The EFSA Journal (2006) 387, 1-33

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1 The EFSA Journal (2006) 387, 1-33 Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on the safety and efficacy of the coccidiostat Elancoban (monensin sodium) as a feed additive for calves for rearing and cattle for fattening in accordance with Regulation (EC) No 1831/2003 (Question N o EFSA-Q ) Adopted on 13 September 2006 SUMMARY Elancoban (Elancoban G200, Elancoban 200, Elancoban G100, Elancoban 100 formulations) is a feed additive intended for the control of coccidiosis, authorized for use for a 10 years period for chickens for fattening, fattening turkeys and chickens reared for laying. The applicant is now seeking for an extension of use of Elancoban for calves for rearing and for cattle for fattening at dose levels of 30 to 45 mg Mo-Na kg -1 complete feed. Elancoban contains monensin sodium (Mo-Na), a polyether ionophore produced by Streptomyces cinnamonensis that exhibits both antibacterial and anticoccidial activities, as the active ingredient. The European Food Safety Authority, in accordance with Regulation (EC) No. 1831/2003, was asked by the European Commission to assess the efficacy and the safety of this product for the target animals, the users, consumers and the environment. Studies on calves with artificial and natural infection as well as a study after natural infection on cattle for fattening demonstrate that 0.9 mg Mo-Na kg -1 bw is effective in controlling coccidiosis. Data after artificial infection with Eimeria spp. indicate a potential effective dose range of mg Mo-Na kg -1 bw. The absence of complete feed intake data in eight out of the nine trials does not allow an exact conversion of mg kg -1 bw data into mg kg -1 complete feedingstuff but only an approximation. Taking into account the reservation above, mg Mo-Na kg -1 complete feedingstuff can be considered as effective dose range. Short duration of all trials (four weeks) prevents the full assessment of efficacy for continuous use. No specific studies on resistance of Mo-Na against bovine Eimeria spp. have been provided. Since the resistance to anticoccidials (including Mo-Na) is a well known phenomenon in poultry which does not seriously affect their efficacy in the field, a similar situation is expected to occur with bovines. The quality of animal products is not influenced by feeding monensin. Tolerance tests indicated that a dose range of 30 to 45 mg kg -1 complete feed can be considered safe for calves for rearing and cattle for fattening, with a margin of safety of approximately 2.5. The margin of safety for accidental overdosing (no longer than 10 days) is considerably higher. Incompatibilities or interactions with feedingstuffs, carriers or other approved additives are not expected considering the known history of Mo-Na. However, the simultaneous use of Elancoban and certain antibiotic drugs (i.e. tiamulin) is contra-indicated. Mo-Na at the dose levels proposed for use in bovines may be toxic to equines.

2 Opinion on Elancoban for calves for rearing and cattle for fattening 2/33 There is no evidence that the use of Elancoban in cattle contributes to the development of cross-resistance by bacteria to antibacterials used in human or veterinary medicine. As in poultry and the rat, Mo-Na is significantly absorbed in bovines, then extensively metabolized to give a similar qualitative pattern of metabolites. It is mainly excreted (unchanged and as metabolites) in the faeces. Unchanged monensin represents 50% of the whole Mo-Na related molecules in faeces. Liver is the target tissue. Unchanged monensin represents less than 10% of the whole residues in liver and disappears very rapidly from all tissues. As already assessed in the previous opinion on Elancoban (EFSA, 2004), Mo-Na from Elancoban shows no evidence of genotoxicity, carcinogenicity, embryotoxicity, fetotoxicity or teratogenicity. An ADI of mg kg -1 bw d -1 has been proposed, applying a safety factor of 100. Mo-Na total residues in tissues have been measured in animals at steady state administered [ 14 C] Mo-Na at a dose of 33 mg kg -1 feed for cattle for fattening and 1.8 mg kg -1 bw for dairy cows. In both cases, Mo-Na is not measurable in the tissues examined (below LOQ, mg kg -1 ) after 12-hour (cattle for fattening) and six-hour (dairy cows) withdrawal. The total exposure of the consumer, calculated on the basis of the highest individual residue levels in the dairy cow study, was mg day -1 representing 93% of the ADI. The FEEDAP Panel considered that the dairy cow study offers the highest safety margin. Since the consumer exposure will not exceed the ADI under these worst case conditions, the FEEDAP Panel does not see the necessity for establishing MRLs and consequently for setting a withdrawal period. The extension of use of Elancoban to cattle does not modify the former opinion of the FEEDAP Panel that monensin is safe for users. The available information suggests that the use of Elancoban for calves for rearing and cattle for fattening at the maximum recommended dose and under conditions typically for the use of a feed additive (continuous use) will pose a risk for soil organisms. Insufficient data was provided to allow the FEEDAP panel to assess the risk for the aquatic environment (groundwater and surface water). Key words: Elancoban, monensin sodium, coccidiosis, ionophore, cattle, calves, Eimeria spp., MRL, consumer safety.

3 Opinion on Elancoban for calves for rearing and cattle for fattening 3/33 TABLE OF CONTENTS Summary... 1 Table of contents... 3 Background... 4 Terms of reference... 4 Assessment Introduction Physical and chemical properties Mode of action Stability Analytical methods for control purposes Efficacy Controlled trials with artificial infections Calves for rearing Controlled field trials with natural infection Calves Cattle Meta-analysis Studies on the development of resistance in Eimeria spp Study on the quality of animal products Conclusions on efficacy Safety studies on target species Tolerance studies Acute toxicity of monensin Repeated dose studies Interactions Microbiological safety of the additive Antimicrobial spectrum and MIC studies Cross-resistance with other antibiotics Effects on colonisation and shedding of enteropathogens Conclusion Metabolism Residues Conclusion Safety studies on laboratory animals Safety evaluation for the human consumer Proposal for an Acceptable Daily Intake (ADI) Maximum Residue Limit (MRL) Responses to the additional information submitted by the applicant Proposal for MRL and a withdrawal period User safety assessment Safety for the environment Predicted environmental concentrations (PEC) Risk characterisation Risk for soil Risk for groundwater and surface water Risk for secondary poisoning Conclusion...26 Conclusions and Recommendations...26 Documentation provided to EFSA...28 References...29 Panel Members...30 Appendices...31

4 Opinion on Elancoban for calves for rearing and cattle for fattening 4/33 BACKGROUND Regulation (EC) No 1831/ establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lies down that any person seeking an authorisation for a feed additive or for a new use of a feed additive shall submit an application in accordance with Article 7. The European Commission received a request from Eli Lilly and Company Limited, Elanco Animal Health 2 for authorisation of the product Elancoban, a coccidiostat consisting of monensin sodium, to be used as a feed additive for calves for rearing and cattle for fattening under the conditions described in Table 1. According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the application to the European Food Safety Authority (EFSA) as an application under Article 4.1 (authorisation of a feed additive or new use of a feed additive). EFSA received directly from the applicant the technical dossier in support of this application. According to Article 8 of that Regulation, EFSA, after verifying the particulars and documents submitted by the applicant, shall undertake an assessment in order to determine whether the feed additive complies with the conditions laid down in Article 5. The particulars and documents in support of the application were considered valid by EFSA as of 7th of October, The additive Elancoban is a preparation of monensin sodium which has been already authorised (E 757) at Community level under Regulation (EC) No 1356/2004, 3 as Elancoban and Elancogran, for chickens for fattening, chickens reared for laying and turkeys, until 30 July The Scientific Committee on Animal Nutrition (SCAN) has issued three reports in where the monensin sodium has been assessed. The first one was issued on 1979 on specific questions about the safety for consumer and the environment of the combined use of the active substance with flavophospholipol. The second report, issued on 1981, evaluated the safety for the consumer and the environment of monensin sodium when used as a coccidiostat in feedingstuffs for poultry. A third report issued on 1983, assessed the metabolism of monensin sodium in feedingstuffs for turkeys, in the light of additional data provided. The Scientific Panel on Additives and Products or Substances used in Animal Feed (FEEDAP Panel) issued an opinion (EFSA, 2004) on the re-evaluation of Elancoban for chickens for fattening, chickens reared for laying and turkeys, in accordance with article 9G of Council Directive 70/524/EEC. TERMS OF REFERENCE According to Article 8 of Regulation (EC) No 1831/2003, EFSA shall determine whether the feed additive complies with the conditions laid down in Article 5. Therefore, EFSA shall deliver an opinion on the safety for the target animals, consumer, user, and the environment, and on the efficacy of the product Elancoban, based on monensin sodium, when used under the conditions described in Table 1. 1 OJ L268, , p.29 2 Eli Lilly and Company Limited. Elanco Animal Health. Kingsclere Road, Basingstoke, Hamphshire RG21 6XA United Kingdom 3 OJ L251, , p. 6

5 Opinion on Elancoban for calves for rearing and cattle for fattening 5/33 Table 1. Register entry as proposed by the applicant Additive Registration number (EC No) Category of additive Functional group of additive Elancoban E757 Coccidiostat N/A Composition, description per kg Granular monensin equivalent to 100g Antidusting oil: 10-25g Limestone granular: g Rice hulls or limestone granular, up to 1000g Description Elancoban G100 Chemical formula 2-(5-ethyltetrahydro-5- (tetrahydro-3 -methyl-5-(tetrahydro-6-hydroxy- 6-(hydroxymethyl)-3,5-dimethyl- 2Hpyran-2-y1)-2-furyl)-2-furyl) 9-hydroxy-ß-methoxy-a,g,2,8- tetramethyl-1,6-dioxaspiro (4.5)decane-7-butyric acid sodium salt. Purity criteria (if appropriat e) Monensin Activity: Not less than 92.5 g/kg and not more than g/kg Method of analysis (if appropriate) HPLC Composition, description per kg Granular monensin equivalent to 200g Antidusting oil: 10-25g Rice hulls or limestone granular, up to 1000g Description Elancoban G200 Chemical formula 2-(5-ethyltetrahydro-5- (tetrahydro-3 -methyl-5-(tetrahydro-6-hydroxy- 6-(hydroxymethyl)-3,5-dimethyl- 2Hpyran-2-y1)-2-furyl)-2-furyl) 9-hydroxy-ß-methoxy-a,g,2,8- tetramethyl-1,6-dioxaspiro (4.5)decane-7-butyric acid sodium salt. Purity criteria (if appropriat e) Monensin Activity: Not less than 185 g/kg and not more than 215 g/kg Method of analysis (if appropriate) HPLC Trade name (if appropriate) Name of the holder of authorisation (if appropriate) Elancoban 100, Elancoban G100, Elancoban 200, Elancoban G200 Eli Lilly and Company Limited, Elanco Animal Health Species or category of Maximum Condition of use Minimum content Maximum content Withdrawal period animal Age mg kg -1 of complete feedingstuffs (if appropriate) Chickens for fattening 35 days days Chickens reared for laying 16 weeks days Turkeys 16 weeks days

6 Opinion on Elancoban for calves for rearing and cattle for fattening 6/33 Calves for rearing 4 months days Cattle for fattening 36 months days Specific conditions or restrictions for use (if appropriate) Specific conditions or restrictions for handling (if appropriate) Other provisions and additional requirements for the labelling Dangerous for equines. This feedingstuff contains an ionophore: simultaneous use with certain medicinal substances (e.g. tiamulin) can be contraindicated. When mixing and handling Elancoban use protective clothing, impervious gloves and a dust mask. Operators should wash thoroughly with soap and water after handling. If accidental eye contact occurs, immediately rinse thoroughly with water. Post market monitoring (if appropriate) Usage monitoring The company will perform follow up monitoring with three key European customers during the first two years of use. This will include investigation into safety, efficacy and appropriate use of Elancoban in the treatment of bovine coccidiosis. Pharmacovigilance Post marketing monitoring will be conducted using an already established pharmacovigilance system. All adverse events (human, animal environmental, residues violations, suspected lack of efficacy) occurring during the use of the product, independent of whether or not the product is considered to be related to the event, which are reported to the company, are recorded and entered into a data base. Specific conditions for use in complementary feedingstuffs (if appropriate) Dangerous for equines. This feedingstuff contains an ionophore: simultaneous use with certain medicinal substances (e.g. tiamulin) can be contraindicated. Maximum Residue Limit (MRL) (if appropriate) Marker residue Species or category of animal Target tissue(s) or food products Maximum content tissues Monensin Chickens for fattening Skin/fat 50µg/kg Monensin Chickens reared for laying Skin/fat 50µg/kg Monensin Turkeys Skin/fat 50µg/kg Monensin Calves for rearing Liver 50µg/kg Monensin Cattle for fattening Liver 50µg/kg in

7 Opinion on Elancoban for calves for rearing and cattle for fattening 7/33 ASSESSMENT 1. Introduction Elancoban (Elancoban G200, Elancoban 200, Elancoban G100, Elancoban 100 formulations) is a feed additive containing monensin sodium (Mo-Na) as the active ingredient. It is authorized for use until 30 July 2014 for chickens for fattening ( mg Mo-Na kg -1 feed), fattening turkeys ( mg kg -1 ) and chickens reared for laying ( mg kg -1 ) (EC, 2004). The same applicant seeks an extension of use of Elancoban (Elancoban G200, Elancoban 200, Elancoban G100 and Elancoban 100 formulations) to control coccidiosis in calf and cattle. The applicant applies for dose levels of 30 to 45 mg Mo-Na kg -1 complete feed for calves for rearing and for cattle for fattening. The product should be administered to cattle for fattening up to 36 months. The applicant proposes that the product could be administered without withdrawal period. Bovine coccidiosis is increasingly recognised as an important protozoal disease. A recent article published by Daugschies and Najdrowski (2005) on Eimeria spp. in cattle, points out that the oocysts are found ubiquitously in the environment making an infection of calves and young cattle, the most susceptible age group, almost inevitable. Of the more than a dozen species, Eimeria bovis and Eimeria zuernii only are responsible for the several clinical symptoms characterized by haemorrhagic diarrhoea with sometimes a lethal outcome. To a lesser extent, Eimeria alabamensis also can cause clinical disease. Mixed infections are the rule. Because of the damage of the intestinal tissue, the digestive process and overall homeostasis can become severely affected, even in the absence of clinical symptoms. The subsequent economic losses for the cattle industry are thus substantial Physical and chemical properties The physico-chemical and chemical properties of Elancoban and monensin sodium submitted in the dossier have already been fully described and assessed by the FEEDAP Panel in its previous Opinion on Elancoban (EFSA, 2004) Mode of action The mode of action of monensin sodium against coccidia species affecting the chicken and turkey, as described previously (EFSA, 2004), applies also to Eimeria bovis and Eimeria zuernii that affect the bovine. Monensin sodium acts as an ionophore, i.e. a chemical substance which complexes monovalent cations with the lipophilic structure that facilitates the transport of the bound ion through biological membranes. Monensin sodium is primarily coccidiocidal in action. Coccidial sporozoites exposed to monensin sodium in the intestinal lumen exhibit considerable swelling, large vacuoles, pitting and holes in the surface suggesting extreme osmotic damage which is potentially lethal. Development of a sporozoite that successfully invades a host cell is inhibited as the ionophore continues its destructive process. Monensin sodium selectively destroys intracellular sporozoites while remaining relatively noninjurious to the host cell (Chapman, 1993). It has been shown that monensin sodium has an effect on second generation merozoites but not upon developing gametocytes. In summary, monensin has an opportunity to kill coccidia parasites and disrupt the life cycle at three different stages. These include the sporozoites at approximately days 1-5 of the

8 Opinion on Elancoban for calves for rearing and cattle for fattening 8/33 cycle, the first generation of merozoites on about day 16 and the second generation of merozoites at about day 19 of the life cycle. From the history of the active substance as growth promotor in cattle, it is well known that monensin affects rumen microbial metabolism. In fact, the production of propionic acid (glucose precursor) is favoured, while acetic acid (and butyric acid) is depressed. Also the formation of methane is reduced, which leads to an increase in metabolizable energy. Monensin also depresses feed intake by reducing rumen fermentation rate and therefore improves while not influencing growth rate predominantly feed efficiency. The use of Elancoban in controlling bovine coccidiosis can therefore not be considered separately from its action on rumen microbial metabolism Stability The stability of Elancoban 200 and Elancoban 100 has been already assessed by the FEEDAP Panel in its previous Opinion on Elancoban (EFSA, 2004). The new data supplied by the applicant include additional studies. The additive can be incorporated homogenously in premixtures, complete and complementary cattle feed 4 independent from the carrier of the additive (rice hulls or limestone) Elancoban is stable at 25 C in premixtures, mash and pelleted feed for 16 weeks. 5 Stability in pelleted feed at 40 C was demonstrated but limited to eight weeks storage Analytical methods for control purposes EFSA has verified the report submitted by CRL and the executive summary of the CRL report on the assessment of the method(s) of analysis for Elancoban is attached as Annex A. 2. Efficacy In this section, only the trials performed with a dosage similar to the dose range claimed by the applicant were considered. All trials submitted give monensin dose in terms of mg kg -1 bw instead of mg kg -1 complete feed. Exact feed intake data (including roughage data) was not provided in the dossier as only the intake of complementary feed containing the additive was measured. Therefore the FEEDAP Panel had to convert the data provided to the level present in the whole feed. A feed intake of 2% of body weight was assumed. On that basis, 0.9 mg Mo-Na kg -1 bw would correspond to 45 mg kg -1 complete feed (12% water) Controlled trials with artificial infections Calves for rearing A first study 7 (Germany, 2004) was conducted on a total of 40 male calves (78 to 85 kg bw, i.e. 9 to 12 weeks old, at the start) kept in single pens. Five groups of eight calves received different dose levels of Mo-Na (0, 0.15, 0.3, 0.6 and 0.9 mg kg -1 bw) incorporated in the concentrate feed over a period of 28 days followed by a seven-day withdrawal period. The experimental infection with approximately 75,000 oocysts of E. bovis and 100,000 oocysts 4 Section II, Volume 33, Attachment 16; Section II, Volume 34, Attachments 18, 21 and 22 5 Section II, Volume 33, Attachment 16; Section II, Volume 34, Attachment 17 6 Section II, Volume 33, Attachment 16 7 Section III, Volume 37, Attachment 21

9 Opinion on Elancoban for calves for rearing and cattle for fattening 9/33 of E. zuernii for each calf, was initiated on day 4. Efficacy was measured by faecal oocyst counts, daily faecal scores, weight gain and clinical condition of the calves. Faecal oocyst counts as well as faecal scoring were carried out weekly during the pre-study period, on day 1, day 7 and daily from day 14 onwards until the end of the study. No calves died during the studied period. The two highest dose groups showed significantly lower oocyst counts than the control (day 20 to day 30) (Figure 1). Weight gain of all groups up to day 21 was similar. However, at day 35 the cumulative weight gains were 15.6, 18.6, 22.5, 30.3 and 29.4 kg for the groups with 0, 0.15, 0.3, 0.6 and 0.9 mg Mo-Na kg -1 bw, respectively. They were significantly higher for the two highest dose groups. Only 0.9 mg Mo-Na kg -1 bw prevented diarrhoea completely, whereas in the group treated with 0.6 mg kg -1 bw one out of eight animals showed liquid faeces. In the last two weeks of the trial, one calf of the control group and one of each of the two lowest treatment groups showed a tendency for reduced faeces consistency Total oocyst counts Total oocyst counts C 0.15 mg kg-1 bw 0.3 mg kg-1 bw 0.6 mg kg-1 bw 0.9 mg kg-1 bw Days Figure 1. Total oocyst counts in faeces of calves for rearing treated with Mo-Na (first study) A second study 8 (UK, 2005) was conducted with five groups of 16 calves (eight males and eight females, 6.5 weeks old, 90 to 91 kg bw at the start) kept in single pens. Four groups received different dose levels of monensin (0.3, 0.6, 0.9, 1.2 mg kg -1 bw) incorporated into the concentrate feed daily over a period of four weeks. The remaining group served as an untreated control. Monensin was mixed into calf concentrate at a dose of 100 mg monensin kg -1 concentrate (analysed value 96.0 mg monensin kg -1 ). The feeding of supplemented concentrate started three days before the experimental infection of each calf with approximately 100,000 oocysts of E. bovis and 50,000 oocysts of E. zuernii. Efficacy was measured by faecal oocyst counts, daily faecal scores, weight gain and clinical condition of the calves. Faecal samples were taken on day 18 to 28 and day Section III, Volume 37, Attachment 22

10 Opinion on Elancoban for calves for rearing and cattle for fattening 10/33 There was a highly significant difference in oocyst counts for the two groups treated with 1.2 and 0.9 mg monensin kg -1 bw compared with the untreated control and 0.3 mg monensin kg -1 bw. The dose group 0.6 mg monensin kg -1 bw performed significantly better than the control and 0.3 mg monensin kg -1 bw. However, the differences in oocyst counts between 1.2 and 0.9 mg monensin kg -1 bw but also the untreated control and lower dose groups were not significant (Figure 2). From day 14 to day 35 the median of faecal consistency scores increased from 0.09 in the 1.2 mg monensin kg -1 bw group to 1.47 in the controls. Three calves died during the study. One died accidentally and two (untreated control on day 27, 0.3 monensin kg -1 bw on day 29) had alimentary signs and intestinal lesions but no histology was performed. During the fourth week, when most oocysts were shed, the weight gains were negative in the untreated control and the lowest monensin dose group but the other groups showed increases related to the amount of monensin present in the ration Total oocyst count Total oocyst counts C 0.3 mg kg-1 bw 0.6 mg kg-1 bw 0.9 mg kg-1 bw 1.2 mg kg-1 bw Days Figure 2. Total oocyst counts in faeces of calves for rearing treated with Mo-Na (overall treatment effect, P<0.0001) (second study) A third study 9 (USA, 1996) was conducted with four groups of male calves (9.5 weeks old, 68 kg bw at the start) kept in single pens. Three groups received different dose levels of monensin incorporated into a concentrate feed (90% dry matter basis) daily over a period of four weeks post challenge. The remaining group of 22 calves served as an untreated control. Monensin was mixed into the concentrate at a dose calculated as 11, 22 and 33 mg monensin kg -1 (analysed value 8.8, 17.9 and 27.3 mg monensin kg -1 ) which corresponded to 0.31, 0.61 and 0.93 mg monensin kg -1 bw, respectively. One week after the beginning of the treatment with Mo-Na each calf was challenged with a total of 300,000 oocysts (approximately 231,000 oocysts of E. bovis, 54,000 oocysts of E. zuernii and 15,000 oocysts of miscellaneous Eimeria species). 9 Section III, Volume 35, Attachment 14

11 Opinion on Elancoban for calves for rearing and cattle for fattening 11/33 Efficacy was measured by faecal oocyst counts, daily faecal scores, feed intake, weight gain and clinical condition of the calves. Between three and four weeks after oocyst challenge, a total of three calves died: one calf of the control group due to coccidiosis and two monensin-treated calves (0.61 and 0.93 mg monensin kg -1 bw) due to bloat. Daily body weight gain was significantly improved for the groups treated with 0.31 mg monensin kg -1 bw (P<0.05) and 0.61 mg monensin kg -1 bw (P<0.1) compared with the untreated control (Table 2). Total and E. bovis oocyst counts were significantly reduced at the 0.31 mg monensin kg -1 feed dose level, and significant further reductions were detected with higher monensin doses. E. zuernii count was significantly reduced at a dose level of 0.31 mg monensin kg -1, and no further reduction was observed with higher monensin doses (Table 2). Table 2. Summary of total oocyst counts by week (log10 basis) and of average daily gain (four weeks) Untreated Control Monensin group, 0.31 mg kg -1 bw Monensin group, 0.61 mg kg -1 bw Monensin group, 0.93 mg kg -1 bw Week 1 Week 2 Week 3 Week Daily weight gain, g a, b P<0.05 A, B P< bb 844 aa 748 aba 721 abab 2.2. Controlled field trials with natural infection Five controlled field trials were included in the dossier, all completed post Calves In a first trial 10 (UK, 2005), 20 male and 20 female calves, six to 12 weeks old, were randomly allocated to eight pens of five calves. The trial lasted 28 days. The treated calves received monensin sodium at 0.9 mg kg -1 bw in a calf rearing unit from day 1 to day 28 inclusive. Elancoban was in the feed at an inclusion rate of 40 mg Mo-Na kg -1, and the quantities fed to the calves were based on weekly weighing. Faecal oocyst counts were examined from day 18 to day 25 inclusive. Monensin significantly reduced (P=0.0064) the total oocyst count over the sampling period. The mean overall weight gain was 2.15 kg lower in the monensin treated group than in the untreated group. The difference in weight gain was not significant (Table 3). In a second trial 11 (Germany, 2004), 48 female calves of 86 to 115 days of age were randomly allocated to four pens of 12 calves. The trial lasted 29 days. The treated calves received monensin sodium at 0.9 mg monensin kg -1 bw in the concentrate (2.5 kg) plus hay and silage (amount not measured) from day 1 to day 29 inclusive. Faecal oocyst counts were examined from day 1 to day 29 inclusive. The treated animals showed lower oocyst excretion (P=0.1349), less diarrhoea and higher weight gain (P=0.0002) (Table 3). 10 Section III, Volume 36, Attachment Section III, Volume 37, Attachment 19

12 Opinion on Elancoban for calves for rearing and cattle for fattening 12/33 In a third trial 12 (Germany, 2004), 40 male calves ranging from seven to nine weeks old were randomly allocated to two pens of 20 calves. The trial lasted 28 days. The treated calves received monensin sodium at 0.9 mg monensin kg -1 bw in the concentrate from day 1 to day 28 inclusive. Faecal oocyst counts were examined from day 1 to 27 inclusive. The monensin treated animals as compared to the control showed significantly lower oocyst excretion (P=0.0020). The incidence of diarrhoea in both treatments was comparable, while the monensin treated group showed a numerically higher weight gain (Table 3). In a fourth trial 13 (UK, 2005), 16 male and female calves aged two to two and half months were randomly allocated to eight pens of one calf. There were two time block trials involving eight calves per time and using different pens in the two studies. The treated calves received monensin sodium at 0.9 mg monensin kg -1 bw in the concentrate feed plus hay and water ad libitum from day 1 to day 28 inclusive. Faecal oocyst counts were examined from day 18 to 29 inclusive. Monensin treated animals showed significantly lower oocyst excretion (P=0.0118) than control animals. The mean overall weight gain by day 28 was 6.0 kg higher in the monensin treated group than in the untreated control group. The difference in weight gain was significant (P=0.0007) (Table 3) Cattle In a trial 14 (Germany, 2005), 80 animals of four to seven months of age were randomly allocated to eight pens of ten cattle. Randomisation was undertaken following ranking by weight. The amount of medicated feed was recalculated on a pen weight basis weekly. The trial lasted 28 days. The treated cattle received monensin sodium at 0.9 mg monensin kg -1 bw in the feed from day 1 to day 28 inclusive. Total feed consumption was measured daily on a pen basis. The difference in feed intake between the monensin treated group and the control group was minimal. Faecal oocyst counts were examined from day 1 to 25 inclusive. Monensin treated animals showed significantly lower oocyst excretion (P=0.0037), less diarrhoea and a significantly higher weight gain at the 10% level (P=0.0928) than control animals (Table 3) Meta-analysis A meta-analysis was performed on the basis of the data obtained from the five field trials performed with calves and cattle, undertaken using 224 animals, two to seven months old and comparing a dose of 0.9 mg monensin kg -1 bw in the feed for 28 days against an untreated control. Efficacy was measured by counting oocysts per gram of faeces and weight gain. In all trials the use of monensin produced a large reduction in the numbers of oocysts. The overall effect of monensin treatment for log10 AUS pen mean oocyst count was highly significant (P<0.0001). During the studies there was an overall weight gain of 21 kg in the medicated group which was 2 kg higher than in the controls. The difference in weight gain was not significant (P=0.2169). 12 Section III, Volume 36, Attachment Section III, Volume 36, Attachment Section III, Volume 37, Attachment 20

13 Opinion on Elancoban for calves for rearing and cattle for fattening 13/33 Table 3. Effect of monensin (0.9 mg Mo-Na kg -1 bw) on body weight gain and oocyst excretion (oocyst/gram of faeces) Control Monensin Trial 1, 15 UK, 2003, initial bw 76 kg Body weight gain in four weeks (kg) Faecal oocyst counts, day 18 Faecal oocyst counts, day 25 Trial 2, 16 G, 2004, initial bw 112 kg Body weight gain in four weeks (kg) Faecal oocyst counts, day 18 Faecal oocyst counts, day 25 Trial 3, 17 G, 2004, initial bw 68 kg Body weight gain in four weeks (kg) Faecal oocyst counts, day 18 Faecal oocyst counts, day 25 Trial 4, 18 UK, 2004, initial bw 93 kg Body weight gain in four weeks (kg) Faecal oocyst counts, day 18 Faecal oocyst counts, day 25 Trial 5, 19 G, 2004, initial bw 147 kg Body weight gain in four weeks (kg) Faecal oocyst counts, day 18 Faecal oocyst counts, day 25 Trial 1, 2, 3, 4: P<0.05 Trial 5; P< , Studies on the development of resistance in Eimeria spp. No studies on resistance of bovine specific Eimeria spp. to Mo-Na were submitted by the applicant. Resistance to anticoccidials (including Mo-Na) is a well known phenomenon in poultry as stated in the previous Opinion on Elancoban (EFSA, 2004), however, it has not seriously affected the efficacy of monensin in the field Study on the quality of animal products The effect of Mo-Na on carcass characteristics was examined in 19 feedlot efficacy experiments. At dose levels of 5.5 to 44 mg kg -1 feed, Mo-Na had no significant effects on the parameters measured. In addition, chemical analysis of tissue from the rib-eye muscle showed no treatment related changes in chemical composition. In another four studies, 20 feeding Mo-Na at doses ranging from 0 to 88 mg kg -1 feed was shown to have no significant or no effect on carcass measurements or proportions of fat, lean and bone in the edible portion of the carcass. Mo-Na had no effect upon the moisture, 15 Section III, Volume 36, Attachment Section III, Volume 37, Attachment Section III, Volume 36, Attachment Section III, Volume 36, Attachment Section III, Volume 37, Attachment Section III, Volume 37, Attachment 23

14 Opinion on Elancoban for calves for rearing and cattle for fattening 14/33 fat and protein of rib-eye muscle. An analysis of the data from over 100 trials 21 at concentrations of /- 7.5 mg kg -1 dry matter showed that carcass characteristics (dressing percentage, marbling score, rib eye area, fat depth, quality grade and yield grade) were not influenced by the presence of Mo-Na Conclusions on efficacy Studies on calves with artificial and natural infection demonstrate that 0.9 mg Mo-Na kg -1 bw is effective in controlling coccidiosis. Data after artificial infection with Eimeria spp. indicate a potential effective dose range of mg Mo-Na kg -1 bw. Moreover, the absence of complete feed intake data in eight out of the nine trials does not allow exact conversion of mg kg -1 bw data into suitable mg kg -1 complete feedingstuffs but only an approximation. Therefore, and taking into account the reservation made above, mg Mo-Na kg -1 complete feedingstuff, as submitted by the applicant, can be considered as an effective dose range. Although there is only one trial on cattle for fattening, the FEEDAP Panel does not see reasons to doubt on the efficacy of Mo-Na at the same dose range against the same Eimeria species in older bovines. However, the short duration of all trials (four weeks) prevents the full assessment of efficacy for continuous use. No specific studies on resistance of Mo-Na against bovine Eimeria spp. have been provided. Since the resistance to anticoccidials (including Mo-Na) is a well known phenomenon in poultry which does not seriously affect their efficacy in the field, a similar situation is expected to occur with bovines. The quality of animal products is not influenced by feeding monensin. 3. Safety studies on target species 3.1. Tolerance studies Acute toxicity of monensin Two studies were presented, both with mycelial monensin administered once by rumen intubations. In study 1, 22 doses of 12.5, 20, 32 and 50 mg Mo-Na kg -1 bw were given to a total of 16 beef cattle with a body weight range of kg. In study 2, beef cattle with a body weight range of kg were treated with 12.5, 22.4 and 39.8 mg Mo-Na kg -1 bw. Intoxication was indicated by diarrhoea and anorexia in both studies. Necropsy findings showed an apparent relationship between ingestion of monensin sodium and myocardial necrosis and haemorrhage (study two). The LD50 was 21.9 ± 3.4 mg kg -1 bw in study one and 35.8 ± 13.5 mg kg -1 bw in study two. In a publication of Todd et al., (1984) the LD50 for cattle is given with 26.4 mg monensin kg - 1 bw. In another study, 24 with monensin doses equivalent to 0, 1.6, 2,4, 3.9, 7.6, and 15.5 mg kg - 1 bw conducted for seven days with five beef steers per group ( kg bw). The highest 21 Section III, Volume 37, Attachment Section IV, Volume 38, Attachment 6 23 Section IV, Volume 38, Attachment 7 24 Section IV, Volume 38, Attachment 8

15 Opinion on Elancoban for calves for rearing and cattle for fattening 15/33 non-lethal dose was estimated to be 3.9 mg monensin kg -1 bw per day. However, the groups with 3.9 mg kg -1 bw (and higher doses) showed complete anorexia. Cattle receiving 1.6 and 2.4 mg consumed less feed and showed mild or delayed onset of diarrhoea. Clinical and pathological alterations (after acute oral doses of 25 or 40 mg monensin kg -1 bw) were also studied in beef calves (Van Vleet et al., 1983). Administration of toxic doses was characterised by anorexia and diarrhoea. Subsequent development of myotoxicity involving cardiac and skeletal muscles (40 mg) was detected by increase in serum activity of muscle-origin enzymes and ECG abnormalities Repeated dose studies Calves Four groups of eight calves (four males, four females, body weight range: kg on day 1) were dosed at levels of 0.0, 0.9 (corresponding to the maximum dose claimed), 2.7 (3- fold) and 4.5 (5-fold) mg kg -1 bw day -1, for 28 days. Two further groups of eight cattle were dosed at levels of 0.0 and 0.9 mg kg -1 bw day -1, for 56 days. 25 The mean body weight of the groups at start (day 1) varied between 97 and 104 kg and 140 kg for the group treated with the highest dose (4.5 mg monensin kg -1 bw) (Table 4). Table 4. Zootechnical data on feed consumption and body weight in calves Monensin (mg kg -1 bw) Feed* consumption (kg) In 28 days In 56 days Body weight (kg) At start (day 1) /29 days** days weeks gain (% of initial bw) (15.0) * Concentrate only ** 28 days for the two 8 weeks groups, 29 for the other groups The statistical analysis indicated that 4.5 mg monensin kg -1 bw resulted in a depression of body weight gain, and that already 2.7 mg kg -1 bw reduced feed intake. Also body weight gain showed a slight but insignificant depression at the three-fold monensin dose. Incorporation of monensin premix has no detrimental effect on calves on the bodyweight, weight gain and feed consumption of calves at the minimum dose level claimed. However, feed consumption is difficult to assess because monensin dosing according to body weight was reached by offering concentrates (with different monensin concentrations) weekly adjusted to body weight. Therefore, feed supply was higher for better growing groups. Also body weight is difficult to compare for the relatively short experimental period because of the differences at start. Monensin was determined to have no significant effect on clinical chemistry (total protein, albumin, globulin, albumin/globulin ration, total bilirubin, cholesterol, urea and uric acid, ALT, AST, CK, γ-gt, inorganic P, Na, Cl, K), haematology or faecal occult blood parameters of calves. At necropsy no treatment related abnormalities were seen in any of the animals. Cattle 25 Section IV, Volume 38, Attachment 11

16 Opinion on Elancoban for calves for rearing and cattle for fattening 16/33 Four groups of ten beef cattle (five steers, five heifers initially averaging 328 kg and 376 kg, respectively) were fed rations containing 0, 22, 66 and 110 mg monensin kg -1 complete feed, respectively, for 160 days. 26 Table 5. Zootechnical results (160 days) on feed consumption and body weight gain in cattle Monensin (mg kg -1 feed) Average daily gain (kg) Feed consumption (kg day -1 ) Feed/gain (kg kg -1 ) All monensin levels reduced feed intake. But 22 and 66 mg monensin kg -1 feed did not affect daily gain, so that a superior feed efficiency resulted from the use of monensin. However, 110 mg monensin kg -1 complete feed resulted in weight gain reduction of about 20% indicating a lower tolerance against this dosage. The effects of monensin on the following parameters were studied at 0 time (pretreatment), 56, 112 and 160 days after the beginning of the treatment: haematology (haemoglobin, haematocrit, RBC, WBC (differential counts), prothrombin time), serum chemistry (glucose, urea nitrogen, Na, K, AP, AST and sulfobromophthalein), urine analysis (ph, specific gravity, albumen, glucose, and occult blood); at necropsy, gross pathology and histopathology was performed, and weights of heart, liver, kidney and adrenals were determined. Pathology including organ weights did not show differences which could be attributed to the treatment. Significant differences in haematological parameters influenced by sex and treatment interaction were observed, but no differences which could be considered indicative for the treatment. After 160 days of treatment, serum chemistry showed significantly reduced glucose and potassium in the 110 mg monensin group and reduced potassium also in the 66 mg group. Total protein was elevated for the group treated with 22 and 66 mg monensin, but not for 110 mg, also BUN in the 66 mg monensin group. Potassium was significantly lower after 112 days of treatment for the 66 mg and the 110 mg monensin groups, but not after 160 days. None of the changes described above can be considered as serious signs of toxicity, because they were not of sufficient magnitude or were comparable to values of other sample periods. However, the reduction of glucose could be seen as related to the depressed feed intake, whereas the reduction of potassium could be interpreted as a result of the inability of the homeostatic regulation mechanisms of the body to compensate for the high ionophore load provided by monensin. Both findings show that cattle are less tolerant to the dose of 110 mg monensin kg -1 feed, as it was shown above with the weight gain reduction. A total of eight Hereford steers, with 33 mg and 330 mg monensin kg -1 feed, were offered to fasted and unfasted animals in a crossover design 27 to simulate inadvertanly overdosing, due for example to a mixing error. From this experiment it can be concluded that a significant reduction in feed intake (for some individuals to zero on day 2 of feeding) will be the first sign of intoxication. Watery diarrhoea was observed when feeding 330 mg monensin kg Section IV, Volume 38, Attachment Section IV, Volume 39, Attachment 13

17 Opinion on Elancoban for calves for rearing and cattle for fattening 17/33 These findings could be confirmed in a second experiment. Cattle reacted with a reduction of feed intake until complete anorexia, when rations highly overdosed in monensin (330 instead of 33 mg kg -1 for 12 days) were offered. 28 Feed consumption returned to normal following the feeding (seven days) of unsupplemented feed. Wentink and Vente (1981) described (accidental) poisoning of dairy cows after ingestion of a feed with 366 mg monensin kg -1 diet (equivalent to 6.5 mg per kg bw) Interactions Clinically important interactions between the ionophore anticoccidials and other antibiotics (i.e. tiamulin) are well known and frequently documented in the literature. The FEEDAP Panel has already reviewed this issue in two previous opinions: Elancoban for chickens for fattening, chickens reared for laying and turkeys and Coxidin for chickens for fattening and turkeys for fattening (EFSA, 2004 and 2005). In a study on Hereford cattle different doses of monensin and tylosin (33 and 11, 99 and 33, and 165 mg monensin kg -1 and 55 mg tylosin kg -1 feed, respectively) were fed together for approximately 160 days. 29 The applicant concludes from the study that 33 mg monensin kg -1 and 11 mg tylosin kg -1 can be fed together with a good margin of safety. Conclusive data for the absence of interactions could not be presented, because only the combinations of the two mentioned substances were fed and studied Microbiological safety of the additive The antimicrobial spectrum of monensin sodium and MIC studies, as well as the crossresistance of monensin sodium with other antibiotics have been already assessed by the FEEDAP Panel in the previous Opinion on Elancoban for chickens for fattening, chickens reared for laying and turkeys (EFSA, 2004). Only the new data supplied by the applicant have been analyzed and assessed Antimicrobial spectrum and MIC studies The sensitivity pattern of bacteria from bovine faecal origin was equivalent to that of bacteria isolates from other animal species, which confirms that Mo-Na is mainly active against Gram-positive bacteria and does not show antimicrobial activity against Gramnegative pathogens Cross-resistance with other antibiotics Adaptation of rumen bacteria to Mo-Na has been described in several studies. It is a population-based phenomenon with an increase in the fraction of cells less susceptible to monensin. It is not the result of acquired resistance but of a modification of the distribution of sub-populations with different monensin susceptibilities. The adapted culture does not show cross-resistance with antibiotics used in human and veterinary medicine. Exposure of intestinal micro-organisms to Mo-Na does not result in the development of resistance to the antibiotics tested which may be used for the prevention or treatment of disease (EFSA, 2004). The effect of monensin on the number and antimicrobial resistance level of faecal E. coli has been assessed in two experimental studies with three groups of six steers, aged about 28 Section IV, Volume 39, Attachment Section IV, Volume 39, Attachment 20

18 Opinion on Elancoban for calves for rearing and cattle for fattening 18/33 eight months, treated for 84 days with 0, 20 and 40 mg Mo-Na kg -1 feed. 30 There was no evidence of any effect of monensin on the number and antimicrobial resistance of faecal E. coli from treated animals against seven other antimicrobials (neomycin, ampicillin, nitrofurantoin, streptomycin, tetracycline, chloramphenicol and trimethoprim: sulphadiazine). Another study 31 was conducted with cattle treated with 0 and 40 mg Mo-Na kg -1 ration found a significant increase in the number of bacteroides in the treated group. However, this increase in number was not accompanied by any increase in resistance to clindamycin, erythromycin and tetracycline Effects on colonisation and shedding of enteropathogens A number of studies considered the effect of Mo-Na on the carriage and excretion of human enteric pathogens, notably Salmonella spp. and E. coli O157:H7, in the calf and cattle. An in vitro study (Callaway et al., 2003) was performed to determine the effect of monensin on four Salmonella strains and two E. coli 0157:H7 strains cultured in pure and mixed ruminal fluid cultures. The results show that bacterial growth rates were not affected by monensin at concentrations up to ten times the approximate rumen monensin concentration under normal feeding conditions. An experiment 32 was designed to determine the effect of Mo-Na at 30 mg kg -1 feed on the colonisation and shedding of Salmonella Typhimurium in experimentally infected calves. The eight weeks exposure of Salmonella -challenged calves to Mo-Na did not affect the colonization as measured by the number of Salmonella spp. excreted and number of shedding days. No change in the resistance pattern of Salmonella spp. isolates tested occurred. In a study (Van Baale et al., 2004), twelve rumen cannulated cattle, adapted to forage or grain diets with or without monensin, were used to investigate the effects of diet and monensin on the concentration and duration of ruminal persistence and faecal shedding of E. coli O157:H7. Cattle were ruminally inoculated with a strain of E. coli O157:H7. Ruminal and faecal samples were collected for 11 weeks then cattle were euthanized and necropsied and digesta from different gut locations were collected. In high-grain fed cattle, monensin supplementation significantly increased the shedding (p<0.05) of E. coli O157:H7. In a longitudinal study (165 days) (Lefebvre et al., 2005), the effect of growth-promoting agents (200 mg monensin day -1 per head and trenbolone acetate-estradiol) and an antibiotic (oxytetracycline) was investigated on the incidence of Escherichia coli O157 in feedlot steers, including antibiotic-resistant and hypermutable isolates. Eighty steers in 16 pens were treated with eight combinations of promoters, and each treatment was duplicated. Faecal samples were collected at nine different sampling times for detection of E. coli O157. Overall, 50 E. coli O157 isolates were detected in treated animals, and none were found in untreated animals. Compared with untreated controls, there was a significant association between the utilization of growth-promoting agents or antibiotics and the shedding of E. coli O157 at day 137 (P=0.03), when a prevalence peak was observed and 50% of the isolates were detected. No specific association between the use of monensin and the shedding of E. coli O157 could be statistically established. 30 Section IV, Volume 26, Attachment Section I, Volume 1, Summary 32 Section IV, Volume 26, Attachment 173