The EFSA Journal (2004), 90, 1-44

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1 1 The EFSA Journal (2004), 90, 1-44 Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on a request from the Commission on the re-evaluation of efficacy and safety of the coccidiostat Monteban G100 in accordance with article 9G of Council Directive 70/524/EEC (Question N o. EFSA ) Adopted on 27 July 2004 Summary Monteban G100 is a feed additive intended for the control of coccidiosis, a debilitating protozoal infection in poultry. In common with a number of other coccidiostats, the additive is due for evaluation to comply with statutory requirements agreed at the EU-level. The European Commission asked the EFSA to evaluate the product Monteban G100 and advise the Commission on its efficacy and safety. Within EFSA this task was allocated to the Scientific Panel on Additives and Products or Substances used in Animal Feed (FEEDAP Panel). The data provided in the dossier to support community authorisation proved insufficient to give conclusive answers to several of the questions raised by the European Commission. Monteban G100 containing not less than 10 % of narasin activity as the active substance is effective as coccidiostat for chickens for fattening at a dose range of mg narasin kg -1 complete feed, as shown in recent floor pen studies. Previous dose response studies (battery-cage and floor pen studies) indicate that mg narasin kg -1 feed, is an effective dose range. However, no recent studies have been presented in order to prove narasin is still efficacious at the dose range. A full assessment of the efficacy was not possible, as no recent field studies have been carried out. No unusual resistance of Eimeria spp to narasin is to be expected in the field. The development of resistance against coccidiostats including narasin is a well-known phenomenon effectively counteracted in practice by rotation or by shuttle programs. Tolerance test showed that Monteban G100 is safe for the target animal with a small margin of safety (about 1.4). Incompatibilities or interactions with feedingstuffs, carriers or other approved additives are not to be expected based on the known history of the additive. There exists a well-known interaction with some medicinal substances (i.e. tiamulin) justifying a warning label on the product that the simultaneous use of these substances may be contraindicated. Monteban G100 at the use level for chickens is dangerous to horses, turkeys and rabbits. Narasin is active against Gram-positive bacteria, while Enterobacteriaceae are resistant. There is no cross-resistance to other antimicrobials except to salinomycin. Increased shedding of Salmonella is unlikely to occur at the dose used under practical conditions. Narasin, at the levels used for treatment of coccidiosis, is also effective in the prevention of necrotic enteritis in chickens. Narasin is absorbed to an unknown extent and excreted rapidly by the chicken. The excretion routes are not established in the chicken whereas faecal excretion prevails in the rat. The main metabolic pathway in the chicken and rat involves oxidative processes leading to the formation of very similar metabolites in both species in terms of chemical structure and biological activity.

2 Opinion on the additive MONTEBAN 2/44 Narasin metabolites in tissues and excreta are qualitatively similar. The liver is the target tissue. Unchanged narasin is not detectable in tissues after six hours withdrawal, with the exception of the skin/fat (until 12 hrs) where it represents the major fraction. A great number of narasin metabolites represent each less than 10 % of the total tissue residues. For food control purposes narasin could be retained as a practical marker residue and skin/fat as marker tissue. Narasin is not genotoxic. No indication of carcinogenicity or developmental toxicity was found at the doses tested in the mouse, rat and rabbit. The lowest NOEL identified in the oral toxicity studies was 0.5 mg kg -1 bw day -1 for the neuropathy seen in a one-year dog study and the ADI (acceptable daily intake) set by FEEDAP Panel is mg kg -1 bw (equal to 300 µg day -1 for a person of 60 kg bodyweight) and the withdrawal time of 1 day would be considered sufficient. A uniform MRL (maximum residue limit) for all tissues is proposed as 0.05 mg narasin kg -1 wet tissue. Validated methods are available which allow monitoring of narasin in premixes and complete feedingstuffs and the determination of the marker residue, narasin, in the liver, kidney, muscle and skin/fat of the chicken. Monteban G100 can cause irritation to the eyes but not to the skin. Inhalation studies in dogs showed that narasin is potentially highly toxic by the inhalation route, compared with the oral route. However, Monteban G100 is formulated as granules with a low dusting potential. For this reason, it is expected that workers will not be exposed by inhalation to toxic levels of narasin dust as a result of its handling. Monteban G100 has sensitisation potential by skin contact and by inhalation. Due to the sensitising properties of Monteban G100, FEEDAP Panel recommends the use of appropriate personnel protective equipment for the workers. Based on the provided information on the toxicity, fate and behaviour of narasin it cannot be excluded that the use of Monteban G100 at the recommended dose range poses a risk for soil organisms. Insufficient data was provided to allow the FEEDAP Panel to assess the risk for the aquatic environment and secondary poisoning. Key words: Monteban, coccidiostat, feed additive, narasin, ionophore, anticoccidial efficacy, microbiological risks, target animal safety, consumer safety, ADI, MRL, worker safety, environmental safety.

3 Opinion on the additive MONTEBAN 3/44 Background According to article 9g of Directive 70/524/EEC as amended by Directive 96/51/EC, additives subject to authorisation linked to a person responsible for putting them into circulation, included in Annex I before 1 st January 1988 should be re-evaluated. In accordance with article 9g of Directive 70/524/EEC the person responsible for each product had to provide a new application for the authorisation for its product, including a monograph and an identification note before the 1 st October Furthermore, a dossier, as referred to in article 4 of Directive 70/524/EEC, had to be submitted not later than 1 st October The Directive requires that the re-evaluation of the dossiers be completed 3 years after the submission of the dossier, this means before 1 st October Fifteen dossiers have been submitted before the deadline of 1 st October Each Member State rapporteur, and the other Member States, checked the dossiers for their compliance with the Guidelines for the assessment of additives in animal nutrition laid down in Council Directive 87/153/EEC as amended by Commission Directive 94/40/EC. The outcome of Member States' check was endorsed at the meeting of the Standing Committee for Animal Nutrition on 29 January Seven dossiers for products of the category "Coccidiostats and other medicinal substances" fulfilled the requirements of the guidelines and have therefore been retained for re-evaluation. Coccidiostats Decoquinate (DECCOX ) Halofuginone (STENOROL ) Lasalocid sodium (AVATEC 15% ) Monensin sodium (ELANCOBAN ) Narasin (Monteban ) Salinomycin sodium (SACOX 120 micro-granulate ) Robenidine hydrochloride (CYCOSTAT 66G ) The Standing Committee for Animal Nutrition started the re-evaluation of the safety and the efficacy of these products on 29 January Terms of reference The Commission requests EFSA to consider each of the above mentioned products and to advise it on their efficacy and their safety. In assessing the product on the basis of the dossiers presented, the EFSA is requested to answer the following questions. Under the conditions proposed for its use as additive in feed, Is the efficacy of the product as described in the Table 1 demonstrated? For the product considered, can its use result in the development of resistance in bacteria to prophylactic or therapeutic preparations? Is the product and its metabolites safe for the target animals, the user, the consumers, the environment? Can the product be monitored? This opinion deals with the product Monteban (narasin).

4 Opinion on the additive MONTEBAN 4/44 Annex inscriptions Table 1. Proposed conditions of use of the additive Additive (trade name ) Narasin (Monteban) Composition, chemical formula, description. C43H72O11 (Polyether monocarboxylic acid produced by Streptomyces aureofaciens) Species or category of animal Chickens for fattening Maximu m age Minimum Maximum content content mg of active substance kg -1 of complete feedingstuff Other provisions Use prohibited at least 5 days before slaughter Indicate in the instructions for use: "Dangerous for equines" "This feedingstuff contains an ionophore: simultaneous use with certain medicinal substances (e.g. tiamulin) can be contra-indicated"

5 Opinion on the additive MONTEBAN 5/44 Table of contents ASSESSMENT INTRODUCTION Physical and chemical properties Mode of action Stability Control methods EFFICACY Dose titration and confirmation studies Controlled floor pen studies Controlled field trials Studies on the development of resistance in Eimeria SAFETY STUDIES ON TARGET SPECIES Tolerance studies Interactions/compatibility Microbiological safety of the additive Metabolism Studies Residues STUDIES ON LABORATORY ANIMALS Acute toxicity Repeat Dose Sub-Chronic Toxicity Mutagenicity Carcinogenicity Reproduction toxicity SAFETY EVALUATION FOR THE HUMAN CONSUMER Effects of narasin on (human) intestinal bacterial isolates Proposal of the Acceptable Daily Intake (ADI) Proposal for MRL Proposal for the withdrawal time WORKER SAFETY Irritation and sensitisation Inhalation Safety Safety Data from Worker Exposure Conclusions on worker safety ENVIRONMENT Exposure assessment Effect assessment... 35

6 Opinion on the additive MONTEBAN 6/ Risk Characterisation CONCLUSIONS AND RECOMMENDATIONS...37 SCIENTIFIC PANEL MEMBERS...41 ACKNOWLEDGEMENTS...41 ANNEX...42

7 Opinion on the additive MONTEBAN 7/44 ASSESSMENT 1 INTRODUCTION Monteban G100 is a feed additive used to control coccidiosis in chickens for fattening (broilers), it contains by specification not less than 10 % of narasin activity. The final formulation of Monteban G100 is summarised below in Table 2. Table 2. Final formulation of Monteban G100 Ingredients Quantity g kg -1 Narasin Granulated Antidusting agent (soybean oil or parafin oil) 20 Anti-caking agent (vermiculite and montmorillonite) 20 Diluent (rice hulls or soybean mill run) The product is presently incorporated in feed for chickens at a final dose of mg narasin kg -1 feed with a withdrawal period of five days. Narasin is a polyether ionophore that exhibits both antimicrobial and anticocidial activities. It is used to prevent coccidiosis in poultry. It is not used in humans. Narasin A [α-ethyl-6-[5-[2-(5-ethyltetrahydro-5-hydroxy-6-methyl-2h-pyran-2-yl)-15-hydroxy- 2,10,12-trimethyl-1,6,8-trioxadispiro[ ]pentadec-13-en-9-yl]-2-hydroxy-1,3-dimethyl-4- oxoheptyl]tetrahydro-3,5-dimethyl-2h-pyran-2-acetic acid]; C43H72O11; CAS-No: ] belongs to the polyether monocarboxylic acid class of ionophores (Figure 1). Figure 1. Structural formula of narasin A R2 R3 R1 Structural variants of Narasin R1 R2 R3 A OH CH3 C00H B =O CH3 C00H D OH C2H5 C00H I OH CH3 C00CH3

8 Opinion on the additive MONTEBAN 8/44 Narasin is produced by fermentation of Streptomyces aureofaciens, strain NRRL 8092 deposited at the Agricultural Research Services Culture Collection (USA) 1. At the end of the fermentation process, the ph is adjusted using potassium or sodium hydroxide and the broth is concentrated using azeotropic distillation (water/amyl alcohol). Amyl alcohol wet solids are then added (clay (montmorillonite) as carrier/anticaking and dipotassium hydrogen phosphate as stabilizer) then dried. The granulation step involves the addition of antioxydant (BHT) and other diluents (corn meal, soybean grits, polyethylene glycol PEG powdered or calcium carbonate). The final granules are treated with an anti-dusting agent (soya bean oil or paraffin oil). According to the notifier at least 85 % of the narasin activity should be due to narasin A. The notifier presented an HPLC analysis of the composition of one lot as an example, indicating that the narasin material in this lot was composed of 96 % narasin A, 1 % narasin B, 2 % narasin D and 1 % narasin I. The relative biopotency of the structural variants of narasin is 1, 0.25, 1.4 and 0.01 respectively. The final product does not contain live cells from the fermentation broth. Routine analysis of clay ensures that only clay without dioxin is used in the formulation of Monteban G100. The analysis of Monteban G100 indicates low levels of undesirable substances (arsenic 1.3, cadmium 0.3, lead 4.9 and mercury <0.04 mg kg -1 ) 2. The final product was demonstrated to be within European Pharmacopoeia limits for preparations for Oral Administration Containing Raw Materials of Organic Origin. However, Monteban G100 is mixed with rice hull or soybean mill run which have been detected as a source of contamination due to the presence of aerobic bacterial flora. Therefore this is controlled by treating the rice hull with formaldehyde (at a concentration of mg kg -1 into the final feed). This treatment should not constitute a hazard to the animals at the low levels found in the finished product Physical and chemical properties Table 3. Summary of physical and chemical properties of narasin Parameter Value Condition Ref Molecular weight 764 g mol- 1 Water solubility 102 mg L -1 ph=7 Water solubility 681 mg L -1 ph=9 pka % DMF Log Kow 4.87 Based on a shake flask method Dorulla, >6.2 (estimated values: ) Based on HPLC determination (see text) Hogg 2002 Log Koc Based on HPLC determination Hogg Particle size In granulated Monteban G100, 15 % of particles are smaller than 100 µm and less than 0.05 % are smaller than 10 µm Mode of action Narasin, as other polyether ionophores, is effective against sporozoites and early and late asexual stages of coccidia in the intestine of the chicken. The biological activity of ionophores/narasin is based on their ability to form lipid soluble and dynamically reversible 1 Supplementary data: Responses to questions from Member States. July 2002, Volume 2, Reference Supplementary data: Responses to questions from Member States. May 2003, Reference 9. 3 Supplementary data: Responses to questions from Member States. May 2003, Reference 8. 4 Volume 29, Section IV, Reference 2 5 Supplementary data: Responses to questions from Member States. July Volume 4, Reference 5. 6 Monteban Monograph, Volume 1 Section I, Chapter 2, pages 3-5.

9 Opinion on the additive MONTEBAN 9/44 complexes with cations, preferably monovalent cations such as the alkaline ions K +, Na + and Rb +. They function as carriers by mediating an electrically neutral exchange-diffusion type of cation transport across the membranes. The resultant changes in transmembrane ion gradients and electrical potentials produce critical effects on cellular function and metabolism of coccidia. 1.3 Stability Narasin, in Monteban G 100, has a shelf life of 24 months at room temperature. The stability of narasin at 80 g kg -1 in premixtures was studied in three experiments, where samples were stored at different temperatures (4ºC, 25ºC, 40ºC and 60ºC) and the remaining narasin measured at different intervals. Losses did not exceed 10 %, when samples were stored for three years at 25º C or up to 18 months at 40ºC 7. The stability and homogeneity (narasin at 70 mg kg -1 ) were studied in expanded and pelleted feed. Good homogeneity and no loss of activity as a result of expanding and pelleting were observed after a minimum of three months storage at 25ºC for feedingstuffs. 1.4 Control methods Determination of narasin in chicken premixes and feeds A high performance liquid chromatography (HPLC) method with post-column derivatization with vanillin has been developed and validated for the quantification of narasin in chickens premix and feeds 8. The chromatographic conditions were selected in order to separate narasin from matrix components and other polyether ionophores. The limit of detection (LOD) and limit of quantification (LOQ) for narasin A are 0.05 mg kg -1 and 0.1 mg kg -1 respectively. Intra-laboratory analysis using several lots of feed ration indicated a RSD of 2.4 to 5.6 % in feed ration, the recovery being around %. A microbiological assay using Streptococcus faecium and a turbidimetric measurement are also available Determination of narasin residues in chicken tissues A validated HPLC analytical method for the determination of narasin in tissues is available 10. For all tissues the limit of quantification (LOQ) for narasin was mg kg -1 wet tissue and the limit of detection (LOD) was 0.01 mg kg -1 tissue. 2 EFFICACY Commission Directive 2001/79/EC requires efficacy data on three stages of target animal experimentation: (a) controlled battery-cage experiments (single and mixed infections), (b) controlled floor pen studies (simulated use conditions), and (c) controlled field trials (actual use conditions). The continuous use of coccidiostats over a long time as well as progress in breeding may result in the selection of resistant variants of Eimeria spp. The above-mentioned Directive also outlines: The dossiers must enable an assessment to be made of the additives based on the present state of knowledge. In the re-evaluation of Monteban G100, FEEDAP Panel therefore took into consideration only efficacy studies, which were conducted not earlier than about Volume 2, Section II, Reference 8. 8 Volume 2, Section II, Reference Volume 2, Section II, Reference Supplementary data: Responses to questions from Member States. August 2003, Reference 3.

10 Opinion on the additive MONTEBAN 10/44 FEEDAP Panel, however, makes exception for battery-cage experiments which serve for the principal discovery or confirmation of the anticoccidial efficacy of an additive against single or mixed infections or for dose titration studies. 2.1 Dose titration and confirmation studies The efficacy of narasin against Eimeria spp. was tested by a single inoculation or multiple inoculations in 104 trials in the U.S.A divided in three groups of experiments according to the type of Eimeria spp. inoculated. 37 trials with E. tenella 28 trials with E. tenella plus one or more species 39 trials with intestinal species (10 with E. acervulina, 2 trials with E. brunetti, 8 trials with E. maxima, 9 trials with E. necatrix and 10 trials with mixed species) The Eimeria isolates were either of laboratory origin or from the field. The efficacy of narasin was studied using a dose range of 0, 40, 60, 80, 100 and 120 mg kg -1 feed. In all experiments eight-day-old male chickens for fattening were distributed to groups of five birds per pen (4-6 pens per treatment, minimum 3 and maximum 20). Treatment was initiated 48 hr prior to oral inoculation of the Eimeria suspensions. Most experiments were terminated on the 6 th or 7 th day post inoculation. The battery trials showed that narasin was effective in preventing coccidiosis. Lesion scores, body weight gain and feed conversion were improved by 60 mg kg -1, compared to 40 (in most cases significantly), and 80 mg kg -1 was superior (in some cases significantly). However, higher doses reduced lesions but did not improve weight gain or feed conversion. Seventeen-dose response studies were conducted in different locations in Europe (1978) 11 in order to study the response of chickens with 0, 40, 60, 70, 80 and 100 mg narasin kg -1 feed. Results from these experiments confirmed that all test groups showed significant (P< 0.05) improvement in final weight, feed conversion and control of coccidial lesions. Based on these results a dose range of mg narasin kg -1 feed is proposed by the Notifier. 2.2 Controlled floor pen studies Two published floor pen studies were included in the dossier post 1990 (Guneratne and Gard, 1991, Watkins and Bafundo, 1993). In the study of Guneratne and Gard (1991) nine trials were conducted in seven countries using a common protocol design. Two trials were conducted both in Colombia and South Africa, and one in Brazil, Malaysia, Thailand, Taiwan and Venezuela. Results were pooled for statistical analysis. A total of day-old broiler chickens in 240 floor pens in open houses were used. Pen sizes ranged from 30 to 180 birds per pen. The concentration of the anticoccidials was analysed and found to be within the acceptable limits of ± 15 % of the intended level. Pen broiler chicken weights and feed consumption were recorded at the end of the starter and grower period. In 7 of 9 trials chickens were exposed to coccidia by introducing old litter from a previous trial or from local farms to pens. In the first Columbian and in the Venezuelian trial sporulated oocysts were administered in the feed. At the end of the starter period 2-5 birds from each pen were randomly selected within sex for coccidial lesion scoring. All data were statistically evaluated. Results are shown in Table 4 and Table Volume 3, Section III, page 20

11 Opinion on the additive MONTEBAN 11/44 Treatment Table 4. Performance and lesion score means at the end of the starter period (pooled date for nine trials). Number of pens Performance Lesion Score (%) Lesion Score (%) Body Feed: Gain Number of Small Number of Caeca weight g kg kg -1 pens intestine pens Untreated control a a a a Narasin 60 mg kg b b b a Narasin 70 mg kg ab ac b a a, b, c: Column means having different superscripts are significantly different (P<0.05). Table 5. Performance parameter (treatment means at termination; n = 30), at the end of the starter period (including 4-7 day withdrawal period). Starter (27/28 days)/ Grower (17/18 days) Mortality % Body weight, g Feed conversion (kg kg -1 ) Untreated - controls 3.89 a 1562 b a Narasin 60 mg kg a 1674 a b Narasin 70 mg kg a 1666 a b a-b: Column means having different superscripts are significantly different (P<0.05). All anticoccidial treatments decreased significantly the small intestinal lesion scores when compared to untreated controls. The orthogonal evaluations that compared group differences showed that all anticoccidial programs reduced significantly the feed to gain ratio. Narasin at 60 mg kg -1 feed improved significantly body weight and feed:gain at the end of the starter period. Both concentrations of narasin (60 and 70 mg kg -1 feed) in the continuous anticoccidial program and narasin used in the four shuttle programs (data not shown) improved significantly body weight and feed conversion at termination. Two trials are presented by Watkins and Bafundo (1993). The trials were undertaken to study the effects of 16 anticoccidial programs on the broiler performance during a mild coccidial challenge. Four replicates of 100 chicks (50 of each sex) each of two identical trials were assigned randomly to each treatment group. All birds were fed starter feed from 1 to 20 days, grower feed from 21 to 40 days, and unsupplemented feed from 41 to 47 days. The concentrations of the anticoccidials were analysed and confirmed. In previous studies, this floor pen facility had been seeded for experimental purposes with field strains of E. acervulina, E. maxima and E. tenella. Prior to the start of the present study, fresh pine shavings were placed in each pen after a complete cleanout. This procedure has historically resulted in a mild coccidial challenge in this facility. These conditions allowed an evaluation of the effects of anticoccidial programs on bird performance. The treatment groups were comprised of four continuous ionophore programs and 12 shuttle programs. Because the treatments were similar for both trials, data were pooled and statistically analysed. Control groups (non-infected non-treated group, infected non-treated group) were not included in this study. As continuous (day 1 40) ionophore programs, 63 and 72 mg narasin kg -1 were compared to 100 mg monensin and 60 mg salinomycin, followed by a 7 day withdrawal period. The same ionophore programs were tested in the grower period (day 21-40) alone after administering (shuttle program) a combination of equal parts of narasin and nicarbazin (72 and 90 mg kg -1, respectively) or nicarbazin alone (113 mg kg -1 ) in the starter period (day 1-20). The results are shown in Table 6.

12 Opinion on the additive MONTEBAN 12/44 Table 6. Effects of continous ionophore programs (Day 1-47; data are means of eight replicates of 100 chickens each) Program Mortality % Body weight g Feed conversion (kg kg -1 ) Monensin* 100 mg kg Narasin* 63 mg kg Narasin* 72 mg kg Salinomycin* 60 mg kg Rations contained roxarsone and bacitracin No significant differences were observed in mortality, body weight and feed efficiency between birds on the four continuous ionophore programs Controlled field trials No controlled field studies undertake after 1990 are presented Studies on the development of resistance in Eimeria Studies submitted by the Notifier Three published studies on the development of resistance were submitted. Zhu and McDougald (1992) propagated a field isolate of E. tenella several times in chickens treated with commonly used dietary polyether ionophores (monensin 100, 150, 200 and 250 mg kg -1, salinomycin 60, 90, 120, 150 mg kg -1, lasalocid 90, 135, 180 mg kg -1, narasin 70, 105, 140, 175 mg kg -1, maduramicin 5, 7.5, 10, 12.5 mg kg -1 ). In a laboratory test with two weeks old chickens, the strain was resistant to three of the ionophores given at double the use level and was resistant to two other ionophores at the normal use level including narasin. In comparison, a laboratory strain extremely pathogenic to chickens was controlled by the normal use level of each anticoccidial. All five ionophores improved body weight gain, oocyst production was greatly reduced or eliminated and caecal lesion scores also were reduced relative to untreated control. In contrast, the field strain was not as pathogenic in untreated groups. No mortality was observed in these groups. Narasin did not improve body weight gain but decreased the lesion scores and oocyst counts. The spectrum of resistance to seven currently used anticoccidial drugs (clopidol 125 mg kg -1, decoquinate 30 mg kg -1, lasalocid 100 mg kg -1, monensin 121 mg kg -1, narasin 80 mg kg -1, robenidine 33 mg kg -1, salinomycin 66 mg kg -1 ) in isolates of Eimeria obtained from farms from two broiler complexes was examined (Chapman and Hacker, 1994). A total of 720 chickens for fattening were used in the experiment. Salinomycin and monensin had been used for many years at both complexes, narasin only in one of them, and lasalocid had never been used. Birds were inoculated with oocysts after first receiving two days anticoccidial treatment. According to the criteria applied by the authors all isolates were resistant to narasin. Coccidia were isolated from a commercial broiler farm with a history of suspected drug resistance (Daugschies et al., 1998). The sensitivity profiles of the Eimeria spp. against six anticoccidials at the recommended dose levels were conducted in three battery trials (B1, B2, B3) corresponding to three field studies (F1, F2, F3) previously run at farm level with different shuttle ionophore programmes. Sporulated oocysts of each of the isolates were used for separate battery trials. Non-sexed broiler chicken were kept in groups of 20 in plastic cages and fed with anticoccidial free feed until an age of six days. Thereafter, they were assigned to groups of 10 or 11 birds according to body weight. One group served as a non-infected and non-treated control and a second group as infected non-treated control. The infected groups received 5.5 x 10 5 of a mixed isolate of E. acervulina and E. tenella (B1) or 5.0 x 10 5 oocysts of E. acervulina (B2, B3). The birds in the treated groups were fed with feed containing the anticoccidials including narasin

13 Opinion on the additive MONTEBAN 13/44 (70 mg/kg feed) ad libitum from day 2 before infection until the termination of the experiment (day 6 or 7). Feed consumption and body weight were recorded daily and feed conversion and mortality at the end of each trial. At the end of the trials surviving birds were killed and subjected to parasitological and pathological examination. Results for the narasin groups are shown in Table 7. Table 7. Results (mean values) of the battery trials (B1, B2, B3) Group B1 B2 B3 OI LS WG% F:G OI LS WG% F:G kg:kg OI LS WG% F:G NNC 0 a 0 a 100 a kg 2.09 kg a 0 aa 0 a 100 a 2.48 a 0 a 0 a 100 a 2.29 kg kg a INC 2.6 b 1.8 b 45 b 3.82 b 3.4 b 2.7 b 63 b 2.94 b 2.7 b 1.7 b 42 b 4.35 b Narasin 1.1 ab 0.9 ab 80 ab 2.35 ab 2.7 a 1.9 ab 120 a 2.35 a 2.4 b 1.4 a 115 ab 2.29 a NNC = non-infected non-treated control; INC = infected non-treated control; OI = oocyst index (0 = no, 1 = 1-10, 2 = 11-20, 3 = 21-50, 4 = , 5 = >100 oocysts/field); LS = duodenal lesion score; WG% = relative body weight gain in percentage; F:G = feed:gain. Column means within a period having different superscripts are significantly different (P<0.05). All parameters differed significantly between noninfected and infected non treated groups. Narasin supplementation showed a tendency of improvement of all parameters compared to infected untreated group, but only a few differences were significant (study 2: body weight gain and feed conversion; study 3: lesion scores and feed conversion). Despite the reduced Eimeria sensitivity narasin proved to be efficacious with respect to body weight gain and feed conversion when compared to the other anticoccidials tested Other published studies Resistance to anticoccidials is a widespread phenomenon, which occurs even with the more recent additives. To describe the resistance situation of Eimeria spp. clinically important in the production of chickens for fattening in Europe, FEEDAP Panel reviewed the results of four studies published in the last decade. The study of Peeters et al. (1994) was performed with coccidia from 122 Belgian broiler farms without clinical coccidiosis, where shuttle programs were most commonly used. 146 E. acervulina, 65 E. maxima and 88 E. tenella isolates were tested in 17 sensitivity profiles. Results were related to the anticoccidial program that had been in use. The data clearly indicate that recorded anticoccidial sensitivity is widespread and that most ionophore did not control isolates significantly. The differences between the ionophores tested confirm according to authors - earlier data of incomplete cross-resistance to polyether ionophorous drugs. The most recent publication (Peek and Landman, 2003) studied the Eimeria resistance situation in Dutch poultry production. Four isolates from 1996 were selected from farms with clinical coccidiosis problems, four isolates from 1999 and seven isolates from 2001, both originating from farms with subclinical disease were also selected in the study. The tests were conducted according to Chapman (1998) as in vivo anticoccidial sensitivity tests. The sensitivity profile is based on the reduction of lesion scores compared to the infected untreated control. Eimeria acervulina was more or less resistant against all coccididostats tested in 1996 (diclurazil, halofuginone, lasalocid, meticlorpindol / methylbenzoquate, monsensin, narasin and nicarbazin), and 3 of 4 strains against maduramycin and salinomycin (1 showed reduced sensitivity). In 1999 the same species presented a similar resistance pattern, one strain (of four) showed sensitivity against monensin and narasin. In 2001 increased sensitivity was found. Higher sensitivity was found for meticlorpindol/methylbenzoquate (7/7), salinomycin and narasin (4/7), followed by nicarbazin (3/7) and monensin (2/7). Resistance as found for lasalocid (5/7), nicarbazin (4/7), diclurazil, monensin, narasin and salinomycin (all 2/7). An E. acervulina reference strain, tested in 1999, showed full sensitivity towards all anticoccidial

14 Opinion on the additive MONTEBAN 14/44 additives tested. The differences between the results of 1996 and 1999/2001 may also reflect the origin of the isolates (1996 from flocks with clinical disease and therefore more virulent strains), but also higher inoculation doses have been applied. In the broiler farms participating in the Dutch coccidiosis monitoring programme, the incidence of cocidiosis (E. acervulina) was approximately 70 (68) % in 1996, 91 (84) % in 2000 and 73 (67) % in Despite the obvious resistance, an increase in clinical problems was not observed. The authors suggest that a form of spontaneous vaccination might occur in field Studies on the quality of animal produce The effect of narasin on the quality of animal produce has been studied in two experiments. In the first study 12, the effect of diets containing 70 mg narasin kg -1 on the flavour of cooked chicken and on fat, protein and water content of chicken was investigated. The sensory tests and chemical analyses did not show differences between test and control samples. In the second study a consumer preference test was performed to compare meat samples from broilers fed a commercial ration without an anticoccidial agent to those from broilers receiving the same ration containing 80 mg narasin kg -1 (Rhorer et al., 1984). No flavour differences (P>0.05) were detected by the sensory panel with freshly cooked or roasted chicken. The consumer panel forced-choice test indicated no preference (P>0.05) Conclusions on the efficacy Earlier confirmation/dose response studies (104 battery cages trials and 17 floor pen trials) showed that narasin is effective in preventing coccidiosis in chickens for fattening. A range of dose between mg kg -1 feed improved intestinal lesions score, body weight and feed conversion. No data post 1990 s were presented to demonstrate the efficacy at the full dose range between 60 and 80 mg narasin kg -1 has been retained. The results of recent floor pen studies indicate that narasin at a dose range of mg kg -1 feed is an effective anticoccidial for chickens for fattening. However the design of the studies did not follow the requirements of Directive 2001/79/EC (lack of uninfected control groups, low number of animals studied). Consequently no firm conclusion can be drawn. In addition, no recent field studies were included in the dossier; therefore the efficacy of the product cannot be established on the basis of the data provided. Reduced sensitivity of field isolates of Eimeria to anticoccidials was demonstrated in different countries. No significant differences between narasin and other synthetic anticoccidials or polyether ionophores in the control of field isolates of E. acervulina, E. maxima and E. tenella were observed. FEEDAP Panel concludes that despite the prevailing (partial) resistance of Eimeria spp. against nearly all coccidiostats the benefits of their use are not essentially jeopardized under field conditions. The recent data also confirm that the development of resistance can successfully be counteracted in practice by rotation (the alternation of coccidiostats from run to run) or by shuttle programmes. Monteban G100 at recommended concentrations in feed does not influence the organoleptic and nutritional quality of chicken meat. 3 SAFETY STUDIES ON TARGET SPECIES 3.1 Tolerance studies The tolerance of narasin has been studied in three experiments, conducted with chickens fed commercial diets containing narasin at doses of 0, 80, 240 and 400 mg kg -1 feed 13, 0, 70, 80, 120 and 210 mg kg -1 feed 14 and 0, 80, 240 mg kg -1 feed Volume 13, Section 3, Reference Volume 14, Section IV, Reference 8.

15 Opinion on the additive MONTEBAN 15/44 These experiments were performed in chickens from day 1 of age until slaughter or one day before slaughter. The effects of narasin were determined by examinations for clinical signs, gross pathology at necropsy, mortality, prothrombin time, body weight gain, feed consumption, feed conversion, litter condition and feather condition. The series of tolerance studies showed that chickens fed with 120 mg narasin kg -1 feed and higher levels had a reduction in body weight gain, an increased mortality and a reduction of animal performance. From these studies only 80 mg narasin kg -1 has been shown to be safe. However in previous dose range finding studies 100 mg narasin kg was tested without adverse effects have been observed. Consequently the FEEDAP Panel concludes that the margin of safety is 100/70 (1.4). The product, like other ionophores, has a very narrow tolerance factor. 3.2 Interactions/compatibility The notifier has not generated and reported information relating to the co-administration of narasin and tiamulin and their interaction. However, published data from three experiments (Lazsay et al., 1989) on the compatibility of narasin with tiamulin, erythromycin, tylosin, kitasamycin, flumequine, sulfachorpyrazine or sulfaquinoxaline confirmed the incompatibility of narasin with tiamulin, erythromycin, sulfachorpyrazine and sulfaquinoxaline. No incompatibilities were found with tylosin, kitasamycin and flumequine. Clinically important interactions between the ionophore anticoccidials and the antibiotic tiamulin are well known phenomena in chickens, turkeys and other species. (Hanrahan et al., 1981; Umemura et al., 1985; Van Vleet et al., 1987; Szucs et al., 2000a, 2000b). The interaction depends on the dose (Meingassner et al., 1979; Lehel et al., 1995; Weisman et al., 1983a, 1983b) and the ionophore itself (co-administration of tiamulin and lasalocid being without adverse effects (Comben, 1984). It was assumed that tiamulin reduces metabolic degradation and excretion of monensin (Meingassner et al., 1979). Later, also further toxic interactions with polyethers (mainly monensin) became known for sulphonamides (Frigg et al., 1983), chloramphenicol (Broz and Frigg, 1987), erythromycin, oleandomycin and furazolidone (see review by Anadón and Martinez-Larrañaga, 1990), Anadón and Reeve-Johnson 1999). Recent data allow to relate this interaction to the inhibition of cytochrome P-450 isoenzymes, which play an important role in the oxidative and reductive metabolism of numerous endogenous and exogenous compounds by tiamulin (Witkamp et al., 1994, 1995, 1996) and macrolide antibiotics (Larry et al., 1983; Watkins et al., 1986). Compounds capable of binding or inhibiting these isoenzymes could therefore be expected to give rise to toxic interactions with the polyether ionophore(s). 3.3 Microbiological safety of the additive Antibacterial activity of narasin The antimicrobial spectrum of narasin is mainly limited to Gram-positive bacteria, including enterococci, staphylococci and Clostridium perfringens. MICs for Staphylococcus aureus and Enterococcus faecalis ranged (based on agar dilution method) respectively from 0.39 to 100 µg ml -1 and from 0.2 to 100 µg ml -1. MICs for Clostridium ranged from 0.1 to 4.0 µg ml -1. Gram-negative bacteria are, in general, resistant to narasin with MICs >512 µg ml In addition, several studies on MICs of strains isolated from various sources have been conducted (Marounek and Rada, 1995; Watkins, et al.1997). Isolates of Staphylococcus, Streptococcus have MICs ranging from to 8.0 µg ml and Enterococcus MICs from 14 Volume 14, Section IV, Reference Volume 14, Section IV, Reference Volume 3, Section III, page Volume 15 Section IV, Reference McLaren I.M. Volume 15, Section IV, Reference 3.

16 Opinion on the additive MONTEBAN 16/ to 4.0 µg ml For Clostridium perfringens, isolated from chickens suffering from avian necrotic enteritis, the MICs range from 0.03 to µg ml From the data in the European monitoring programmes and the literature, it can be concluded that development of resistance to narasin is common among enterococci of poultry origin. The notifier presented several papers in relation to the effect of narasin on the reduction of growth of C. perfringens (Elwinger et al., 1992, Elwinger et al., 1994). That is important following the withdrawal of antibiotic growth promoters, as chickens are less protected against C. perfringens. The presence of C. perfringens can cause an intestinal problem, known as enteritis necrotic (NE) Additional studies (Brennan et al 2001; Brennan et al 2003) have shown a significant effect of 70 mg narasin kg -1 in feed in reducing mortality due to NE in chickens challenged with Clostridium perfringens Effects of exposure to narasin on the antibacterial resistance among bacteria A study was designed to detect the possible induction of cross-resistance to other antimicrobials by narasin. 21 Seven bacterial species were tested: Staphylococcus aureus (isolated from chicken), Enterococcus faecalis (isolated from chicken), Escherichia coli (two laboratory strains, one harbouring a resistance plasmid encoding tetracycline and streptomycin resistance), Clostridium perfringens (isolated from chicken), Bifidobacterium bifidum (an ATCC strain) and Bacteroides fragilis (human clinical isolate). The strains were passaged 40 times in broth (one passage per day) in the presence of sub- inhibitory narasin concentrations. These concentrations were determined for each strain. In general, there were minor fluctuations in the MIC with either two-fold increases or decreases. For some strains there was a significant increase of MIC (five-fold increase in the MICs of chlortetracycline and tylosin with S. aureus, three-fold increase of chlortetracycline MIC in E. faecalis). These increases were not associated with enhanced narasin resistance, and were found to occur also when the strains were passaged in broth not containing narasin. In another study 22 the development of resistance to narasin in Bacteroides fragilis (10 strains) and Clostridium perfringens (2 strains) was studied using a gradient plate technique. While resistant B. fragilis isolates (MICs > 64 µg L -1 ) could be readily isolated, no resistant clostridia appeared. No cross-resistance to cefoxitin, clindamycin, erythromycin, metronidazole or tetracycline was observed. In a similar experiment 23 with 10 strains of streptococci and 10 S. aureus strains transient resistance to narasin (reverting back to original levels after subculturing in media without narasin) was observed in both organisms. Cross-resistance was checked against ampicillin, benzylpenicillin, cephazolin, chloramphenicol, erythromycin, tobramycin, tetracycline and vancomycin, but none was observed Narasin and Salmonella shedding Two studies on the effect of narasin on the shedding of Salmonella from challenged broiler chickens were presented by the Notifier. In the first study, 24 the experimental Salmonella Typhimurium strain was originally isolated from a chicken. The study was combined with susceptibility screening against narasin, erythromycin, penicillin, sulfamethazine, streptomycin, tetracycline and chloramphenicol. The tests were done on four-week old chickens divided in control and test groups, each with 12 birds. The test group started to receive narasin (100 mg kg -1 feed) one day before being challenged orally with 2.9 x 10 9 cfu Salmonella. The shedding of Salmonella was followed for 19 Volume 15, Section IV, Reference Volume 15, Section IV, Reference Volume 15, Section IV, Reference Volume 15, Section IV, Reference Volume 15, Section IV, Reference Volume 15, Section IV, Reference 10.

17 Opinion on the additive MONTEBAN 17/44 eight weeks. The shedding of Salmonella in the narasin group was significantly more frequent and the duration of the shedding longer than in the controls. However, the antimicrobial susceptibility pattern of the Salmonella strain was not affected, nor were Salmonella detected in the tissue samples of the challenged birds. In the second study 25 the control and test groups consisted each of eighteen day old chickens. The narasin concentration in the diet was 72.6 mg kg -1 (above the recommended maximum dose of 70 mg kg -1 ), and the challenge concentrations of salmonellae x 10 9 cfu per bird. The shedding was followed for 56 days. In this study narasin did not affect the prevalence or duration of Salmonella shedding Field studies to monitor bacterial resistance to the additive. In the last years harmonised antimicrobial resistance surveillance programmes in animals and animal-derived food have been initiated in the European Union. These programmes, recommended by international bodies (WHO, OIE) were based on Danish experience and followed international guidelines (Franklin et al., 2001). Monitoring of antimicrobial resistance in zoonotic bacteria is requested by the zoonosis directive (Common Position (EC) n 0 13/2003) and is part of a surveillance network implemented in the European Union for communicable diseases (Common Decision of July ). In the meantime several European countries have improved existing systems. Countries such as Denmark (DANMAP ), Norway (Norm-VET ) Sweden (SVARM ), France (Sanders et al., 2001), Germany (Guerra et al., 2003), Spain (Moreno et al., 20002) and the UK (Goodyear, 2002) have implemented programmes for monitoring antimicrobial resistance in commensal bacteria such as E. coli and enterococci collected from faeces or caecal contents of healthy animals. The sampling is, in principle, based on the same epidemiological approach to permit international comparisons (Bywater et al., 2003). In the monitoring programmes resistance to the ionophores salinomycin (DANMAP 2002) and narasin (Norm-VET 2002; SVARM 2002) for broiler isolates of E. faecalis and E. faecium is reported. Resistance in the form of a bimodal distribution of narasin MICs was reported (Norm-VET 2002; SVARM 2002). Frequent resistance to narasin and salinomycin in enterococci of poultry origin was reported from Belgium and there was full cross-resistance between narasin and salinomycin. There was no crossresistance between those polyether ionophores and lasalocid and monensin (Butaye et al., 2000) Conclusions The highest tolerated dose in chickens for fattening is 100 mg kg -1 feed, which represents a margin of safety of 1.4 of the recommended dose. The known history of use of narasin has shown that incompatibilities or interactions with feedingstuffs, carriers, or other approved additives are not to be expected. On the other hand it is well known from the literature that severe interactions between the polyether ionophor coccidiostats and the diterpene-antibiotic tiamulin as well as other antimicrobials (mainly macrolides) may occur. Therefore the simultaneous use of Monteban G100 and certain antibiotic drugs (i.e. tiamulin) is contra-indicated. The MICs of narasin for common intestinal bacterial species such as Enterococcus spp. and Clostridium perfringens are basically low but enterococci may develop resistance to narasin. There is no cross-resistance to other antimicrobials except to salinomycin. Narasin may increase Salmonella-shedding, but there is no reason to believe that narasin is different from other polyether ionophores in this respect. There are no data on the influence of narasin on the intestinal microflora other than on Clostridium perfringens and Salmonella Narasin, at the levels used for treatment of coccidiosis, is also effective in the prevention of necrotic enteritis in chickens. Clostridium perfringens is susceptible to narasin and this may have implications in the control of necrotic enteritis in chickens. 25 Volume 15, Section IV, Reference 11.

18 Opinion on the additive MONTEBAN 18/ Metabolism Studies Chickens A non-glp study has been performed 26 in chickens preconditioned on feed containing 80 mg narasin kg -1 that received a single oral capsule dose of [ 14 C]-narasin equivalent to the daily intake through feed. Narasin was excreted rapidly, 85 % of the dose being recovered in the excreta within 48 hours. An experiment was designed to establish the steady state equilibrium of residues in chicken tissues. 27 Chickens (3 male and 3 female, 7-weeks of age) were fed a ration containing 100 mg [ 14 C]-narasin kg -1 and killed after four and six days dosage, with zero-time withdrawal (six hours after treatment). No difference was observed in tissue (kidney, liver, fat and muscle) residue levels between treatment duration, indicating that four days exposure is sufficient to reach the metabolic steady state. The isolation and characterisation of narasin metabolites from chicken excreta was undertaken in a non-glp study in animals (sex and number not specified) dosed for five days with 100 mg [ 14 C]-narasin (labelling position not indicated) kg -1 feed. 28 Parent narasin was the major component in the faeces accounting for about 30 % of the whole excreta radioactivity. Another 25% corresponded to seven metabolites, which were extracted, separated and identified by mass spectrometry. Four metabolites corresponded to dihydroxynarasin and two to trihydroxynarasin isomers. This identified hydroxylation as the main metabolic pathway. The remaining radioactivity corresponded to a great number of metabolites each representing less than 10% of the total. In another study 29 chickens received for five days a diet supplemented with 80 mg [ 14 C]- narasin (labelling position not indicated) kg -1 feed and were slaughtered on the 5 th day. Excreta were collected and analyzed for narasin and its metabolites. Unchanged narasin represented 5 % of the whole radioactivity and six metabolites were characterized. Two corresponded to well characterised tri-hydroxynarasins and represented about 14 % of total radioactivity. Four metabolites corresponded to di-hydroxynarasin and accounted for another 14 %. Other minor (below 10 %) metabolites were separated but not identified. Attempts have been made to establish comparative metabolic profiling of narasin in tissues. In a first study, 30 chickens were fed for five days a feed supplemented with 50 mg [ 14 C]-narasin kg -1 (the labelling was distributed into 10 different positions of the molecule including the C1 carboxyl) then slaughtered with a zero-day withdrawal time. The analysis of the excreta confirmed and completed the preceding results 31 showing that unchanged narasin represented 3% of the whole radioactivity excreted while 15 metabolites, predominently diand tri-hydroxylated narasin A and narasin B as well as tetra-hydroxynarasin A, represented almost 50 % of total radioactivity, the remainder being distributed into a number of minor fractions which could not be identified. When tissues were concerned, the liver exhibited the highest concentration of radioactivity of which up to 75 % was extractable. However, narasin metabolites could not be identified due to the low amount of material available after purification. Radioactivity in fat was isolated and found to be predominantly the parent compound (61 %). In a recent GLP study 32 adult chickens (10 males and 10 females) received [ 14 C]-narasin supplemented feed (labelling identical to that of the former study) at a 71 mg kg - 1 dosage for five consecutive days then were slaughtered after a 6 hr withdrawal period. Narasin and metabolites were extracted from the liver (49 % of the total radioactivity) and excreta (96 %) then separated and identified by LC-MS analysis. The results confirmed the 26 Volume 16, Section IV, Reference Volume. 16, Section IV, Reference Volume 16, Section IV, Reference Volume 16, Section IV, Reference Volume 16 Section IV, Reference Volume 16, Section IV, Reference Responses to questions from Member States. July Volume 3, Section III and IV, Reference 3.