The EFSA Journal (2005) 207, 1-6

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1 The EFSA Journal (2005) 207, 1-6 Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on a request from the Commission on the safety of the product Lactiferm for chickens for fattening for use as a feed additive (Question Nº EFSA-Q ) Adopted on 13 April 2005 SUMMARY The active ingredient of the microbial feed additive Lactiferm are viable cells of Enterococcus faecium NCIMB (M74). This product has been provisionally authorised as a feed additive for use in calves and piglets. The European Food Safety Authority has been requested by European Commission to assess the safety of micro-organism product "Lactiferm", for the chickens for fattening, the human consumer, the user of the product and the environment, when this product is used as feed additive at the dose 2.5 x x 10 9 cfu kg -1 of complete feedingstuff. The five-weeks tolerance study was performed with 253 broiler chickens fed with ten-times the maximum recommended dose of the additive (150 x 10 9 cfu kg -1 feed). The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP Panel) concludes from the data provided that the product is well tolerated by chickens for fattening. In one experiment the effect of the product on gut microbiota was analysed, enumerating enterococci and E. coli of birds fed with E. faecium NCIMB at levels lower than the maximum recommended dose. The inadequate design and the low number of bacterial species investigated did not allow conclusions to be drawn. However, the FEEDAP Panel recognises that that E. faecium is a normal constituent of the gut microbiota of the production species. The MIC (Minimum Inhibitory Concentration) values of Enterococcus faecium NCIMB for clinically relevant antibiotics were below or at the same level as the SCAN breakpoints indicating the absence of resistance determinants against antibiotics of human and veterinary importance. The E. faecium strain NCIMB was shown to be free from known virulence determinants. The FEEDAP Panel concludes that the product is well-tolerated by the target species, and is safe for consumers and users. The FEEDAP Panel also agrees with the previous SCAN opinion that the environmental risk assessment is not necessary in this case. Keywords: Lactiferm, Enterococcus faecium NCIMB 11181, chickens for fattening, probiotic, safety assessment

2 Opinion on the additive Lactiferm for chickens for fattening 2/6 BACKGROUND Council Directive 70/524/EEC establishes rules governing the Community authorisation of additives for animal nutrition and in particular defines the conditions that a substance/product should meet to get authorisation. The product Lactiferm is already provisionally authorised in Council Directive 70/524/EEC as a feed additive under the category Micro-organism, for use in calves and piglets until 6 January 2004 (first authorisation: Commission Regulation (EC) Nº 2690/1999). The Scientific Committee on Animal Nutrition (SCAN) issued a favourable opinion on the safety of Lactiferm for the mentioned animal categories. Company Medipharm AB is seeking provisional Community authorisation of its microorganism product Lactiferm as a feed additive intended for use in chickens for fattening under category "Micro-organism" (Table 1). Table 1. Description of the product Product category: Micro-organism Trade name: Lactiferm Description: Enterococcus faecium M74 (NCIMB 11181) Target animal category: Chickens for fattening Applicant: Medipharm AB Member State Rapporteur: Sweden Type of request: Provisional authorization In accordance with the above legislation, the Commission received a request from this company, through Sweden, the Member State Rapporteur chosen by the company, to authorise its product under the conditions set out in Table 2. Supporting data produced by the company were compiled in a dossier and accompanied that application. All Member States received the dossier, which was introduced at the Standing Committee on the Food Chain and Animal Health at its meeting of 24/25 September This dossier is made available to the European Food Safety Authority (EFSA) directly by the company, via its Member State rapporteur.

3 Opinion on the additive Lactiferm for chickens for fattening 3/6 Table 2. Annex entry, as proposed by the company Additive Chemical formula, description Species or category of animal Maximum age Minimum content Maximum content cfu kg -1 of complete feedingstuff Other provisions Enterococcus faecium NCIMB Preparation of Enterococcus faecium containing a minimum of: Powder form: 4x10 11 cfu g -1 additive Coated form: 5x10 10 cfu g -1 additive Chickens for fattening 45 days 2.5x x10 9 In the directions for use of the additive and premixture, indicate the storage temperature, storage life and stability to pelleting. TERMS OF REFERENCE In view of the above, the Commission asks the European Food Safety Authority (EFSA) to deliver an opinion on the safety of micro-organism product "Lactiferm", for the target animals, the human consumer, the user of the product and the environment, when used under the conditions set out in Table 2 above, taking into account the background and the information submitted by the applicant in the dossier. ASSESSMENT The product Lactiferm, based on the strain Enterococcus faecium NCIMB 11181, has been previously assessed for its safety when included in feed for piglets and calves by the SCAN 1. In the course of these assessments the safety for users of the additive and for consumers of products derived from animals fed the additive was considered and found not to be of concern. It was also concluded that no environmental risk assessment was necessary. Consequently, the focus of this assessment is the safety of the additive for the additional target species, chickens for fattening, focusing on the tolerance to an overdose of the product and the effects of the product at the recommended dose on the flora of the digestive tract. However, FEEDAP Panel in considering the previous assessment of the product noted a need to revise elements of the assessment taking account of more recent scientific developments. Therefore FEEDAP Panel sought data from the Notifier on data demonstrating the absence of genes encoding for virulence factors and more recent data on antibiotic resistance. 1. General description The active ingredient in the additive Lactiferm is the bacterial strain Enterococcus faecium NCIMB The product is intended for use in diets for chickens for fattening at a level of 2.5 x x 10 9 cfu kg -1 feedingstuff. E. faecium NCIMB was isolated from a 1

4 Opinion on the additive Lactiferm for chickens for fattening 4/6 healthy child in 1974 and is deposited at "The National Collections of Industrial and Marine Bacteria Ltd", Aberdeen, Scotland, where it is registered under the number NCIMB Studies concerning the safety of use of the additive 2.1. Tolerance study in chickens for fattening A 35-day tolerance study including a negative control group (242 broiler-chickens, breed JVA) and two supplemented groups fed with the maximum recommended dose (15 x 10 9 cfu kg -1 feed, 253 broiler chickens) and ten-times the maximum recommended dose (150 x 10 9 cfu kg -1 feed, 253 broiler chickens) of Lactiferm was reported. The dose was confirmed by microbial enumerations. The animals were housed in an experimental floor husbandry for poultry, divided in three compartments with four subdivisions each. Following the directions of GLP, complete disinfection was performed 10 days before housing of the animals. Wood shavings were used as litter. Chickens were offered ad libitum feed based on wheat, maize and soybean with or without the additive supplementation. The parameters studied included zootechnical parameters (body weight monitored once a week, feed intake once a day), scoring of faeces consistency (once a week), parasitological examinations in faecal samples (once a week). Histopathological studies were not performed. Data were checked using t-test if normality in distribution or Man-Whitney-Rank-Sum. The proportional differences in mortality were analysed by Chi-square analysis. Results indicate that the maximum and ten-times the maximum recommended doses of Lactiferm did not lead to negative effects on chickens for fattening. The two treated groups showed significantly increased live weight and improved feed conversion rate in comparison to control. Mortality was low, ranging between 0.8% and 1.8%. The incidence of liquid faeces increased in all groups during the experiment, but it was more pronounced in the group with the highest concentrations of Lactiferm. However no disturbances in growth parameters and in general health were related to these changes Effects on gut microflora The influence on the intestinal microflora was investigated in an old study (1982): 40 broiler chicks were divided in five groups, a control group and four groups given Lactiferm at different doses (5, 10, 15 and 20 x 10 8 cfu kg -1 feedingstuff). No data were presented with animals fed with E. faecium NCIMB at the maximum recommended dose (15x10 9 cfu kg -1 feedingstuff). At day 56 the small intestinal content was sampled and enterococci and E. coli were enumerated. No relevant numerical differences were present among control and treated groups. No information was provided on other relevant bacterial genera which can be cultivated from gut microbiota. The poor design and the insufficient data set did not allow conclusions on the effect of Lactiferm on gut microbiota to be drawn Antibiotic susceptibility of Enterococcus faecium NCIMB E. faecium NCIMB strain was susceptible to ampicillin, chloramphenicol, ciprofloxacin, gentamicin, linezolid, rifampicin, streptomycin, synercid, tetracycline, trimethoprim and vancomycin. The absence of vancomycin resistance genes has been

5 Opinion on the additive Lactiferm for chickens for fattening 5/6 confirmed in PCR experiments. The MIC of erythromycin (4 µg ml -1 ) and kanamycin (1024 µg ml -1 ) were at the level of breakpoints defined by SCAN guidelines 2. The absence of erm(a), erm(b) and erm(c) genes, encoding for erythromycin resistance and of apha3 gene, conferring resistance to kanamycin, has been demonstrated Virulence determinants in Enterococcus faecium M 74 The presence of genes coding for virulence determinants in E. faecium NCIMB has been assessed using a PCR approach. Primers complementary to gene coding for surface proteins (agg, esp, efaafs and efaafm), gelatinase (gele), and pheromone production have been used. The PCR based screening for virulence factors in E. faecium NCIMB did not reveal presence of virulence factors. CONCLUSIONS In the view of the FEEDAP Panel, the consumer and user safety of this product has been previously addressed. Only the new issues since that assessment are addressed in this current opinion. In a tolerance test performed with ten-times the maximum recommended dose for five weeks no adverse effects were observed in the chickens for fattening. Despite the lack of haematological and blood-chemistry data, it appears unlikely that E. faecium NCIMB represents a hazard for the target species even when supplied in overdose. However, the presented microbiological data do not allow an assessment of the effect of Lactiferm on gut microbiota at the maximum recommended dose and thus no conclusions on this potential hazard can be drawn. MIC values for all the tested antibiotics tested were below or at the same level as the SCAN breakpoints indicating the absence of resistance determinants against antibiotics of human and veterinary importance. The E. faecium strain NCIMB was shown to be free from known virulence determinants. The FEEDAP Panel concludes that the product is well-tolerated by the target species, and is safe for consumers and users. Although data on the effects of the product on the gut flora are inadequate, the Panel recognises that E. faecium is a normal constituent of the gut microbiota of the production species. FEEDAP Panel also agrees with the previous SCAN opinion that the environmental risk assessment is not necessary in this case. DOCUMENTATION PROVIDED TO EFSA 1. Enterococcus faecium M74 Extension dossier for broiler. Version Submitted by Medipharm AB, Sweden. October Supplementary information on Enterococcus faecium M74, (NCIMB 11181, E 1708) for broiler. Supplement to the answers on the opinions from the Member States and the EFSA concerning the application for an extension of the authorisation of Enterococcus faecium M74 as a feed additive according to 70/524/EC with the 2 Opinion of the scientific committee on animal nutrition on the criteria for assessing the safety of microorganisms resistant to antibiotics of human clinical and veterinary importance. 24 January

6 Opinion on the additive Lactiferm for chickens for fattening 6/6 target group chicken for fattening (broiler). Submitted by Medipharm AB, Sweden. December SCIENTIFIC PANEL MEMBERS Arturo Anadón, Margarita Arboix Arzo, Georges Bories, Paul Brantom, Joaquim Brufau de Barberà, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël Dierick, Gerhard Flachowsky, Anders Franklin, Jürgen Gropp, Ingrid Halle, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Kimmo Peltonen, Guido Rychen, Pascal Sanders, Amadeu Soares, Wilhelm Windisch and Pieter Wester ACKNOWLEDGEMENTS The Scientific Panel on Additives and Products or Substances used in Animal Feed wishes to thank Professor Atte von Wright for the contribution to the draft opinion.

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