats (and histomonostats) for use as feed additive Article 3 of Council Directive No (EC) 70/524 concerning additives in feedingstuffs 5 provides that no additive may be put into circulation unless a Community authorisation has been granted. This Community authorisation can only be granted if, taking into account the 4 OJ L 35, , p. 1 5 OJ L 270, , p.1 The EFSA Journal (2008) 716, 5-31

6 conditions of use, it does not adversely affect human or animal health or the environment, nor harm the consumer by impairing the characteristics of animal products. Diclazuril is authorised for use as feed additive in accordance with the provisions of Council Directive 70/524/EEC (see Table 1). Based on the recent assessments of the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) (EFSA, 2008a,b), it can be expected that it will be considered in the near future to expand the table to also include rabbits for fattening with the same maximum content in complete feed. Regulation (EC) No 1831/ of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition is applicable since 19 October 2004 and repeals Directive 70/524/EEC with effect from that date. Table 1. Species or category of animals for which the use of diclazuril is authorised (target animal), and authorised maximum content in complete feed Cocciodiostat (active substance) Diclazuril Species or category of animals for which the use of coccidiostats is authorised (target animal) Chickens for fattening Chickens reared for laying (max 16 weeks) Turkeys for fattening (max 12 weeks) Authorised maximum content of active substance in complete feed 1 mg/kg (Clinacox) 1 mg/kg (Clinacox) 1 mg/kg (Clinacox) 4. Unavoidable cross-contamination (under practical conditions) Diclazuril is authorised for use as a feed additive for the production of feedingstuffs for target animal species according to the conditions of authorisation. However the production of feed containing diclazuril can result in cross-contamination to feedingstuffs for non-target animal species. Of major importance is the application by the feed operator of good manufacturing practices to avoid to the largest extent possible, the cross-contamination of residues of the coccidiostat in subsequent batches of compound feedingstuffs. However, even if all prevention measures are applied, including the use of rinsing batches, the cross-contamination of residues is unavoidable under practical conditions. 5. Tolerances Therefore, the possibility to set tolerances for these in practice unavoidable residues of coccidiostats in feedingstuffs for non-target species should be considered in the frame of 6 OJ L 268, , p The EFSA Journal (2008) 716, 6-31

7 Directive (EC) No 2002/32 of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed 7. Such tolerances in feedingstuffs for non-target animals could be set following the ALARA principle (As Low As Reasonably Achievable) taking into account good manufacturing practices. According to information received from professional organisations, levels of crosscontamination of 3-10% with a majority at 5% or lower can be achieved after implementing thorough actions to reduce cross-contamination. Such tolerances in feedingstuffs for non-target animals should not have any pharmacological activity and should not threaten animal health and public health, as in some cases the tolerances for feedingstuffs for non-target animals could result in presence of residues in foodstuffs of animal origin. TERMS OF REFERENCE AS PROVIDED BY THE REQUESTOR In accordance with Article 29 (1) (a) of Regulation (EC) No 178/2002 the Commission asks EFSA to provide an opinion of the risks involved for animal health and public health as the consequence of undesirable cross-contamination of diclazuril authorised as feed additive into non-target feeds. The assessment should take into account hypothetical carry-over rates of 2%, 5% and 10% from feed produced with the highest authorised dose of diclazuril into the afterwards produced non-target compound feed (for non-target animal species). The EFSA is requested to provide an opinion whereby - the animal health risk for non-target species (food producing farm animals) will be assessed, - the adverse effects as a consequence of cross-contamination of diclazuril into feed for non-target animals, - on the basis of the available information, an estimate of the level of residues present in food of animal origin from non-target species as the consequence of cross-contamination is performed, - the possible risks for human health as the consequence of the presence of such residues in food of animal origin (eggs, milk, meat, edible offal) from non-target species are assessed. 7 OJ L 140, , p. 10. Directive as amended by Directive (EC) No 2005/6 (OJ L 24, , p. 33) The EFSA Journal (2008) 716, 7-31

8 ACKNOWLEDGEMENTS The European Food Safety Authority wishes to thank the members of the Working Group for the preparation of this opinion: Arturo Anadón, Alexander Feil, María Luisa Fernández-Cruz, Johanna Fink-Gremmels, Kimmo Peltonen, Derek Renshaw, Pascal Sanders and Pieter Wester. GLOSSARY OF TERMS USED BY THE PANEL IN ITS OPINIONS ON COCCIDIOSTATS Considering the current EU legislation, the following terms will be applied in the Opinion: Coccidiosis: Coccidiosis is a common protozoan infection in farm animals, affecting predominantly young animals. Under common farm conditions, herd health management cannot exclude coccidial infections in large poultry and rabbit units and the use of coccidiostatic agents (coccidiostats) remains necessary to control animal health and welfare, and to avoid substantial losses due to acute and often lethal coccidiosis. Coccidiostats: Currently, in the EU 11 coccidiostatic substances are authorised for the prevention of coccidiosis in one or more animal species. Authorisation is given for a minimum and maximum level to be included as feed additive into the animal s diet, and may prescribe the animal species as well as the species categories (as for example chickens for fattening and chickens reared for laying) and in some cases withdrawal periods. Of the million tonnes of feed produced annually for chickens for fattening, turkeys and rabbits, approximately million tonnes is manufactured with the addition of a coccidiostat (IFAH, 2007, document provided to EFSA). Various coccidiostats exert also a distinct antibacterial effect and are licensed in Third Countries (countries outside the EU) as growth promoting agents in fattening ruminants (lambs or cattle) and fattening pigs. Target animal species: Animal species or animal category within a species for which the compound under consideration is authorised for use as a coccidiostat. This term also covers chickens reared for laying or turkeys until the age of 12 or 16 weeks (as defined in the authorisation of the specific product). The choice of either 12 or 16 weeks depends on the request made by the applicant and/or the data submitted. The chicken or turkey thereafter turns into a non-target animal species. A hen starts egg laying between 18 and 26 weeks of age. Non-target animal species: Any other animal species or category for which the compound is not authorised. The EFSA Journal (2008) 716, 8-31

9 Feed additive: A substance, micro-organism or preparation, other than feed material and premixtures, which are intentionally added to feed at concentrations up to a defined maximum level (mg/kg feed). Currently, coccidiostats are authorised for use as feed additives according to the provisions of Council Directive 70/524/EEC and Council Regulation No (EC) 1831/2003 that repeals Directive 70/524/EEC (see also the background chapter). According to these provisions, authorisation and prerequisites for use of coccidiostats are defined for individual products (brands) following review by the FEEDAP Panel of data provided by the applicant. Premixture: A mixture of feed additives with feed materials. Premixtures are not intended for direct consumption by animals, and are therefore not addressed in the Opinion. Cross-contamination: Contamination of feeds that are produced after the production of a mixed feed containing additives with residual amounts of the previous feed batch. Levels of cross-contamination: According to the mandate as described in the Terms of Reference, three levels of cross-contamination will be considered, i.e. 2%, 5% and 10% of the maximum concentration authorised for target animal species, respectively. Assessment of animal exposure and adverse health effects in animals: Adverse health effects occurring in non-target animal species are described. A distinction is made between the likelihood of adverse health effects that are associated with an accidental consumption of feeds prepared for a target animal species by a non-target animal species, and the involuntary exposure of non-target animal species by residual amounts of coccidiostats occurring in feed as a consequence of cross-contamination. ADI values: Acceptable daily intake (ADI) of a substance that can be consumed by a human over a lifetime without adverse health effects. As the CONTAM Panel did not have access to the complete safety (toxicological, pharmacological and microbiological) database available for the individual substances under consideration, the ADI value as derived by the FEEDAP Panel and where appropriate also the ADI(s) derived by other relevant scientific committees (e.g. the CVMP 8 or the JECFA 9 ) is used for the risk characterization and assessment. The CONTAM Panel noted in some cases the divergence between ADI values derived by the FEEDAP Panel and the ADI values derived by the CVMP and/or JECFA. These differences were attributable to the application of different uncertainty factors, or the inclusion of new endpoints, such as antimicrobial activity (antimicrobial no-effect level) in the assessment. The CONTAM Panel decided to consider both values in the presentation of its risk assessment for non-target animal species. 8 The Committee for Medicinal Products for Veterinary Use of the European Medicines Agency 9 The Joint FAO/WHO Expert Committee on Food Additives (JECFA) is an international expert scientific committee that is administered jointly by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO). The EFSA Journal (2008) 716, 9-31

10 MRL values: Maximum residue limits. The CVMP applied Regulation No (EC) 1055/ amending the Annexes I and III of Regulation No (EC) 2377/90 11 to propose maximum residue limits (MRLs) for a number of coccidiostats. The FEEDAP has also recommended MRLs for some coccidiostats, and the CONTAM Panel considered these recommendations in the evaluation process. Residues of coccidiostats in edible tissues, milk and eggs: According to Directive No (EC) 96/23 12 Member States are obliged to monitor certain substances and residues thereof in animals and animal products. These data are collected by the Commission and a compilation of the results from 2004 and 2005 are used in the human exposure assessment. Equivalents: Where kinetic studies have been conducted with the coccidiostat 14 C- radiolabelled, the concentration of total radioactive residue levels measured in the different tissues are expressed as µg parent coccidiostat equivalents/kg tissue, to indicate that these levels could be the parent compound and/or metabolites. Human dietary exposure: The present assessment is confined to the evaluation of residues of coccidiostats in foodstuffs derived from non-target animals. Where appropriate, total exposure originating from different products including edible tissues, milk and eggs is estimated. Risk characterization: The risk characterization is based on the ADI and MRL values from either the FEEDAP Panel, the CVMP or the JECFA as outlined above. These levels are compared with levels of residues found in tissues and/or products (for example eggs) of nontarget animal species as far as these are available. Where appropriate uncertainties in the establishment of ADI values are discussed. ASSESSMENT 1. Introduction Diclazuril is a synthetic compound of the triazinone family that is used as a feed additive for the control of coccidiosis. The chemical structure of diclazuril is presented in Figure OJ L 192, , p OJ L 224, , p OJ L 125, , p The EFSA Journal (2008) 716, 10-31

11 CN Cl O N Cl N O N Cl Figure 1. Chemical structure of diclazuril. The CAS number of diclazuril (2,6-dichloro-a-(4-chlorophenyl)-4-(4,5-dihydro-3,5-dioxo- 1,2,4-triazin-2(3H)-yl) benzeneacetonitrile) is The molecular weight is and the molecular formula C 17 H 9 Cl 3 N 4 O 2. Diclazuril exhibits trace solubility in non-polar solvents (hexane), weak solubility in alcohols (0.29 g/l in methanol) and high solubility in N,N-dimethylformamide (32.6 g/l) and dimethylsulfoxide (48.0 g/l). The pka is 5.92 and the log Kow is 4.54 at ph 8 and 4.00 at ph 5 (EFSA, 2007a). As summarised in the background chapter, diclazuril is authorised as a coccidiostatic feed additive for chickens for fattening, chickens reared for laying (up to 16 weeks of age) and turkeys for fattening (up to 12 weeks of age). The minimum and maximum concentration of diclazuril that is authorised is 1 mg/kg feed and the withdrawal period before slaughter is five days except for chickens reared for laying where no withdrawal period is necessary (Directive No 70/524/EEC concerning additives in feedingstuffs-list of authorised additives in feedingstuffs (2004/C 50/01) as amended by Regulation (EC) No 1519/ ). The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) has in April 2008 published two opinions on diclazuril (EFSA 2008a,b). In one opinion the FEEDAP Panel considers that a zero withdrawal period is acceptable for chickens and turkeys for fattening and it is therefore possible that the provisions on a withdrawal period of 5 days will be deleted in the list of authorised additives in feedingstuffs. In addition, if maximum residue limits (MRLs) should be considered necessary, they propose them to be set at 1.5, 1, 0.5 and 0.5 mg diclazuril/kg wet tissue for liver, kidney, muscle and skin/fat, respectively, in chickens and turkeys for fattening and chickens reared for laying (EFSA, 2008a). In the other opinion, the FEEDAP Panel evaluated the use of diclazuril in rabbits for fattening and breeding, and demonstrated its safety of use at 1 mg/kg feed. The FEEDAP Panel proposed one day withdrawal time and MRLs for rabbit tissues: 2.5, 1, 0.15 and 0.3 mg diclazuril/kg in the liver, kidney, muscle and fat, respectively (EFSA, 2008b). However, the legislation for use of diclazuril as a feed additive in rabbits is not yet in place, and the MRL have therefore not entered into force. 13 OJ C 50, , p. 1 /Amended by OJ L 335, , p. 15. The EFSA Journal (2008) 716, 11-31

12 Diclazuril is also listed in Annex II of Regulation (EC) No 2377/90 14 as a veterinary medicine for oral use in all ruminants and porcine animals 15. The recommended dose regimen is 1 mg diclazuril/kg b.w. for lambs and 5 mg diclazuril/kg b.w. for cattle and pigs as a single oral administration. The authorised veterinary medicinal uses of active ingredients that are placed in Annex II of Council Regulation No 2377/90 are regarded as being sufficiently safe for consumers and therefore no MRLs are needed. Consequently, no MRLs have been set for veterinary medicinal uses of diclazuril in ruminants and porcine animals. CODEX Alimentarius has established international MRLs in sheep, poultry and rabbits of 3, 2, 1 and 1 mg/kg for liver, kidney, muscle and skin/fat, respectively, 16 for the use of diclazuril as a veterinary medicine Biological activities of diclazuril Anticoccidial activity According to the anticoccidial properties of triazinones, diclazuril is active against intracellular development stages of coccidia, namely during schizogony and gametogony. After diclazuril treatment, the first- and second generation schizonts show extensive degenerative changes, which are characterised by the loss of internal structure, the appearance of many intracytoplasmic vacuoles and incomplete merogony. This leads to complete degeneration of schizonts and gamonts (EFSA, 2007a). Antibacterial activity In vitro studies of effects on growth of a limited range of microorganisms have shown that diclazuril is practically devoid of antibacterial and antimycotic properties at concentrations of up to 100 µg/ml (EFSA, 2007a) Previous evaluations of the toxicity and safety of diclazuril The consumer safety of diclazuril has recently been assessed by the FEEDAP Panel as part of the evaluation of applications for authorisation of the feed additive product Clinacox 0.5% (EFSA, 2007a,b; 2008a,b). Previous evaluations of the consumer safety of diclazuril include assessments by the Scientific Committee for Animal Nutrition (SCAN) for the use of diclurazil as a feed additive for chickens, turkeys and rabbits in 1991, 1997 and 2000 (EC, 1991, 1997a,b, 2000) respectively, the European Medicines Agency (EMEA, 1996, 2000) and the Joint FAO/WHO Expert Committee on Food Additives (JECFA) (FAO/WHO, 1996, 14 OJ L 224, , p.1 15 OJ L 211, , p yle=by+substance&domain=vetdrugmrls&language=english&query_form=%2fmrls%2fvetdrugs%2fvetd_ q-e.htm The EFSA Journal (2008) 716, 12-31

13 1999). The SCAN identified a toxicological ADI for diclazuril of 29 µg diclazuril/kg b.w. by applying a 100-fold uncertainty factor to the lowest no observed effect level (NOEL) of 2.9 mg/kg b.w. per day, established from a 2 year oral toxicity/carcinogenicity study in male mice using non specific changes in the liver (swelling of centrilobular hepatocytes and of sinusoidal cells) and, histiocytosis and the presence of pigmented macrophages in the mesenteric lymph nodes. EMEA (1996) and the JECFA (FAO/WHO, 1999) derived an ADI of 30 µg/kg b.w. based on the same data and apparently by rounding the ADI to one significant figure. The FEEDAP Panel retained the NOEL of 2.9 mg/kg per day and derived an ADI corresponding to 1.7 mg/person for a 60 kg person (EFSA, 2008b). The FEEDAP Panel also concluded that diclazuril had very low acute toxicity and was not mutagenic, genotoxic, carcinogenic, embryotoxic, foetotoxic or teratogenic (EFSA, 2007a, 2008b) Cross-contamination of feed batches Feed additives, such as coccidiostats, are marketed as premixtures, intended to be incorporated into mixed feeds during the mixing and production process. Cross-contamination refers to the fact that under the practical conditions in a commercial feed mill, residual amounts of feed materials remain in the production line (see also the background chapter) and may contaminate following feed batches. The degree of cross-contamination depends on the technical facilities and procedures, as well as on product characteristics Factors influencing the rate of cross-contamination Several studies have shown that a completely contamination-free production of premixes and compound feeds in existing multi-product plants is impossible in practice (Strauch, 2003). Various process parameters and the physicochemical characteristics of the product act together to determine the residual amount remaining in the circuit and hence the rate of crosscontamination from one feed batch to the subsequent batches produced in the same production line (Kennedy et al., 1996, 1998a; McEvoy et al., 2003; Harner et al., 1996). In a similar way, the purchased premix that is incorporated into the feed can itself contain traces of contamination of other substances, due to cross-contamination during the production of the premix. The technological equipment in the feed mill can influence the amount of crosscontamination that may occur. The following sites in the circuit have been identified as being places where fractions of feeds can be retained, with the possible consequence of contamination of later batches: - Areas of reduced flow in piping, material ledges, and non-plane surfaces (screw couplings, weld seams, moulded tanks) can lead to a sedimentation of feed materials. The EFSA Journal (2008) 716, 13-31

14 - Oversized and long conveying systems, and non-continuous earthing of parts of the production plant. - In silos or containers, differences in flow rate may cause segregation of the bulk material, which accumulates in dead zones with solidification of the bulk material. - Conveyors which do not empty completely, such as screw conveyors and elevator boots. - Wear of mixing equipment and conveying systems can cause a reduced flow in certain areas at which material can accumulate. - Filter systems may accumulate residues, in particular with material featuring high dusting potential and strong aspiration flow. The physicochemical characteristics of additives can contribute to cross-contamination in the following ways: High dusting potential, low product moisture, adherence due to electrostatic charge, as well as environmental conditions (e.g. adhesions caused by surrounding moisture) contribute to crosscontamination. The more dispersed in air and the lower the density of the components, the more sensitively they react to current fields. Basically, particle sizes <500 µm are dispersible in the air which facilitates the discharge of suitable, airborne components by aspiration air. An accumulation of feed material in filters and incomplete or inappropriate cleaning (see above) can lead to cross-contamination of these components into the next production batch. Also a high electrostatic loading potential as well as higher product moistures can cause adhesions inside production plants and can result in cross-contamination. Finally, it should also be mentioned that activities in or outside the feed mill may contribute to undesired contamination of non-target animal feed, for instance, insufficient rinsing or no rinsing during product changes will result in a greater amount of cross-contamination. The beneficial effect of using rinsing batches can be reduced considerably if the residual material adhering to the equipment cannot be fully removed by the material flow of the rinsing batch (McEvoy et al., 2003; Noser et al., 2006; Dorn et al., 1988). Further cross-contamination can occur at the feed plant during conveying (contaminated conveying equipment) and on-farm (e.g. during storage and transport to the feeding location) Assessing cross-contamination in feed mills In investigations involving the majority of German compound-feed plants (approximately 450), more than half of the examined production plants had a level of cross-contamination of less than 4% (Strauch, 2002). A survey of Belgian compound-feed production companies showed similar values for pelleted products (OVOCOM, 2004, document provided to EFSA). The EFSA Journal (2008) 716, 14-31

15 Similar results were achieved with mashed (not pelleted) feeds (approx. 69% containing less than 5% cross-contamination). The investigations described above, refer to a general technical control of the mixing facilities used by commercial feed mills. Comparable investigations on the behaviour of coccidiostats during compound-feed production have not been carried out. As yet, analytical controls of the produced feeds for the presence of coccidiostats were only conducted in cases for which residual amounts of the coccidiostatic agents were found in food obtained from accidentally exposed animals. Systematic investigations of the behaviour of coccidiostats at compoundfeed production companies have been carried out for lasalocid, narasin, nicarbazin and monensin (Kennedy et al., 1996, 1998a,b; McEvoy et al., 2003; Noser et al., 2006). From these investigations it can be concluded that: - Cross-contamination can be reduced significantly by suitable measures. - Contamination by coccidiostats was detected in several rinsing batches Specific data for diclazuril-based feed additive products No information has been identified for diclazuril concerning cross-contamination and physical parameters. 2. Methods of analysis for diclazuril 2.1. Analysis of diclazuril in premixes and animal feeds Fully validated methods of diclazuril in chicken feed were reported by Mortier and coworkers (2005a). Feed samples were extracted with methanol and after evaporation extracts were analysed with high pressure liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS) using multiple reaction monitoring mode and electrospray ionization. At the concentration range of 0-2 mg/kg the regression analysis demonstrated a mean slope of 3.29 (R 2 value 0.99) and 3.23 (R 2 value 0.997), respectively. The decision limits (CCα) 17 and the detection capabilities (CCβ) were 2.1 and 0.5 and 2.2 and 0.6 µg/kg feed. 17 Definitions of limit of detection (LOD), limit of quantification (LOQ), decision limit (CCα) and detection capability (CCβ): Commission decision 2002/657 of 12 August 2002 implementing Directive No (EC) 96/23 concerning the performance of analytical methods and the interpretation of results (OJ L 221, , p. 8-36) define the performance of analytical methods used for residue control and the interpretation of results. CCα means the limit at and above which it can be concluded with an error probability of α that a sample is non-compliant. CCβ means the smallest content of the substance that may be detected, identified and/or quantified in a sample with an error probability of β. CCα is equivalent to the LOD defined by IUPAC guidance (IUPAC, 1995). The LOQ (ISO, 1997) is defined by the relative standard deviation of the estimated quantity. Generally, it corresponds to the lowest concentration tested with a relative standard deviation below the performance value needed, such as the performance for repeatability defined by Decision No (EC) 2002/657 (OJ L 221, , p. 8 36). The EFSA Journal (2008) 716, 15-31

16 In EU legislation, an official method for the determination of diclazuril in feed and premixtures has been established by Directive 1999/27/EC 18. By this method the content of diclazuril is determined by ternary gradient reversed-phase HPLC using a ultra-violet (UV) detector. The limit of detection (LOD) of the method is 0.1 mg/kg and the limit of quantification (LOQ) is 0.5 mg/kg Analysis of diclazuril residues in animal products According to the list of methods used by the National Reference Laboratories (NRL) for residue control, edited by the Community Reference Laboratory (CRL) (Bohm et al., 2005), diclazuril residues methods are reported for muscle and liver tissues by 4 and 2, respectively and for eggs by 5 out of the 20 NRLs within the EU. The Member States used different methods such as LC-MS or LC-MS/MS for screening and confirmatory purposes. The decision limits ranged between 0.3 and 25 µg diclazuril/kg tissue. A specific analytical method (HPLC) with a LOQ of 0.05 mg/kg wet tissue liver, kidney and muscle, and 0.1 mg/kg fat, has also been provided by industry to the FEEDAP Panel (EFSA, 2007, 2008). No MRLs have been established in eggs or other animal tissues for the use of diclazuril as a feed additive, however, MRLs for rabbit tissues are expected to be established shortly (see Chapter 1) Screening methods Dubois et al. (2004) described a multi-residue qualitative method based on LC-MS/MS for determining in muscle and eggs, nine coccidiostats. The method uses extraction in acetonitrile followed by a clean-up on solid phase extraction (SPE). Two mass transitions (m/z and m/z ) were monitored. For diclazuril residue in muscle, extraction recovery was 60% and CCα was 0.3 µg/kg. The method is also applicable to eggs Quantitative and confirmatory methods Mortier et al. (2003) described a multi-residue method based on LC-MS/MS. After acetonitrile extraction, the extract was concentrated and filtrated before analysis. One mass transition (m/z ) was monitored. Diclazuril recovery was 113% and CCα was 6 µg/kg. 18 Commission Directive 1999/27/EC of 20 April 1999 establishing Community methods of analysis for the determination of amprolium, diclazuril and carbadox in feedingstuffs and amending Directives 71/250/EEC, 73/46/EEC and repealing Directive 74/203/EEC. OJ L 118, , p. 36 The EFSA Journal (2008) 716, 16-31

17 A method based on LC-MS/MS for quantification of diclazuril in poultry muscle and liver was described by Mortier et al. (2005b). Diclazuril residues were extracted with acetonitrile. The extract was evaporated to dryness. The sample was redissolved in acetonitrile/water before analysis. Two mass transitions (m/z and m/z ) were monitored. Linearity was determined in the concentration range µg/kg and µg/kg for meat and liver, respectively. Recoveries were close to 95% for meat and 100% for liver. A CCα value of 0.5 µg/kg was determined for diclazuril in muscle. A method based on LC-MS/MS in electrospray negative mode was developed for analysis of diclazuril residue in eggs (Daeseleire et al., 2005). After liquid-liquid extraction with acetonitrile, the extract was injected for liquid chromatography. With a rheodyne 6-way valve, it can be programmed that the flow only enters the mass spectrometer when the compound is expected to elute. Splitting off the flow can reduce pollution of the mass spectrometer. Diclazuril-bis (a structural analogue of diclazuril) was used as internal standard. Two mass transitions (m/z and m/z ) were monitored. The concentration range was µg/kg. The mean slope was 0.71±0.07 (r 2 =0.998). The CCα and CCβ were 0.5 and 0.6 µg/kg respectively. Recoveries ranged from 95.5% at 5 µg/kg to 108.1% at 100 µg/kg. 3. Occurrence of diclazuril 3.1. Occurrence of diclazuril residues in feed materials for non-target animal species Data on cross-contamination of feed are scarce. The Czech Republic reported the results of 254 analyses that were performed during No residues of diclazuril were found with a LOQ of 0.5 mg/kg. There have been no notifications from the Rapid Alert System for Food and Feed (RASFF) 19 concerning feed containing diclazuril between April 2002 and April Occurrence of diclazuril residues in animal products derived from non-target animal species Residues of diclazuril in non-target animal tissues and eggs can arise from crosscontamination but also if a non-target animal is given feed formulated for target animal species, intentionally or accidentally. Eggs, muscle and liver from different animal species are analysed by the Member States according to requirements in Directive (EC) No 96/ However, the results from the 19 For more information on the RASFF system: The EFSA Journal (2008) 716, 17-31

18 Member States were very different in terms of LODs and the definition of compliant and noncompliant. The levels at which a result is defined as non-compliant are not harmonised within the Member States, but some countries report results above 10 µg diclazuril/kg tissue as noncompliant. For diclazuril, combined results of 2004 and 2005 show that 19 non-compliant samples were found amongst 3511 samples of different animal tissue. Thirteen non-compliant samples were found in poultry (5 out of 659 in muscle and 8 out of 418 in liver), six non-compliant samples were found in eggs out of 274 samples analysed. The LOD ranged from 0.8 to 25 µg/kg. Belgium has provided individual data of 972 samples of foods that were analysed for diclazuril in 2005 and Six samples of poultry muscle contained residues of diclazuril that were greater than the Belgian defined non-compliant limit of 10 µg/kg. One sample of poultry muscle and one of farmed game contained residues of diclazuril between the LOD of 5 µg/kg and the Belgian non-compliant limit of 10 µg/kg (data provided to EFSA). In the large annual survey on coccidiostat residues in food in UK, diclazuril was not included. Incidence of residues of diclazuril in eggs has been studied by Mortier et al. (2005c). From 320 eggs, 26 were positive whereof 92% of the samples containing concentrations of diclazuril <1 µg/kg. The highest concentration found was 3.1µg/kg and the CCα was 0.5 µg/kg. 4. Toxicity of diclazuril 4.1. Mechanisms of toxicity Although the mode of action of diclazuril is not precisely known, its effect on the asexual or sexual stages of coccidiosis blocks the excretion of oocysts inducing an interruption of the life cycle of the parasites (EMEA, 1996) Toxicity of diclazuril in target animal species Chicken Diclazuril was administered to chickens at the maximum authorised level in feedingstuffs (1 mg/kg), and was well tolerated with no toxicity observed. Identical conclusions were drawn from experiments performed on female and male breeding broilers, fed with a diet at 5 mg/kg of diclazuril for 28 and 42 days respectively. Finally, at 25 times (25 mg/kg) the maximum authorised concentration of 1 mg/kg, chickens for fattening exposed from birth to day Directive No (EC) 96/23 of 29 April 1996 on measures to monitor certain substances and residues thereof in live animals and animal products. OL L 125, , p The EFSA Journal (2008) 716, 18-31

19 showed no signs of intolerance. This was concluded to indicate a very large safety margin for diclazuril in chickens (EC, 1991) Turkey Tolerance studies with diclazuril were performed on male and female growing turkeys under semi-field conditions.the animals were fed for 16 weeks with diets containing 1, 12.5 and 25 mg diclazuril/kg feed. No effects in terms of body weight gain, feed consumption, feed conversion, clinical signs, mortality, haematology, serum biochemistry, and histopathology of liver, spleen, kidney, heart, bursa fabricii, brain, thymus, bone and bone-marrow, testes and ovary compared to controls. At 10 and 100 mg diclazuril/kg feed administered for 7 consecutive days, no clinical signs of toxicity nor drug or dose-related lesions were observed (EC, 1997b) Rabbits Efficacy trials demonstrated that treatments with 1, 2 or 3 mg diclazuril/kg of complete feedingstuffs were equivalent in terms of coccidiosis control and did not show undesirable effects (EC, 2000). Mortality rate and hepatic damage were reduced in animals on the diclazuril diet compared to controls. Weight gain and feed conversion index were improved. Recently, tolerance studies in rabbits for fattening and breeding demonstrated that diclazuril was tolerated at six-fold the highest dose authorised for use in chickens and turkeys for fattening (1 mg/kg feed) (EFSA, 2007a) Toxicity of diclazuril in non-target animal species Ducks Groups of 10 mallard ducks received diclazuril at a level of 0, 10, 30, 100, 300 or 1000 mg/kg of feed for 28 days (data provided by the industry). Feed consumption, body weight, mortality, morbidity, and gross pathology were reported. Other parameters such as egg production, shell thickness, egg quality and hatchability were also recorded. Ducklings were monitored for weight gain, mortality and gross pathology at 14 days of age. Treatment with diclazuril did not affect body weight, feed consumption, mortality, clinical signs or reproduction in adult ducks. Body weights and survival of hatchlings were unrelated to treatment with diclazuril. Groups of 50 Peking ducks (one day old unsexed) received diclazuril at a dose of 10 mg/kg of feed for 56 days (data provided by the industry). No adverse effects were reported and live The EFSA Journal (2008) 716, 19-31

20 weight gain, feed consumption and mortality in the treated birds remained comparable to the control group. Groups of 10 Peking ducks received diclazuril at a dose of 0, 10 or 100 mg/kg of feed for 7 days (data provided by the industry). All birds were observed daily and body weight and feed consumption recorded at the end of treatment. Birds after sacrifice were necropsied and blood samples collected for haematology and blood chemistry. The measured end-points included clinical signs, body weight gain, feed consumption or feed conversion rate. There were no gross pathology abnormalities or blood parameter changes and no differences were observed between animals receiving diclazuril, and untreated animals Quails and guinea fowls The tolerance of diclazuril in Japanese quail and guinea-fowl was investigated at a level of 0, 1, 12.5 or 25 mg/kg of feed (data provided by the industry). Day-old unsexed quails (n=452) and guinea-fowl (n=223) were each randomly divided into 4 groups. Quail were dosed for 31 days and in guinea-fowl for 85 days. Any adverse effects on, mortality, growth performance, blood clinical chemistry, gross pathology and histopathology were recorded. Diclazuril was well tolerated in both bird species. Blood clinical chemistry parameters, gross pathology and histopathology did not reveal any treatment related changes. Japanese quail received diclazuril at level of 0, 1, 5, 10, 20 and 50 mg/kg of feed for two weeks. The following fertility parameters were evaluated: egg count, egg weight, cracked eggs and wind-eggs, fertilization rate, chick embryos with an early or late death and hatchability. No changes in reproductive performance were found in any group and no side effects were observed even at a feed inclusion rate of 50 mg/kg Horses Two healthy adult horses received diclazuril orally at a daily dose of 20 mg/kg b.w. per day for 21 days (data provided by the industry). Body weight, clinical examinations (heart rate, rectal temperature, respiratory rate, appetite, presence of faeces), haematology and blood clinical chemistry were observed. There were no treatment-related effects on these parameter values. Diclazuril was administered at a dose of 1 mg/kg b.w. per day for 6 days as a top dressing in feed to one horse (data provided by the industry). There were no alterations in clinical signs, bodyweights, haematology and blood clinical chemistry. The EFSA Journal (2008) 716, 20-31

21 Calves Groups of young calves received diclazuril, as an oral suspension, at daily doses of 1, 3 or 5 mg/kg b.w. per day for 3 days (data provided by the industry). There were no changes in clinical examinations, body weight, haematology and blood clinical chemistry between the treated animals. Gross and microscopic examination did not reveal treatment-related effects Sheeps Groups of 2 Merino sheep were given diclazuril as a 0.25% oral suspension (an oral drench ) at doses of 20, 40 or 60 mg/kg b.w. (data provided by the industry). No adverse reactions were recorded at any of the dose levels tested. In tolerance studies, neither drug-related abnormal clinical observations nor side-effects were observed in sheep after treatment at 1, 3 or 5 times the proposed therapeutic dose of 1 mg diclazuril/kg b.w. (EMEA, 1996) Goats Groups of Angora goats were dosed with a 0.25% oral solution of diclazuril at a dose level of 20, 40 or 80 mg/kg b.w. (data provided by the industry). No toxic effects were observed Piglets Two groups of 8 piglets were given an oral administration of diclazuril at 10 mg/kg b.w. per day for 3 days (data provided by the industry). The piglets were monitored for a period of 10 days after the first administration for clinical signs, changes in body weight and feed consumption. No adverse effects occurred, and there were no differences in performance parameters between the treated group and an untreated control group. 5. Kinetics and tissue disposition 5.1. Kinetics of diclazuril in the target animal species Chickens In a single-dose study, 14 C-labelled diclazuril was administered to chickens for fattening in a lactose mixture at a dose of 1 mg/kg b.w.. Maximum radioactivity concentrations of µg diclazuril equivalents (total radioactive residues)/ml were attained in plasma 6 hours after administration. The clearance from plasma had a half-life of approximately 50 hours. Equilibrium between plasma and tissue levels was rapidly achieved. The tissue concentrations were 2 to 10 times lower than the corresponding plasma concentrations. Maximum concentrations of 1260, 1070, 170 and 140 µg diclazuril equivalents/kg in liver, kidney, The EFSA Journal (2008) 716, 21-31