SCIENTIFIC OPINION. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3

EFSA Journal 2013;11(4):3175 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of Provita LE (Enterococcus faecium and Lactobacillus rhamnosus) as a feed additive for calves for rearing 1 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3 ABSTRACT European Food Safety Authority (EFSA), Parma, Italy Provita LE is a preparation of Enterococcus faecium and Lactobacillus rhamnosus authorised for use in piglets and calves. EFSA was requested to re-evaluate Provita LE when used with calves for rearing up to four months in the range of 1 10 9 to 5 10 9 CFU/kg feedingstuffs. The identity of both strains has been established and no resistance to clinically relevant antibiotic detected. L. rhamnosus satisfies the requirements for the qualified presumption of safety approach to safety assessment, and is presumed safe for the target animals, consumers and the environment. Safety of the complete additive for the target species at the proposed dose range was demonstrated in a tolerance study in which no adverse effects were seen when a 100-fold overdose of the additive was administered to calves. The enterococcal strain was shown not to contain marker genes typical of hospital-associated isolates responsible for clinical infections and does not raise additional safety concerns. Since no other sources of concern were identified, Provita LE is considered safe for consumers. No data on skin/respiratory sensitisation were provided. In the absence of data, the potential of the additive to be a skin/respiratory sensitiser cannot be excluded. The dustiness of the additive tested indicated a potential for users to be exposed via inhalation. Given the proteinaceous nature of the active agents, the additive should be considered to have the potential to be a skin/respiratory sensitiser and treated accordingly. E. faecium is a natural component of the gut microbiota and its use as Provita LE in animal feeding is not expected to pose any risk to the environment. The data from three trials presented showed that Provita LE has the potential to improve the zootechnical performance of calves for rearing at 1 10 9 CFU/kg milk replacer or complementary feed. European Food Safety Authority, 2013 KEY WORDS Zootechnical additive, Provita LE, Enterococcus faecium, Lactobacillus rhamnosus, calves for rearing, safety, efficacy 1 On request from the European Commission, Question No EFSA-Q-2012-00421, adopted on 13 March 2013. 2 Panel members: Gabriele Aquilina, Alex Bach, Vasileios Bampidis, Maria De Lourdes Bastos, Gerhard Flachowsky, Mikolaj Antoni Gralak, Christer Hogstrand, Lubomir Leng, Secundino López-Puente, Giovanna Martelli, Baltasar Mayo, Derek Renshaw, Guido Rychen, Maria Saarela, Kristen Sejrsen, Patrick Van Beelen, Robert John Wallace and Johannes Westendorf. Correspondence: FEEDAP@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the Working Group on Micro-Organisms including Andrew Chesson, Pier Sandro Cocconcelli and Ingrid Halle, for the preparatory work on this scientific opinion. Suggested citation: EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP); Scientific Opinion on the safety and efficacy of Provita LE (Enterococcus faecium and Lactobacillus rhamnosus) as a feed additive for calves for rearing. EFSA Journal 2013;11(4):3175. [14 pp.] doi:10.2903/j.efsa.2013.3175. Available online: www.efsa.europa.eu/efsajournal European Food Safety Authority, 2013

SUMMARY Provita LE is a preparation of a strain of Enterococcus faecium and of Lactobacillus rhamnosus in a 7:3 ratio. This product is authorised for use in piglets and calves. The company is requesting the reevaluation of the product Provita LE when used as a feed additive for calves for rearing up to four months at a minimum dose of 1 10 9 and a maximum dose of 5 10 9 of complete feedingstuffs. The identity of E. faecium and L. rhamnosus has been established and no resistance to clinically relevant antibiotic detected. L. rhamnosus satisfies the requirements for the qualified presumption of safety (QPS) approach to safety assessment, and thus is presumed safe for the target animals, consumers of products derived from animals fed the additive and the environment. Safety of the complete additive for the target species at the proposed dose range was shown by the results of a tolerance study in which no adverse effects were seen when a 100-fold overdose of the additive was administered to calves. The E. faecium strain was shown not to contain marker genes typical of hospital-associated isolates responsible for clinical infections and does not raise additional safety concerns. In addition, no other sources of concern have been identified in the additive. Consequently, the FEEDAP Panel considers the use of the Provita LE safe for consumers. Provita LE is not irritant to skin or eyes. The dustiness of the additive indicated that users may be exposed via inhalation. Given the proteinaceous nature of the active agents, the additive should be considered to have the potential to be a skin/respiratory sensitiser and treated accordingly. E. faecium is a natural component of the gut microbiota and its use in conjunction with L. rhamnosus as Provita LE in animal feeding would not be expected to pose any risk to the environment. Five studies were performed in which calves for rearing were administered milk replacer with or without the additive at the minimum recommended dose. In all five trials, calves given the additive had a higher final body weight and showed a greater weight gain than control animals. These changes reached significance in three of the five trials. Therefore, the FEEDAP Panel concludes that Provita LE has the potential to improve the zootechnical performance of calves for rearing at the dose of 1 10 9 CFU/kg milk replacer or complementary feed. EFSA Journal 2013;11(4):3175 2

TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 3 Background... 4 Terms of reference... 4 Assessment... 7 1. Introduction... 7 2. Characterisation... 7 2.1. Characterisation of the active substances... 7 2.1.1. Antibiotic susceptibility... 7 2.1.2. Virulence of Enterococcus faecium DSM 7134... 7 2.2. Manufacture and characterisation of the product... 7 2.3. Stability and homogeneity... 8 2.4. Conditions of use... 8 2.5. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) 9 3. Safety... 9 3.1. Safety for the target species... 9 3.2. Safety for the consumer... 9 3.3. Safety for the user... 9 3.4. Safety for the environment... 10 4. Efficacy... 10 5. Post-market monitoring... 12 Conclusions... 12 Documentation provided to EFSA... 12 References... 12 Appendix... 14 EFSA Journal 2013;11(4):3175 3

BACKGROUND Regulation (EC) No 1831/2003 4 establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 10(2) of that Regulation also specifies that for existing products within the meaning of Article 10(1), an application shall be submitted in accordance with Article 7, at the latest one year before the expiry date of the authorisation given pursuant to Directive 70/524/EEC for additives with a limited authorisation period, and within a maximum of seven years after the entry into force of this Regulation for additives authorised without a time limit or pursuant to Directive 82/471/EEC. The European Commission received a request from the company Lactosan GmbH & Co.KG 5 for reevaluation of the product Provita LE (Enterococcus faecium DSM 7134 and Lactobacillus rhamnosus DSM 7133), when used as a feed additive for calves for rearing (category: zootechnical additive; functional group: gut flora stabiliser) under the conditions mentioned 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 10(2) (reevaluation of an authorised feed additive). EFSA received directly from the applicant the technical dossier in support of this application. 6 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 5 July 2012. Provita LE is a preparation of Enterococcus faecium (DSM 7134) and Lactobacillus rhamnosus (DSM 7133). This product is authorised for use in piglets 7 and calves. 8 The strain Enterococcus faecium (DSM 7134) alone is authorised (as Bonvital) for weaned piglets, pigs for fattening, sows and chickens for fattening. 9 The Scientific Committee on Animal Nutrition (SCAN) issued an opinion on the safety for pigs for fattening and calves, the consumer, user and environment of the microbial product containing Enterococcus faecium (DSM 7134) and Lactobacillus rhamnosus (DSM 7133) (EC, 1997, updated 2003) and another opinion on the safety of Enterococcus faecium (DSM 7134) for piglets, pigs for fattening and sows (EC, 2003). EFSA published an opinion on the safety of Bonvital (Enterococcus faecium DSM 7134) for chickens for fattening (EFSA, 2004), an opinion on the safety and efficacy for piglets and pigs for fattening (EFSA, 2006), an opinion on the safety and efficacy for sows (EFSA, 2007a), and two opinions on Bonvital when used as a feed additive in chickens for fattening (EFSA, 2009 and 2010). 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. EFSA shall deliver an opinion on the safety for the target animals, consumer, user and the environment and the efficacy of the product 4 Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition. OJ L 268, 18.10.2003, p. 29. 5 Lactosan GmbH & Co.Kg, Industriestrasse West 5, 8605 Kapfenberg, Austria. 6 EFSA Dossier reference: FAD-2010-0305. 7 Commission Regulation (EC) No 2148/2004 of 16 December 2004 concerning the permanent and provisional authorisations of certain additives and the authorisation of new uses of an additive already authorised in feedingstuffs. OJ L 370, 17.12.2004, p. 24. 8 Commission Regulation (EC) No 1288/2004 of 14 July 2004 concerning the permanent authorisation of certain additives and the provisional authorisation of a new use of an additive already authorised in feedingstuffs. OJ L 243, 15.7.2004, p. 10. 9 Commission Regulation (EC) No 538/2007 of 15 May 2007 concerning the authorisation of a new use of Enterococcus faecium DSM 7134 (Bonvital) as a feed additive. OJ L 128, 16.5.2007, p. 16. EFSA Journal 2013;11(4):3175 4

Provita LE, Enterococcus faecium DSM 7134 and Lactobacillus rhamnosus DSM 7133, when used under the conditions described in Table 1. EFSA Journal 2013;11(4):3175 5

Table 1: Description and conditions of use of the additive as proposed by the applicant Additive Registration number/ec No/No Category(-ies) of additive Functional group(s) of additive Enterococcus faecium DSM 7134 and Lactobacillus rhamnosus DSM 7133 E 1706 Zootechnical additive Gut flora stabiliser Composition, description Mixture of Enterococcus faecium DSM 7134 and Lactobacillus rhamnosus DSM 7133 having a minimum activity of 1 x 10 10 CFU/g of the additive (E. faecium 7 x 10 9 CFU/g and L. rhamnosus 3 x 10 9 CFU/g) Trade name Name of the holder of authorisation Chemical formula Provita LE Description Lactosan GmbH & Co.KG Purity criteria Impurities: Fungi < 100 CFU/g Clostridia < 10 CFU/g Enterobacteria <10 CFU/g Salmonella not detectable in 25 g Method of analysis Quantification of L. rhamnosus (according ISO 15787:2009) and E. faecium (according ISO 15788:2009) Species or category of animal Maximum Age Conditions of use Minimum content Maximum content CFU/kg of complete feedingstuffs Withdrawal period Calves for rearing 4 months 1 x 10 9 5 x 10 9 N/A Other provisions and additional requirements for the labelling Specific conditions or restrictions for use Specific conditions or restrictions for handling Post-market monitoring Specific conditions for use in complementary feedingstuffs N/A The directions for use must indicate storage temperature, shelf life Lactosan GmbH & Co.Kg will conduct post-market monitoring in compliance with EU law on feed hygiene, namely by use of HACCP and treaceability systems, and formal monitoring of customer feedback through product or service complaints. N/A Marker residue Maximum Residue Limit (MRL) Species or category of animal Target tissue(s) or food products Maximum content in tissues N/A N/A N/A N/A EFSA Journal 2013;11(4):3175 6

ASSESSMENT 1. Introduction The microbial feed additive Provita LE is a preparation of Enterococcus faecium (DSM 7134) and Lactobacillus rhamnosus (DSM 7133). This product is authorised for use in piglets and calves. The strain Enterococcus faecium (DSM 7134) alone is authorised for weaned piglets, pigs for fattening, sows and chickens for fattening under another trade name. The applicant is now seeking a renewal of the authorisation for calves and has specified calves for rearing. 2. Characterisation 2.1. Characterisation of the active substances The product is composed of a strain of Enterococcus faecium and of Lactobacillus rhamnosus in a 7:3 ratio. These strains, originally isolated from silage, are deposited in Deutsche Sammlung von Mikroorganismen und Zellkulturen, under the accession numbers DSM 7134 (E. faecium) and DSM 7133 (L. rhamnosus). 10 The taxonomic identification of the two strains was achieved by the full 16S rrna gene sequence and pulse-field gel electrophoresis (PFGE) analysis. 11 Strain-specific identification and genetic stability analysis for both strains is based on the use of random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) typing approach. 12 Using this method, the master cultures are routinely compared with working cultures used to inoculate fermentation batches. No differences in the resultant patterns have been observed to date. 2.1.1. Antibiotic susceptibility Both strains were tested for antibiotic susceptibility using two-fold broth dilutions. 13 The battery of antibiotics tested included all of those recommended by EFSA (EFSA, 2012a). As all minimum inhibitory concentration (MIC) values were equal to or lower than the corresponding cut-off values defined by the FEEDAP Panel, no further investigation is required. 2.1.2. Virulence of Enterococcus faecium DSM 7134 The MIC of ampicillin for E. faecium DSM 7134 was 0.5 mg/l and the PCR assays demonstrated the absence of the genetic determinants IS16, hyl Efm and esp, typical of hospital-associated strains. 14 Therefore, the FEEDAP Panel concludes that E. faecium NCIMB 10415 does not contain marker genes typical of hospital-associated isolates responsible for clinical infections. 2.2. Manufacture and characterisation of the product The two strains of the additive are produced separately by fermentation in a typical industrial medium for lactic acid bacteria. After fermentation, cells are harvested using centrifugation, cryoprotectants are added (lactose, glutamate, citrate, lactate and mannose) and the product is freeze dried and ground to a fine powder. The additive is produced by blending the concentrated cell mass of the two strains with whey protein to give a product containing a minimum of 1 10 10 CFU/g of the additive (E. faecium 7 10 9 CFU/g and L. rhamnosus 3 10 9 CFU/g) with an approximate composition of 89 94 % whey proteins and 5 10 % cell mass. 10 Technical dossier/section II/ Annexes 2_2-1 to 2. 11 Technical dossier/section II/ Annexes 2_2-3 to 7. 12 Technical dossier/section II/ Annexes 2_2-8 to 9. 13 Technical dossier/ Section II and Supplementary information/annexes II.2-16 to 17 and Antibiotic. 14 Technical dossier/ Section II and Supplementary information /Annexes II.2-11 to 14 and Virulence. EFSA Journal 2013;11(4):3175 7

Data from five batches of the additive showed that the minimum specification was met in all cases (range 1.3 1.7 10 10 CFU/g additive) with the expected ratio (7:3) between the two strains. 15 The resulting additive is a pale, free-flowing powder consisting of particles with a mean diameter of 97 µm as determined by laser diffraction. 16 Approximately 10 % of the product has a particle size of diameter less than 10 μm, 30 % less than 50 μm and 50 % less 100 μm. The dusting potential of the additive was found to be 3.9 g/m 3 using the Heubach dustometer. 17 The product is routinely analysed for microbial contamination. 18 Specifications are set in the final product for Enterobacteriaceae (< 1 000 CFU/g), Escherichia coli (< 10 CFU/g), yeast and filamentous fungi (< 1 000 CFU/g) and Salmonella (absence in 25 g). Analyses of five batches demonstrated compliance with the specifications for Enterobacteriaceae and fungi and analysis of a single batch demonstrated compliance with the specifications for E. coli and Salmonella. The product is not routinely analysed for heavy metals, arsenic and mycotoxins because of the composition of the growth medium and excipients. However, a single batch of the final product was analysed for its contents of heavy metals, arsenic and the most commonly encountered mycotoxins. 19 All values were below the level of detection, confirming the approach taken by the applicant. 2.3. Stability and homogeneity The stability of Provita LE was investigated in a long-term storage experiment by examining the differential counts of the two strains of the additive when stored at 4, 25 and 40 C in its original packaging. 20 Data from three batches of Provita LE demonstrated the stability of the products at 4 C over 24 months, at 25 C over 12 months and at 40 C for up to two months. Owing to the nature of the additive (hygroscopic, lyophilised powder), Provita LE must be stored in a packaging that protects the additive against environmental conditions (light, moisture, oxygen). Stability (one batch) in three dry milk replacers and one complementary feed was monitored for three months when stored at 20 C. 21 No significant losses of either strain were recorded over the experimental period. To measure the stability of Provita LE in reconstituted milk replacer, differential counts of Provita LE were determined at several time points up to four hours when stored at 37 and 55 C. 22 Neither of the strains was affected by the temperature used to prepare the milk replacer and cell numbers remained essentially unchanged for a further four hours. The ability of the additive to homogeneously mix with dry milk replacer was tested in a study of 10 subsamples. 23 Differential counts indicated that the product could be homogeneously dispersed and that no segregation of the strains occurred. 2.4. Conditions of use Provita LE is intended for use in milk replacer and complementary feed for calves for rearing up to four months at a minimum dose of 1 10 9 and a maximum dose of 5 10 9 CFU/kg. 15 Technical dossier/section II/ Annex II.1-4. 16 Technical dossier/section II/ Annex II.1-8. 17 Technical dossier/section II/ Annex II.1-9. 18 Technical dossier/section II/ Annexes II.1-5 to 6. 19 Technical dossier/section II/ Annex II.1-7. 20 Technical dossier/section II/ Annex II.4-1. 21 Technical dossier/section II/ Annex II.4-3. 22 Technical dossier/supplementary information October 2012/ Annex I. 23 Technical dossier/section II/ Annex II.4-4. EFSA Journal 2013;11(4):3175 8

2.5. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) EFSA has verified the EURL report as it relates to the methods used for the control of the active agent in animal feed. The Executive Summary of the EURL report can be found in the Appendix. 3. Safety One strain of the product, L. rhamnosus, is considered by EFSA to be suitable for the qualified presumption of safety (QPS) approach to safety assessment (EFSA, 2007b, 2012b). In the view of the FEEDAP Panel, the identity of the strain has been established and the antibiotic susceptibility qualification met. Consequently, this strain is presumed safe for the target species, consumers of products from the target animals and the environment. However, since the E. faecium strain does not qualify for the QPS approach, some studies aiming to demonstrate the safety of Provita LE have been presented and are described below. 3.1. Safety for the target species In a tolerance study, 30 Holstein Friesian bull calves (body weight 46.7 kg, average age of 18 days) were divided in three groups and received a milk replacer, concentrate and hay. 24 The calves were weighed at the beginning, after four weeks and at the end of the trial. Milk replacer was given individually, and concentrate and hay intake in each group was recorded weekly. The calves were divided into a control group and treatment groups receiving milk replacing containing Provita LE at 1 10 9 CFU/kg (the minimum recommended dose) or 5 10 11 CFU/kg (100 times the maximum recommended dose). The duration of the trial was 58 days. The statistical analysis of the data was performed with linear mixed models (t-test). Veterinary interventions against gastrointestinal events, cough and fever were reported. They were, however, evenly distributed among the groups and did not include antibiotics. No animals died during the trial. There were no significant differences in the mean live weight between the groups at the end of the trial (mean calf weight 85.9, 86.4 and 87.3 kg in the control group and the two treatment groups, respectively). The incidence of diarrhoea (number of days) was low and similar in the different groups (control, 44.5 days; 1 group, 34 days; and the 100 group,38.5 days). 3.2. Safety for the consumer The L. rhamnosus strain qualifies for the QPS approach to safety assessment and is presumed safe for consumers. The second strain, E. faecium, does not contain marker genes typical of hospital-associated isolates responsible for clinical infections and is not resistant to clinically relevant antibiotics. The metabolic pathways of E. faecium are well known and, when the potential for infection is excluded, no other harmful metabolites or substances are expected to be produced during fermentation. The additive does not contain excipients of concern. Consequently, Provita LE is presumed safe for consumers when used under the conditions specified. 3.3. Safety for the user Studies of acute dermal irritation/corrosion with the two strains separately, each mixed with whey as carrier, were performed following a protocol that conformed to OECD Guideline 404 and using 0.5 g of test material. 25 Since, over an observation period of 72 hours, no skin reactions were seen in any of the animals with either strain, it is concluded that the mixture Provita LE would be unlikely to cause skin irritation. Studies of acute eye irritancy were performed with the same materials as above and using groups of three male New Zealand White rabbits, following a protocol that conformed to OECD Guideline 405 24 Technical dossier/section III and Supplementary information October 2012/Annexes III.1-1 and 2. 25 Technical dossier/section III /Annexes III.3-3 and III.3-4. EFSA Journal 2013;11(4):3175 9

and using 0.1 g of the test materials. 26 The test item including L. rhamnosus DSM 7133 produced conjunctival redness in two animals and that containing E. faecium DSM 7134 did so in one case. All events were fully reversible after 72 hours. The overall results indicate that Provita LE does not require classification as an eye irritant under Regulation (EC) No 1272/2008. 27 No data on skin sensitisation were provided. The dustiness of the additive tested indicated a potential for users to be exposed via inhalation. Given the proteinaceous nature of the active agents, the additive should be considered to have the potential to be a skin/respiratory sensitiser and treated accordingly. 3.4. Safety for the environment The strain L. rhamnosus qualifies for the QPS approach to safety assessment and is presumed safe for the environment. E. faecium is a natural component of the gut microbiota and its use as Provita LE in animal feeding would not be expected to pose any risk to the environment. 4. Efficacy Six studies were performed to demonstrate the efficacy of Provita LE in calves for rearing, all carried out in the same Member State. However, one was disregarded because the duration was insufficient. 28 The first study involved 30 calves (mixed sex, Red Holstein Friesian Flecked cattle, eight to nine days of age) kept in individual pens during the first week. 29 From week 2 until the end of the trial, the calves were housed three or four to a pen and supplied with individual feeders. They were divided into two groups, a control and a treatment group, matched for sex, age and weight. Animals were fed milk replacer twice a day containing 0 or approximately 9.8 10 8 CFU Provita LE/kg (confirmed by analysis). Hay and concentrate were offered separately ad libitum from the start and maize silage was also fed ad libitum from trial week 6 to week 7. The weekly intake of milk replacer per calf was measured. The weekly intakes of concentrate, hay and silage intake were also measured in each group. At the end of the trial, final weights were measured and daily weight gain calculated. The differences between means were tested with analysis of variance (ANOVA). There were no significant differences in any of these parameters between groups (Table 2). The incidence of diarrhoea was low and similar between groups. In the second study, 80 bull calves (flecked cattle, 38 days of age) were divided into four treatment groups (control, Provita LE, zinc bacitracin and zinc bacitracin + Provita LE). 30 However, the last two groups were not further considered. The calves were housed in pens containing four or five animals. The additive was administered via milk replacer (at 1 10 9 CFU/kg, confirmed by analysis), which was fed twice daily to each calf. Hay and concentrate were offered up to a maximum of 0.5 kg and 2 kg per calf per day, respectively. Maize silage was also fed ad libitum from the second week. Feed intake (other than milk replacer) was measured per pen. At the end of the trial, final weights were measured and daily weight gain calculated. The differences between means were tested for significance by the Student Newman Keuls test. There were no significant differences between groups in any of the measured parameters (Table 2). The third study involved 20 Holstein Friesian bull calves (16 days of age) individually housed. 31 Milk replacer (with or without the additive at 1 10 9 CFU/kg, confirmed by analysis) was fed twice daily to 26 Technical dossier/section III/Annexes III.3-5 and III.3-6. 27 Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006 (Text with EEA relevance). OJ L 353, 31.12.2008, p. 1. 28 Technical dossier/ Section IV/Annex IV-2. 29 Technical dossier/ Section IV/Annex IV-3. 30 Technical dossier/ Section IV/Annex IV-4. 31 Technical dossier/ Section IV/Annex IV-5. EFSA Journal 2013;11(4):3175 10

each calf. Hay was fed ad libitum in the first two weeks, concentrate was fed to a maximum of 2 kg/day from the second week and grass/maize silage was also fed ad libitum from the eighth week. Concentrate and silage intake was measured per calf per week, while hay intake was measured per group per week. At the end of the trial, final weights were measured and daily weight gain calculated. The data were analysed by ANOVA and difference of means tested using the multiple range test. At the end of the experimental period the final body weight and daily weight gain in the Provita LE group were significantly higher than in the control group (Table 2). The fourth study was conducted in two consecutive batches involving a total of 102 bull calves (Holstein Friesian; 16 days of age). 32 Animals were housed individually for the first two weeks and subsequently kept in groups of five. Milk replacer, with or without the additive (at 1 10 9 CFU/kg, confirmed by analysis), was fed individually, while maize silage and Lucerne straw were fed ad libitum. From the second week onwards feed concentrate was also fed and intake measured. At the end of the trial, final weights were measured and daily weight gain calculated. Normality was tested with the Kolmogorov Smirnov test and homogeneity using Levene s test. Differences between means were tested for significance by ANOVA. The results show that the final body weight and daily weight gain were significantly higher in the Provita LE group than in the control group (Table 2). The fifth study involved 60 Holstein Friesian bull calves (19 days of age) randomly allocated according to body weight and age to control and treatment groups, the latter receiving 1 10 9 CFU/kg milk replacer (confirmed by analysis). 33 Animals were distributed in eight boxes (seven or eight calves per box). Milk replacer was fed individually. Feed concentrate was offered at up to 2 kg (weeks 1 8), and hay from week 3 to week 8 ad libitum, both measured per group. At the end of the trial, final weights were measured and daily weight gain calculated. Statistical evaluation was performed with a linear model taking into consideration the group as the fixed effect and the box as the random effect. The results show that the final body weight and daily weight gain were significantly higher in the Provita LE group than in the control group (Table 2). Table 2: Summary of results from the efficacy trials on calves with Provita LE Trial no (duratio n, days) 1 (84) 2 (56) 3 (58) 4 (84) 5 (56) No of animals per treatment 15 15 22 22 10 10 51 51 30 30 Dose (CFU/kg milk replacer) Initial weight (kg) 0 42.8 1 10 9 42.9 0 77.8 1 10 9 78.0 0 47.1 1 10 9 46.8 0 44.3 1 10 9 44.4 0 51.9 1 10 9 50.9 Total feed Final intake weight (kg dry (kg) matter/ head/day) 1 101.7 1.68 104.4 1.73 119.7 1.87 123.4 1.96 90.1 a 1.53 95.8 b 1.75 102.5 a 1.51 107.4 b 1.54 93.1 a 1.57 99.2 b 1.69 Daily weight gain (kg/day) 0.70 0.74 0.75 0.81 0.74 a 0.83 b 0.69 a 0.75 b 0.74 a 0.86 b a,b Treatment means differ significantly from control (P < 0.1). 1 Milk replacer and concentrate only in trials 4 and 5. The results of the three trials presented showed that Provita LE has the potential to improve the zootechnical performance of calves for rearing at the dose of 1 10 9 CFU/kg milk replacer or complementary feed. 32 Technical dossier/ Section IV/ Annex IV-6. 33 Technical dossier/supplementary information October 2012/Annexes Va, Vb and Vc. EFSA Journal 2013;11(4):3175 11

5. Post-market monitoring Provita LE for calves for rearing The FEEDAP Panel considers that there is no need for specific requirements for a post-market monitoring plan other than those established in the Feed Hygiene Regulation 34 and Good Manufacturing Practice. CONCLUSIONS Lactobacillus rhamnosus satisfies the requirements for the QPS approach to safety assessment, and thus is presumed safe for the target animals, consumers and the environment. Safety of the complete additive for the target species at the proposed dose range was shown by the results of a tolerance study in which no adverse effects were seen when a 100-fold overdose of the additive was administered to calves. The Enterococcus faecium strain was shown not to contain marker genes typical of hospital-associated isolates responsible for clinical infections and does not raise additional safety concerns. In addition, no other sources of concern have been identified in the additive. Consequently, the FEEDAP Panel considers the use of the Provita LE safe for consumers. Provita LE is not irritant to skin or eyes. The dustiness of the additive indicated that users may be exposed via inhalation. Given the proteinaceous nature of the active agents, the additive should be considered to have the potential to be a skin/respiratory sensitiser and treated accordingly. E. faecium is a natural component of the gut microbiota and its use in conjunction with the L. rhamnosus as Provita LE in animal feeding would not be expected to pose any risk to the environment. The data of three trials presented showed that Provita LE has the potential to improve the zootechnical performance of calves for rearing at the dose of 1 10 9 CFU/kg milk replacer or complementary feed. DOCUMENTATION PROVIDED TO EFSA 1. Dossier on Provita LE (Enterococcus faecium DSM 7134 and Lactobacillus rhamnosus DSM 7133). November 2010. Submitted by Lactosan GmbH & Co. KG. 2. Dossier on Provita LE (Enterococcus faecium DSM 7134 and Lactobacillus rhamnosus DSM 7133). Supplementary information. July 2012. Submitted by Lactosan GmbH & Co. KG. 3. Dossier on Provita LE (Enterococcus faecium DSM 7134 and Lactobacillus rhamnosus DSM 7133). Supplementary information. October 2012. Submitted by Lactosan GmbH & Co. KG. 4. Evaluation report of the European Union Reference Laboratory for Feed Additives on the Methods(s) of Analysis for Provita LE. 5. Comments from Member States received through the ScienceNet. REFERENCES EC (European Commission), 1997, updated 2003. Opinion on the use of certain microorganisms as additives in feedingstuffs. Available online: http://ec.europa.eu/food/fs/sc/scan/out93_en.pdf EC (European Commission), 2003. Report of the Scientific Committee on Animal Nutrition on the safety of the Micro-organism product Provita E for use as feed additive. Available online : http://ec.europa.eu/food/fs/sc/scan/out107_en.pdf 34 Regulation (EC) No 183/2005 of the European Parliament and of the Council of 12 January 2005 laying down requirements for feed hygiene. OJ L 35, 8.2.2005, p. 1. EFSA Journal 2013;11(4):3175 12

EFSA (European Food Safety Authority), 2004. 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 product Bonvital (Provita E) for chickens for fattening. The EFSA Journal, 120, 1-4. EFSA (European Food Safety Authority), 2006. Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) on the safety and efficacy of product Bonvital, a preparation of Enterococcus faecium, as a feed additive for piglets and pigs for fattening in accordance with Regulation (EC) No 1831/2003. The EFSA Journal, 440, 1 9. EFSA (European Food Safety Authority), 2007a. Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) on the safety and efficacy of product Bonvital, a preparation of Enterococcus faecium, as a feed additive for sows in accordance with Regulation (EC) No 1831/2003. The EFSA Journal, 521, 1 8. EFSA (European Food Safety Authority), 2007b. Opinion of the Scientific Committee on a request from EFSA on the introduction of a Qualified Presumption of Safety (QPS) approach for assessment of selected microorganisms referred to EFSA. The EFSA Journal, 587, 1 16. EFSA (European Food Safety Authority), 2009. Scientific Opinion of the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) on a request from the European Commission on the safety and efficacy of Bonvital (Enterococcus faecium) as feed additive for chickens for fattening. The EFSA Journal, 990, 1 12. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2010. Scientific Opinion on Bonvital (Enterococcus faecium) as a feed additive for chickens for fattening EFSA Journal, 8(6):1636. 5 pp EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2012a. Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. EFSA Journal, 10(6):2740. 10 pp. EFSA Panel on Biological Hazards (BIOHAZ), 2012b. Scientific Opinion on the maintenance of the list of QPS biological agents intentionally added to food and feed (2012 update). EFSA Journal, 10(12):3020. 84 pp. EFSA Journal 2013;11(4):3175 13

APPENDIX Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for Provita LE 35 In the current application authorisation is sought under Article 10(2) for Provita LE, consisting of two active agents: Enterococcus faecium DSM 7134 and Lactobacillus rhamnosus DSM 7133, under the category/functional group 4(b), ''zootechnical additives/gut flora stabilisers'', according to the classification system of Annex I of Regulation (EC) No 1831/2003. The feed additive will be marketed in a powdered form containing a minimum concentration of 7 x 10 9 CFU/g of Enterococcus faecium DSM 7134 and 3 x 10 9 CFU/g of Lactobacillus rhamnosus DSM 7133, with dextrose anhydrous, maltodextrin and whey powder as carriers. Specifically, authorisation is sought for the use of the feed additive for calves for rearing and it is intended to be mixed at a total concentration of the two microorganisms ranging from 1 x 10 9 to 5 x 10 9 CFU/kg of complete feedingstuffs. For the enumeration of the two microorganisms in the feed additive, premixtures and feedingstuffs the Applicant proposed two internationally recognised ring-trial validated spread plate method issued by the European Committee for Standardization (CEN): (i) for the enumeration of Enterococcus spp (EN 15788), using Bile Esculin Azide Agar and for enumeration of Lactobacillus spp (EN 15787), using MRS agar. The performance characteristics reported for CEN methods after logarithmic transformation (CFU) are: a repeatability standard deviation (s r ) ranging from 0.12 to 0.24 log10 CFU/g, a reproducibility standard deviation (s R ) ranging from 0.23 to 0.41 log10 CFU/g; and a limit of detection (LOD) of 1x10 5 CFU/kg feedingstuffs. Based on the performance characteristics presented, the EURL recommends for official control, the two CEN methods (EN 15788 and EN 15787) for the determination of Enterococcus faecium DSM 7134 and Lactobacillus rhamnosus DSM 7133 in the feed additive, premixtures and feedingstuffs. Molecular methods were used by the Applicant to identify the active agent in the feed additive. The EURL recommends instead for official control Pulsed Field Gel Electrophoresis (PFGE), a generally recognised standard methodology for microbial identification. Further testing or validation of the methods to be performed through the consortium of National Reference Laboratories as specified by article 10 (Commission Regulation (EC) No 378/2005) is not considered necessary. 35 The full report is available on the EURL website: http://irmm.jrc.ec.europa.eu/sitecollectiondocuments/finrep-fad- 2010-0305.pdf EFSA Journal 2013;11(4):3175 14