The EFSA Journal (2008) 696, 1-12 Maximum Residue Limits for Clinacox 0.5% (diclazuril) for turkeys for fattening, chickens for fattening and chickens reared for laying 1 Updated Scientific Opinion of the Panel on Additives and Products or Substances used in Animal Feed (Question No EFSA-Q-2007-191) Adopted on 16 April 2008 PANEL MEMBERS Georges Bories, Paul Brantom, Joaquim Brufau de Barberà, Andrew Chesson, Pier Sandro Cocconcelli, Bogdan Debski, Noël Dierick, Anders Franklin, Jürgen Gropp, Ingrid Halle, Christer Hogstrand, Joop de Knecht, Lubomir Leng, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Miklós Mézes, Carlo Nebbia, Walter Rambeck, Guido Rychen, Atte von Wright and Pieter Wester SUMMARY Following a request from the European Commission, the European Food Safety Authority (EFSA) was asked to complete its former opinion on the maximum residue limits (MRL) of Clinacox 0.5% (diclazuril) for turkeys for fattening, chickens for fattening and chickens reared for laying. The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) assessed the supplementary information supplied by the applicant on the diclazuril impurity/metabolite R070016 and concluded that the compound is devoid of genotoxic activity. Consequently, the FEEDAP Panel is in a position to adopt the already proposed ADI for diclazuril (1.7 mg diclazuril per person per day). The metabolic fate of diclazuril in chicken and turkey is qualitatively similar, diclazuril being the marker residue in tissues. At a zero withdrawal time, the theoretical consumer exposure to the total residues from both chickens and turkeys represents about 13 % of the ADI. Therefore, the FEEDAP Panel considers that applying a withdrawal period of zero day would not compromise consumer safety. Consequently, no MRLs are required for chickens and turkeys for fattening. Should MRLs be considered necessary, the FEEDAP Panel proposes the following values: 1.5, 1, 0.5 and 0.5 mg diclazuril kg -1 wet tissue for liver, kidney, muscle and skin/fat, respectively. Those MRLs are in line with highest measured residues and ensure consumer safety (the worst case calculation representing 30 % of the ADI). 1 For citation purposes: Updated Scientific Opinion of the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) on a request from the European Commission on the Maximum Residue Limits for Clinacox 0.5% (diclazuril) for turkeys for fattening, chickens for fattening and chickens reared for laying. The EFSA Journal (2008) 696, 1-12 European Food Safety Authority, 2008
A suitable analytical method in terms of specificity and sensitivity is available for the determination of diclazuril residues in tissues at the MRL level. Key words: coccidiostats, Clinacox 0.5%, diclazuril, chickens for fattening, turkeys for fattening, chickens reared for laying, MRLs, consumer safety, ADI, withdrawal time The EFSA Journal (2008) 696, 2-12
TABLE OF CONTENTS Panel Members...1 Summary...1 Table of Contents...3 Background as provided by EC...4 Terms of reference as provided by EC...4 Acknowledgements...4 Assessment...5 1. Introduction...5 2. Supplementary information submitted by the applicant...5 3. Proposal for an ADI...6 4. Residues...6 4.1. Total residues...6 4.1.1. Chickens for fattening...6 4.1.2. Turkeys for fattening...7 4.2. Marker residue...7 4.2.1. Chickens for fattening...7 4.2.2. Turkeys for fattening...8 4.3. Ratio marker to total residues...8 5. Withdrawal time...9 6. Proposal for MRLs...9 6.1. Chickens for fattening...9 6.2. Turkeys for fattening...10 7. Evaluation of the analytical methods by the Community Reference Laboratory (CRL)...10 Conclusions...10 Documentation provided to EFSA...11 References...11 Appendices...12 The EFSA Journal (2008) 696, 3-12
BACKGROUND AS PROVIDED BY EC Regulation (EC) No 1831/2003 2 establishes the rules governing the Community authorisation of additives for use in animal nutrition and in particular defines the conditions that a substance/product should meet to modify the authorisation. The company Janssen Animal Health 3 is seeking Community modification of the authorisation of its coccidiostat with the trade name Clinacox 0.5% for turkeys for fattening, chickens for fattening and chickens reared for laying (Table 1). Table 1. Description of the preparation Category of additive Coccidiostats and other medicinal substances Functional group of additive - Trade name Clinacox 0.5% Description diclazuril Target animal category turkeys for fattening, chickens for fattening and chickens reared for laying Applicant Janssen Animal Health Type of request Update opinion On 13 th June 2007, the Scientific Panel on additives and Products or Substances used in Animal Feed of the European Food Safety Authority adopted an opinion on the Maximum Residue Limits for Clinacox 0.5% (diclazuril) for turkeys for fattening, chickens for fattening and chickens reared for laying (EFSA-Q-2006-134). It was concluded that there is not enough data to propose MRLs of this coccidiostats for turkeys for fattening, chickens for fattening and chickens reared for laying. Therefore the Commission gave the possibility to the company to submit complementary information to complete the assessment. The Commission has now received supplementary dossier from the applicant Janssen Animal Health for this coccidiostat with new genotoxicity studies. The data generated by the company and compiled in the above mentioned supplementary dossier have been sent directly to the Authority. TERMS OF REFERENCE AS PROVIDED BY EC In view of the above, the Commission asks to the European Food Safety Authority to deliver an opinion on the safety (MRLs) of this coccidiostats for turkeys for fattening, chickens for fattening and chickens reared for laying, taking into account its earlier opinion of 13 th June 2007 and the new data submitted. ACKNOWLEDGEMENTS The European Food Safety Authority wishes to thank the members of the Working Group on Coccidiostats. 2 OJ L 268, 18.10.2003, p.29 3 Janssen Animal Health, Turnhoutseweg 30, B-2340 Beerse, Belgium The EFSA Journal (2008) 696, 4-12
ASSESSMENT 1. Introduction In its former opinion on the Maximum Residue Limits (MRLs) for Clinacox 0.5% (diclazuril) for turkeys for fattening, chickens for fattening and chickens reared for laying (EFSA, 2007), the FEEDAP Panel concluded that: A specific and major diclazuril impurity and/or metabolite has been identified in rabbit fat which has not been found to occur in laboratory animals. It cannot be ruled out that the accumulation in fat of this lipophilic impurity/metabolite might occur in other species, including the chicken and turkey. The safety of this compound has not been established. Provided the safety issues relating to this impurity/metabolite are satisfactorily addressed then the FEEDAP Panel would be in a position to adopt the already proposed ADI (1.7 mg per person per day) and the safety assessment for the consumer could be completed. In order to assess the safety of this impurity/metabolite (R070016), the applicant was requested to supply a complete battery of genotoxicity tests. 2. Supplementary information submitted by the applicant In response to the FEEDAP Panel question on the safety of the diclazuril impurity/metabolite R070016, the applicant supplied a set of in vitro and in vivo studies: 4 1. An in vitro bacterial reverse mutation genotoxicity test (Ames test). It was performed in triplicate and was carried out on five different Salmonella typhymurium strains (TA98, TA100, TA1537, TA1535, TA102), with and without metabolic activation (Arochlor 1254 induced liver S9-mix has been used as activation system). All the experiments were conducted at the limit of solubility which explains that the top dose has been lowered from 5000 μg to 800 μg per plate. From the study provided, it could be concluded that R070016 has no mutagenic properties towards the different Salmonella typhymurium strains used, both in the absence and the presence of metabolic activation. 2. An in vitro Mouse lymphoma test to investigate mutations at the Thymidine Kinase (tk) locus of the mouse lymphoma L5178Y cells (MLA) using microtitre fluctuation technique (mutations at gene level in eukaryotes). The experimental conditions included concentrations of R070016 up to the solubility limit in two independent experiments, both in the absence and the presence of metabolic activation. The mutant frequencies of the concentrations plated were all inferior to the sum of the mean control mutant frequency plus the global evaluation factor, indicating negative results. 3. An in vivo Micronucleus test on peripheral blood reticulocytes of mice. The induction of structural/numerical chromosome aberrations in peripheral blood reticulocytes was assayed after administration of R070016 in male and female CD1 mice. R070016 was dosed orally once with 500, 1000, and 2000 mg kg -1 at 48 or 72 hours before the collection of blood samples. R070016 did not induce a statistically significant or a biologically relevant increase of micronucleated erythrocytes in male or female CD1 mice. It can be concluded that R070016 at doses at which some signs of bone marrow toxicity were recorded, does not induce statistically significant increase in the percentage of micronucleated erythrocytes. R070016 can be considered as devoid of 4 Supplementary Dossier. November, 2007 The EFSA Journal (2008) 696, 5-12
clastogenic and/or aneugenic properties leading to the formation of micronuclei in the mouse bone marrow micronucleus test. From the outcome of this battery of genotoxicity tests, the FEEDAP Panel concludes that under the conditions of the experiments performed, the impurity/metabolite R070016 of diclazuril is considered as devoid of genotoxic potential. 3. Proposal for an Acceptable Daily Intake Considering the above results, the FEEDAP Panel is now in a position to adopt the already proposed acceptable daily intake (ADI) for diclazuril (1.7 mg diclazuril per person per day) (EFSA, 2007). Other scientific bodies (EMEA, 1996; JECFA, 1999) have proposed the same value. 4. Residues 4.1. Total residues 4.1.1. Chickens for fattening A kinetic study of total residues and unchanged diclazuril was performed in chickens (four weeks of age at start of dosing, four males and four females per group). [ 14 C]-diclazuril was administered by gavage (capsule, twice a day) at a constant dose of 0.118 mg diclazuril per day, corresponding to 1.08 mg diclazuril kg -1 feed (average dose over the whole experimental period, i.e. 1.26 mg kg -1 feed at the beginning and 0.87 at the end, estimated from historical feed intake and growth data). The animals were slaughtered at different times following the last administration. 5 Figures for total residues and diclazuril residues are given in Table 2. Table 2. [ 14 C]-diclazuril residues (TTR (a) ) in chicken tissues following repeated oral administration (14 days) of a constant dose of 0.118 mg diclazuril per bird and day Withdrawal Time (days) Liver Kidney Muscle Skin/fat TRR Diclaz (b) TRR Diclaz TRR Diclaz* TRR Diclaz 0.25 0.386 ± 0.069 0.370 ± 0.052 0.324 ± 0.038 ND (c) 0.087 ± 0.007 0.078 0.193 ± 0.017 0.158 ±0.022 1 0.240 ± 0.057 0.202 ± 0.053 0.199 ± 0.038 ND 0.044 ± 0.005 0.038 0.110 ± 0.016 0.085 ± 0.013 3 0.187 ± 0.031 0.138 ± 0.021 0.133 ± 0.020 ND 0.032 ± 0.005 0.026 0.083 ± 0.011 0.059 ± 0.005 5 0.107 ± 0.017 0.085 ± 0.029 0.079 ± 0.021 ND 0.019 ± 0.006 0.019 0.049 ± 0.010 0.041 ±0.013 (a) TRR = total residual radioactivity expressed as mg equivalent diclazuril kg -1 wet tissue, average ± SD values from four males and four females per withdrawal time point. (b) Diclaz = diclazuril residues (mg kg -1 wet tissue) (c) ND = not determined * M. femoralis, derived from the ratio marker to total residue in M. pectoralis The lack of adjustment of the labelled diclazuril dose results in an underestimation of the total residues to be expected after consumption of feed containing the target dose of 1 mg diclazuril 5 Technical Dossier. Section IV, Volume 1, Reference 7 The EFSA Journal (2008) 696, 6-12
kg -1. However, those values are considered suitable for the calculation of the ratio marker vs. total residues. 4.1.2. Turkeys for fattening Total residues in tissues have been measured in turkeys (three males and three females, 11 weeks of age at start of dosing). 6 [ 14 C]-diclazuril was administered by gavage (capsule, twice a day) for 14 days at a constant daily dose of 0.34 mg per bird (0.069 mg kg -1 bw at start, 0.044 mg kg -1 bw at the end, estimated from historical feed intake and growth data) corresponding to an average of 1.24 mg kg -1 feed. The animals were slaughtered after a 0.25-day withdrawal period (Table 3). Total residues were not determined in skin/fat but in abdominal fat (values taken by the FEEDAP Panel as the worst case situation for skin/fat). Table 3. [ 14 C]-diclazuril residues (TRR a ) in turkey tissues following repeated oral administration (14 days) of a constant dose of 0.34 mg diclazuril per bird and day Withdrawal Liver Kidney Muscle Abdominal fat (b) Time (days) 0.25 0.508 ± 0.121 0.372 ± 0.090 0.055 ± 0.010 0.246 ± 0.093 ( a) TRR = total residual radioactivity expressed as mg equivalent diclazuril kg-1 wet tissue, average ± SD values from three males and three females (b) Taken as a worst case for skin/fat When compared to the situation described for chickens (see Section 4.1.1), the experimental procedure would not have influenced the residue data to the same extent because the corresponding average of feed concentration (1.24 mg kg -1 ) was higher than the target dose (1 mg kg -1 ). Diclazuril has only been determined in the liver and amounted to 0.352 mg kg -1 wet tissue (70 % of total radioactivity). 4.2. Marker residue On the basis of the data supplied concerning the metabolic fate of diclazuril in chicken 7 and turkey, 8 it appears that the substance behaves qualitatively very similarly in those two species, i.e. absorbed and biotransformed at a very limited extent. Diclazuril is the major residue in all tissues tested (70 to 90 % of total residues). The FEEDAP Panel concludes that diclazuril is the marker residue in poultry. This is in agreement with former conclusions from the SCAN (EC, 1991), JECFA (1999) and EMEA (1996). 4.2.1. Chickens for fattening A study on tissue residue kinetics of diclazuril was performed in chicken (three males and three females per group) administered Clinacox 0.5% (1 mg diclazuril kg -1 feed) for 42 days. 9 The animals were slaughtered after different withdrawal periods and diclazuril was measured in liver, kidney, muscle and skin/fat (Table 4). 6 Technical Dossier. Section IV, Volume 2, Reference 13 7 Technical Dossier. Section IV, Volume 1, Reference 6 8 Technical Dossier. Section IV, Volume 2, Reference 11 9 Technical Dossier. Section IV, Volume 2, Reference 9 The EFSA Journal (2008) 696, 7-12
Table 4. Tissue residue kinetics of diclazuril in chickens administered Clinacox 0.5% (1 mg diclazuril kg -1 feed) for 42 days Withdrawal Liver (*) Kidney (*) Muscle (*) Abdominal fat (*) Time (days) 0.25 0.621 ± 0.121 0.467 ± 0.103 0.124± 0.018 0.115± 0.037 1 0.480 ± 0.095 0.412 ± 0.112 0.100 ± 0.017 0.089 ± 0.058 3 0.268 ± 0.075 0.188 ± 0.065 0.058 ± 0.028 0.065 ± 0.026 5 0.148 ± 0.073 0.121 ± 0.055 0.033± 0.015 0.026± 0.017 ( * ) mg kg -1 wet tissue, average values ±SD of three males and three females per withdrawal time point Average diclazuril concentrations in all tissues and withdrawal times were, as expected, higher than the total residues formerly measured (see Section 4.1.1.). As a consequence, the latter values (Table 4) have been retained as reflecting practical farming conditions. 4.2.2. Turkeys for fattening Only one set of data concerning diclazuril residues in the liver of turkeys administered [ 14 C]- diclazuril for 14 days and slaughtered after a 0.25-day withdrawal period is available. 10 They amounted to 0.352 mg kg -1 wet tissue which accounted for about 70 % of the total radioactivity. A study on tissue residue kinetics of diclazuril was performed in turkeys (three males and three females per group) administered Clinacox 0.5% (1 mg diclazuril kg -1 feed) for 115 days. 11 The animals were slaughtered after different withdrawal periods and diclazuril was measured in liver, kidney, muscle and skin/fat (Table 5). Table 5. Tissue residue kinetics of diclazuril in turkeys administered Clinacox 0.5% (1 mg diclazuril kg -1 feed) for 115 days Withdrawal Liver (*) Kidney (*) Muscle (*) Abdominal fat (*) Time (days) 0.25 0.399 ± 0.035 0.285 ± 0.012 0.045± 0.006 0.146± 0.018 1 0.301 ± 0.030 0.268 ± 0.020 0.043 ± 0.004 0.132 ± 0.019 3 0.194 ± 0.008 0.161 ± 0.012 0.024 ± 0.002 0.095 ± 0.011 5 0.122 ± 0.014 0.103 ± 0.014 0.015± 0.001 0.077± 0.017 * mg kg -1 wet tissue, average values ±SD of three males and three females per withdrawal time point 4.3. Ratio marker to total residues The ratios marker/total residues calculated from the data supplied for chickens (Table 2) amounted to 0.96, 0.90 and 0.82 for liver, muscle and fat, respectively, after a 0.25-day withdrawal period. Data supplied do not allow the calculation of the ratio for kidney. For turkey, the data supplied allowed the calculation of the ratio only for liver after a 0.25-day withdrawal time (0.70). 12 Other data taken from a study carried out following an acute oral dosage of 1 mg [ 14 C]-diclazuril kg -1 bw (approximately 20-fold higher than the corresponding recommended dose in feed), allow the calculation of ratios of 0.56, 0.81, 0.77 and 0.38 for 10 Technical Dossier. Section IV, Volume 2, Reference 12 11 Supplementary information. November 2006 12 Technical Dossier. Section IV, Volume 2, Reference 13 The EFSA Journal (2008) 696, 8-12
liver, kidney, muscle and skin/fat after a zero-day withdrawal time. 13 However, those values do not correspond to the metabolic steady state and therefore are only considered indicative. Considering the variable dosing in the chicken study, the incompleteness of the dataset (absence of kidney data for chickens, only liver data for turkeys) and the inadequacy of some of the data supplied (ratios for the relevant turkey tissues determined only after acute dose), the FEEDAP Panel, following a worst case approach, retains a common ratio marker residue vs. total residues of 0.70 for all tissues in both animal species. 5. Withdrawal time Daily exposure of the consumer was calculated using the theoretical consumption figures defined in Directive 2001/79/EC 14 for different tissues and maximised total residues (average plus 2 SD) measured after a 0.25-day withdrawal period in chicken (Table 2) and turkey (Table 3). Considering the limitations of the chicken study with radiolabelled diclazuril (see Section 4.1.1), the total residues have been calculated from the 42-day feeding study (Table 4) taken as a reference and applying the tissue-specific ratios of marker residue vs. total residues taken from the radiolabel study (Table 2). Under these conditions, human exposure is calculated to be 0.226 mg per day, corresponding to about 13 % of the ADI. Comparison of diclazuril residues in chickens (Table 4) and turkeys (Table 5) shows that the turkey data do not exceed chicken data. Therefore, human exposure resulting from turkey tissues consumption would also comply with the ADI. The FEEDAP Panel concludes there is no safety concern from consumption of edible tissues from poultry fed feedingstuffs containing 1 mg diclazuril kg -1 complete feed. As the experimental 0.25-day withdrawal period corresponds to a zero withdrawal in practice, the FEEDAP Panel considers that a zero withdrawal time is acceptable. 6. Proposal for Maximum Residue Limits As the consumer safety is ensured without applying a withdrawal period (zero withdrawal), there is no necessity to establish maximum residue limits (MRLs). This view of the FEEDAP Panel is in agreement with EMEA conclusions (EMEA, 1996). If MRLs are considered necessary, their setting should comply with the following requirements: i) the consumer theoretical exposure resulting from the consumption of chicken tissues containing each the corresponding MRLs, calculated using the ratio marker residue to total residues at the proposed withdrawal time (zero day), should enter the ADI, ii) for control purposes the values retained should be as close as possible to the upper limit of the residue range observed in chicken and turkey tissues after the use of the additive at the highest level authorised, iii) a specific analytical method with sufficient sensitivity should be available. 6.1. Chickens for fattening Kinetic data of diclazuril residues in chicken indicate an exponential decrease in all tissues with the increase of the withdrawal period. The statistical analysis supplied by the applicant (95 % tolerance limit with 95 % confidence) indicates the following upper values (taken from the graph, not exact values) extrapolated to a zero withdrawal: 1.4, 1, 0.35 and 0.45 mg diclazuril kg -1 for liver, kidney, muscle and skin/fat, respectively. These figures support the establishment 13 Technical Dossier. Section IV, Volume 2, Reference 11 14 OJ L 267, 17.9.2001, p.1 The EFSA Journal (2008) 696, 9-12
of MRL values of 1.5, 1, 0.5 and 0.5 mg kg -1, respectively. According to the calculations described in Table 6, the theoretical (worst case scenario) whole exposure of the consumer corresponding to those MRLs would represent 30 % of the ADI. Table 6. Diclazuril: MRLs and consumer exposure from chicken tissues at a zero-day withdrawal time Liver Kidney Muscle Skin/fat Sum MRC (a) (mg kg -1 ) 0.863 0.673 0.160 0.189 --- RMTR (b) 0.7 0.7 0.7 0.7 --- DITR (c) (mg) 0.123 0.010 0.069 0.024 0.226 MRL (d) proposed (mg kg -1 ) 1.5 1 0.5 0.5 --- DITR (e) MRL (mg) 0.214 0.014 0.214 0.064 0.506 (a) Marker residue concentration in individual tissues/products (mg kg -1 ) (b) Ratio MRCi-j to TRCi-j for individual tissues/products (c) Dietary intake for individual tissues/products calculated from total residues (mg) DITRi-j = Qti-j x TRCi-j (d) Maximum residue limit in tissues/products (mg marker substance kg -1 ) (e) Dietary intake calculated from MRLs (mg) of individual tissues/products DITRMRLi-j = Qti-j x MRLi-j RMTRi-j -1 Higher MRL values (3, 2, 1 and 1 mg kg -1 for liver, kidney, muscle and skin/fat, respectively) have been retained by the JECFA (1999), which reflects the use of diclazuril as a veterinary drug. The proposed lower MRL values are in accordance with the use of Clinacox 0.5% as feed additive (diclazuril doses 20-fold lower compared to veterinary application). 6.2. Turkeys The kinetic data of diclazuril residues in turkeys indicate an exponential decrease in all tissues with the increase of the withdrawal period (Table 5). The statistical analysis supplied by the applicant (95 % tolerance limit with 95 % confidence) indicates the following upper values (taken from the graph, not exact values) extrapolated to a zero withdrawal: 0.5, 0.45, 0.07 and 0.25 mg kg -1 for liver, kidney, muscle and skin/fat, respectively. These figures are all below those found in chicken and therefore the same MRLs can be retained, i.e. 1.5, 1, 0.5 and 0.5 mg kg -1. 7. Evaluation of the analytical methods by the Community Reference Laboratory (CRL) EFSA has verified the CRL report as it relates to the methods used for the control of the active substance in tissues in animal feed. The Executive Summary of the CRL report can be found in the Appendix. CONCLUSIONS The FEEDAP Panel concludes that the diclazuril impurity/metabolite R070016 is devoid of genotoxic activity. Consequently, the FEEDAP Panel is in a position to adopt the already proposed ADI for diclazuril (1.7 mg diclazuril per person per day). The metabolic fate of diclazuril in chicken and turkey is qualitatively similar, diclazuril being the marker residue in tissues. At a zero withdrawal time, the theoretical consumer exposure to the total residues from both chickens and turkeys represents about 13 % of the ADI. Therefore, the FEEDAP Panel considers that applying a withdrawal period of zero day would not compromise consumer safety. Consequently, no MRLs are required for chickens and turkeys for fattening. The EFSA Journal (2008) 696, 10-12
Should MRLs be considered necessary, the FEEDAP Panel proposes the following values: 1.5, 1, 0.5 and 0.5 mg diclazuril kg -1 wet tissue for liver, kidney, muscle and skin/fat, respectively. Those MRLs are in line with highest measured residues and ensure consumer safety (the worst case calculation representing 30 % of the ADI). A suitable analytical method, in terms of specificity and sensitivity is available for the determination of diclazuril residues in tissues at the MRL level. DOCUMENTATION PROVIDED TO EFSA 1. Technical Dossier on the establishment of Maximum Residue Limits for Clinacox 0.5% (diclazuril). June 2006. Submitted by Janssen Animal Health. 2. Supplementary information on the proposal of a withdrawal period for Clinacox 0.5% (diclazuril). November 2006. Submitted by Janssen Animal Health. 3. Supplementary Dossier on Clinacox Poultry (diclazuril R064433). Answer to question N EFSA-Q-2006-134. November 2007. Submitted by Janssen Animal Health. REFERENCES EC (European Commission), 1991. Report of the Scientific Committee for animal Nutrition on the use of Diclazuril in feedingstuffs for chickens. <http://ec.europa.eu/food/fs/sc/oldcomm6/antibiotics/09_en.pdf> EFSA (European Food Safety Authority), 2007. Opinion of the Scientific Panel on Additives and Products or Substances used in animal Feed on the Maximum Residue Limits for Clinacox 0.5% (diclazuril) for turkeys for fattening, chickens for fattening and chickens reared for laying. <http://www.efsa.europa.eu/efsa/scientific_opinion/feedap_op_ej505_clinacox_mrl_en.p df> EMEA (European Medicines Agency), 1996. Summary report on Diclazuril (1) by the Committee for Veterinary Medicinal Products. <http://www.emea.europa.eu/pdfs/vet/mrls/008696en.pdf> JECFA (Joint FAO/WHO Expert Committee on Food Additives), 1999. Fiftieth Report. Evaluation of certain veterinary drug residues in food. Geneva. WHO Technical Report Series, 888, pp. 95. The EFSA Journal (2008) 696, 11-12
APPENDIX Executive summary of the evaluation Report of the Community Reference Laboratory (CRL) Clinacox 0.5% is a feed additive authorised as coccidiostat according to Directive 70/527/EEC. Clinacox 0.5% contains 0.5% w/w diclazuril as active substance. The proposed inclusion rate of the active substance in feedingstuff for chickens and turkeys for fattening is 1 mg/kg, corresponding to 200 mg Clinacox 0.5% per kg feed. The recommended withdrawal period is 5 days. In the current application a modification of the authorisation is sought for establishing Maximum Residue Levels (MRLs) of diclazuril in tissues of all poultry species after application of Clinacox 0.5%. The proposed MRLs are: 3000 μg/kg in liver, 2000 μg/kg in kidney, 1000 μg/kg in skin with fat and 500 μg/kg in muscle. Diclazuril is the established marker residue, after application of Clinacox 0.5%. For determination of the marker residue diclazuril in animal tissues several methods have been proposed by the applicant. The procedures are based on high performance liquid chromatography (HPLC), gas chromatography (GC) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In our opinion the LC-MS/MS procedure is satisfactory validated for chicken (broiler) tissues, but in principle also applicable to turkey tissues. The obtained validation results carried out at 100 μg/kg of diclazuril in tissue show a relative within-laboratory standard deviation for reproducibility ranging from 3.7 % to 8.3 % depending on the specific tissue (muscle, liver, kidney, fat/skin). The limit of quantification (LOQ) is 5 μg/kg and therefore well below the target MRLs. According to the validation results the LC-MS/MS method can be considered suitable as a quantitative method. The suitability as a confirmatory method for official control is not fully established, since the applied mass spectrometry parameters do not fulfil the identification criteria as established by Commission Decision 2002/657/EC. However, most probably the LC-MS/MS method can fulfil the required confirmation criteria for diclazuril in the frame of official control when using the confirmatory mass spectrometry conditions proposed by another but similar LCMS/MS method (Mortier, L et al. (2005) Analytica Chimica Acta 529, 229-234), although this method was only described for poultry meat and not for liver, kidney and fat/skin. Besides, in order to establish the suitability for official control, it is strongly recommended that the method will be checked by a second independent laboratory. KEYWORDS Clinacox 0.5%, diclazuril, coccidiostat, feed additive, poultry tissues. The EFSA Journal (2008) 696, 12-12