EFSA Journal 2015;13(7):4158 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of Cylactin (Enterococcus faecium NCIMB 10415) as a feed additive for pigs for fattening, piglets and sows 1 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3 ABSTRACT European Food Safety Authority (EFSA), Parma, Italy Cylactin is the trade name for a preparation of Enterococcus faecium. It is currently authorised for use several animal species. The product is intended for use with sows at a minimum dose of 7 10 8 and a maximum dose of 1.25 10 9 colony-forming unit (CFU)/kg feed and with weaned and suckling piglets and pigs for fattening at a minimum dose of 3.5 10 8 and a maximum dose of 1.0 10 9 CFU/kg feed. The characterisation of the additive was fully described and its safety for consumers, users and the environment was assessed in previous opinions and it was found not to give rise to any concerns. The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) is unaware of any new data which would lead it to revise its conclusions. Therefore, the focus of the present opinion is on the safety and efficacy of Cylactin when used with the target animals. Results on the safety and efficacy apply to all forms of the additive when delivering the same dose. When used at 100 times the maximum recommended dose, Cylactin did not show any adverse effect on the performance or mortality of piglets and sows. Therefore, the additive is considered safe for suckling and weaned piglets, pigs for fattening and sows at the maximum proposed dose. Cylactin has the potential to be efficacious in sows and suckling piglets when supplemented to both sows and suckling piglets at 7.0 10 8 and 1 10 9 CFU/kg feed, respectively. Cylactin also has the potential to be efficacious in weaned piglets when supplemented at the minimum recommended dose of 3.5 10 8 CFU/kg feed. Insufficient data were provided to support the efficacy of Cylactin when used in pigs for fattening. European Food Safety Authority, 2015 KEY WORDS cylactin, Enterococcus faecium, pigs for fattening, piglets, sows, safety, efficacy 1 On request from the European Commission, Question No EFSA-Q-2012-00419, adopted on 17 June 2015. 2 Panel members: Gabriele Aquilina, Vasileios Bampidis, Maria De Lourdes Bastos, Lucio Guido Costa, Gerhard Flachowsky, Mikolaj Antoni Gralak, Christer Hogstrand, Lubomir Leng, Secundino López-Puente, Giovanna Martelli, Baltasar Mayo, Fernando Ramos, 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 Pier Sandro Cocconcelli, Andrew Chesson and Ingrid Halle, for the preparatory work on this scientific opinion. Suggested citation: EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2015. Scientific Opinion on the safety and efficacy of Cylactin (Enterococcus faecium NCIMB 10415) as a feed additive for pigs for fattening, piglets and sows. EFSA Journal 2015;13(7):4158, 18 pp. doi:10.2903/j.efsa.2015.4158 Available online: www.efsa.europa.eu/efsajournal European Food Safety Authority, 2015
SUMMARY Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of Cylactin for sows, piglets and pigs for fattening. Cylactin is the trade name for a preparation of a strain of Enterococcus faecium that is currently authorised for use in chickens for fattening, chickens reared for laying, minor poultry species for fattening and minor poultry species reared for laying, pigs for fattening, piglets, sows, calves, kids, cats and dogs in various formulations. The applicant is now seeking a re-evaluation of the use of the additive in feed for sows at a minimum dose of 7 10 8 and a maximum dose of 1.25 10 9 colony-forming units (CFU)/kg feed and with weaned and suckling piglets and pigs for fattening at a minimum dose of 3.5 10 8 and a maximum dose of 1.0 10 9 CFU/kg feed. The characterisation of the additive was fully described and its safety for consumers, users and the environment was assessed in previous opinions and it was found not to give rise to any concerns. The Panel is unaware of any new data which would lead it to revise its conclusions. Therefore, the focus of the present opinion is on the safety and efficacy of Cylactin when used with the target animals. Results on the safety and efficacy apply to all forms of the additive when delivering the same dose. When used at 100 times the maximum recommended dose, Cylactin did not show any adverse effect on the performance or mortality of piglets and sows. Therefore, the additive is considered safe for suckling and weaned piglets, pigs for fattening and sows at the maximum proposed dose. Cylactin has the potential to be efficacious in sows and suckling piglets when supplemented to both sows and suckling piglets at 7.0 10 8 and 1 10 9 CFU/kg feed, respectively. Cylactin also has the potential to be efficacious in weaned piglets when supplemented at the minimum recommended dose of 3.5 10 8 CFU/kg feed. Insufficient data were provided to support the efficacy of Cylactin when used in pigs for fattening. EFSA Journal 2015;13(7):4158 2
TABLE OF CONTENTS Abstract... 1 Summary... 2 Background... 4 Terms of reference... 5 Assessment... 8 1. Introduction... 8 2. Conditions of use... 8 3. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL)... 8 4. Safety for the target species... 8 4.1. Safety for suckling piglets... 8 4.2. Safety for weaned piglets... 9 4.3. Safety for sows... 9 4.4. Conclusions on the safety for the target species... 10 5. Efficacy... 10 5.1. Efficacy for sows and suckling piglets... 10 5.2. Efficacy for weaned piglets... 12 5.3. Efficacy for pigs for fattening... 14 5.3.1. Conclusions on the efficacy for the target species... 15 6. Post-market monitoring... 15 Documentation provided to EFSA... 15 References... 16 Annex Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for Cylactin... 18 EFSA Journal 2015;13(7):4158 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 DSM Nutritional Products Ltd 5 for re-evaluation of the product Cylactin (Enterococcus faecium NCIMB 10415), when used as a feed additive for pigs for fattening, piglets and sows (category: zootechnical additive; functional group: gut flora stabilisers) 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 2 May 2012. The additive Cylactin is a preparation of Enterococcus faecium NCIMB 10415. This product is authorised for use in chickens for fattening, 7 pigs for fattening, 8 piglets, 9 sows, 10 calves, kids, cats and dogs, 11 chickens reared for laying, minor poultry species for fattening and minor poultry species reared 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 DSM Nutritional Products Ltd. (Switzerland), represented by DSM Nutritional Products Sp. Z.o.o. (Poland). 6 EFSA Dossier reference: FAD-2010-0269. 7 Commission Implementing Regulation (EU) No 361/2011 of 13 April 2011 concerning the authorisation of Enterococcus faecium NCIMB 10415 as a feed additive for chickens for fattening (holder of authorisation DSM Nutritional products Ltd represented by DSM Nutritional Products Sp. z o.o) and amending Regulation (EC) No 943/2005. OJ L 100, 14.4.2011, p. 22. 8 Commission Regulation (EC) No 943/2005 of 21 June 2005 concerning the permanent authorisation of additives in feedingstuffs. OJ L 159, 22.6.2005, p. 6. 9 Commission Regulation (EC) No 252/2006 of 14 February 2006 concerning the permanent authorisations of certain additives in feedingstuffs and the provisional authorisations of new uses of certain additives already authorised in feedingstuffs. OJ L 44, 15.2.2006, p. 3. 10 Commission Regulation (EC) No 1200/2005 of 26 July 2005 concerning the permanent authorisation of certain additives in feedingstuffs and the provisional authorisation of a new use of an additive already authorised in feedingstuffs. OJ L 195, 27.7.2005, p. 6. 11 Commission Implementing Regulation (EU) No 1061/2013 of 29 October 2013 concerning the authorisation of a preparation of Enterococcus faecium NCIMB 10415 as a feed additive for calves, kids, cats and dogs and amending Regulation (EC) No 1288/2004 (holder of the authorisation DSM Nutritional Products Ltd represented by DSM Nutritional products Sp. Z o.o). OJ L 289, 30.10.2013, p. 38. EFSA Journal 2015;13(7):4158 4
for laying. 12 The same strain is also authorised under a different trade name (Oralin) for calves, 13 piglets, 14 chickens for fattening, 15 turkeys for fattening and dogs. 16 The Scientific Committee on Animal Nutrition (SCAN) issued an opinion on the safety for the target animals, consumers, users and environment for this product when used as a feed additive for chickens for fattening, piglets, pigs for fattening and calves (EC, 1997, updated 2003). EFSA has issued several opinions on this product: one on the safety of for dogs and cats (EFSA, 2004a), two on the safety and efficacy for chickens for fattening (EFSA, 2010a,b), one on the safety for dogs and cats (EFSA FEEDAP Panel, 2013a), one on the safety and efficacy for calves, lambs and kids for rearing and for fattening (EFSA FEEDAP Panel, 2013b) and one on the safety and efficacy for chickens for fattening, chickens reared for laying, minor poultry species for fattening and minor poultry species reared for laying (EFSA FEEDAP Panel, 2014a). The SCAN issued two opinions on the safety of Oralin (Enterococcus faecium NCIMB 10415/DSM 10663) for turkeys (EC, 2002) and for pigs for fattening, calves and chickens for fattening (EC, 2003). EFSA issued an opinion on the safety of Oralin for dogs (EFSA, 2004b), one opinion on the safety and efficacy of the same product for cats (EFSA FEEDAP Panel, 2014c) and one for calves for rearing, piglets, chickens for fattening, turkeys for fattening and dogs (EFSA FEEDAP Panel, 2014b). 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 Cylactin (Enterococcus faecium NCIMB 10415), when used under the conditions described in Table 1. 12 Commission Implementing Regulation (EU) 2015/518 of 26 March 2015 concerning the authorisation of the preparation of Enterococcus faecium NCIMB 10415 as a feed additive for chickens reared for laying, minor poultry species for fattening and minor poultry species reared for laying and amending Implementing Regulation (EU) No 361/2011 as regards the compatibility with coccidiostats (holder of the authorisation DSM Nutritional Products Ltd represented by DSM Nutritional products Sp. z o.o). OJ L 82, 27.3.2015, p. 75 13 Commission Regulation (EC) No 255/2005 of 15 February 2005 concerning the permanent authorisations of certain additives in feedingstuffs. OJ L 45, 16.2.2005, p. 3. 14 Commission Regulation (EC) No 1200/2005 of 26 July 2005 concerning the permanent authorisation of certain additives in feedingstuffs and the provisional authorisation of a new use of an additive already authorised in feedingstuffs. OJ L 195, 27.7.2005, p. 6. 15 Commission Regulation (EC) No 1259/2004 of 8 July 2004 concerning the permanent authorisations of certain additives in feedingstuffs. OJ L 239, 9.7.2004, p. 8. 16 Commission Regulation (EC) No 1520/2007 of 19 December 2007 concerning the permanent authorisations of certain additives in feedingstuffs. OJ L 355, 20.12.2007, p. 17. EFSA Journal 2015;13(7):4158 5
Table 1: Description and conditions of use of the additive as proposed by the applicant Additive E. faecium NCIMB 10415 Registration number/ec No/No Category(-ies) of additive E1705 Zootechnical additive Functional group(s) of additive Gut flora stabiliser Composition, description E. faecium NCIMB 10415 Microencapsulated form: Minimum 10 x10 9 CFU/g Granulated form: Minimum 35 x10 9 CFU/g Chemical formula - Description Composition, description E. faecium NCIMB 10415 Microencapsulated form: Minimum 10 x10 9 CFU/g Granulated form: Minimum 35 x10 9 CFU/g Chemical formula - Trade name Name of the holder of authorisation Cylactin, Cernivet DSM Nutritional Products Ltd Conditions of use Species or category of animal Maximum Age Species or category of animal Maximum Age CFU/kg of complete feedingstuffs Species or category of animal Sows in order to have benefits in piglets Piglets (suckling and weaned) Not relevant 0.70 x 10 9 0.35 x 10 9 1.25 x 10 9 1.0 x 10 9 none Fattening pigs 0.35 x 10 9 1.0 x 10 9 Other provisions and additional requirements for the labelling Specific conditions or restrictions for use Specific conditions or restrictions for handling Post-market monitoring E. faecium NCIMB 10415 is a class 1 biological agent. No specific labeling required. No additional requirements further to the need for traceability and recall procedures established by Regulation No 178/2002. EFSA Journal 2015;13(7):4158 6
Specific conditions for use in complementary feedingstuffs None Marker residue Maximum Residue Limit (MRL) Species or category of animal Marker residue Species or category of animal Not relevant Not relevant Not relevant Not relevant EFSA Journal 2015;13(7):4158 7
ASSESSMENT 1. Introduction Cylactin 17 is a zootechnical additive consisting of dehydrated cells of Enterococcus faecium NCIMB 10415. It is marketed in three forms Cylactin LBC ME10 and Cylactin LBC ME20 Plus (coated forms), which contain the micro-encapsulated bacterium at concentrations of 1 10 10 and 2 10 10 CFU/g, respectively, and Cylactin LBC G35, a granulated non-coated form with a guaranteed content of 3.5 10 10 CFU/g. It is presently authorised for use in feed for chickens for fattening, chickens reared for laying, minor poultry species for fattening and minor poultry species reared for laying, pigs for fattening, piglets, sows, calves, kids, cats and dogs. The applicant is now seeking re-evaluation of the additive when used with sows, piglets (suckling and weaned) and pigs for fattening. The characterisation of the additive was fully described and its safety for consumers, users and the environment was assessed in previous opinions and it was found to not give rise to any concerns (EFSA FEEDAP Panel, 2010a, 2013a). The Panel is unaware of any new data which would lead it to revise its conclusions. Therefore, the focus of the present opinion is on the safety and efficacy of Cylactin when used with the target animals. 2. Conditions of use Cylactin is intended to be used in liquid and solid diets for suckling and weaned piglets at the minimum dose of 3.5 10 8 CFU/kg complete feed and the maximum dose of 1 10 9 CFU/kg complete feed. It is also intended for use in feed for pigs for fattening at the minimum dose of 3.5 10 8 CFU/kg complete feed and the maximum of 1 10 9 CFU/kg complete feed and for use in diets for sows, to have a beneficial effect in piglets, in the range of 7.0 10 8 to 1.25 10 9 CFU/kg complete feed. The three forms of the additive Cylactin LBC ME10, Cylactin LBC ME20 and Cylactin LBC G35 are for use in either dry feed or liquid feed. Cylactin LBC ME10 and ME20 Plus can be incorporated in the premix or directly into milk replacer, while Cylactin LBC G35 is suitable only for incorporation in milk replacer. 3. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) The European Food Safety Authority (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 Appendix A. 4. Safety for the target species 4.1. Safety for suckling piglets In a tolerance study, 15 sows (EUROC line) were randomly assigned to three experimental groups with five replicates each. 18 The sows were fed ad libitum a complete feed for lactating sows without additive supplementation. After farrowing, the litter size was corrected to accommodate 10 or 11 piglets per litter (average body weight 1.75 kg/pig). Suckling piglets were fed a standard prestarter diet with no additive (control (C)) or the basal diet containing Cylactin either at the maximum recommended dose (treatment 1 (T1) = 1 10 9 CFU/kg complete feed) or at a 100 overdose (treatment 2 (T2) = 1 10 11 CFU/kg complete feed) from 8 to 28 days of age. All doses were confirmed by analysis. Animals health and performance were monitored by measuring body weight, 17 The Applicant intends to market with other names such as Cernivet. 18 Technical dossier/ Section III/Annex III-3. EFSA Journal 2015;13(7):4158 8
feed intake and prevalence of diarrhoea per pen (four-point scale with 1 denoting normal faeces and 4 denoting watery faeces with colour changes). Data were subject to an analysis of variance (ANOVA) using the litter as the experimental unit. At the end of the experimental period, none of the piglets died or showed signs of disease. The feed intake and the body weight gain of the suckling piglets of the two Cylactin treatment groups were significantly higher than the control piglets (feed intake: C = 444 g/pig, T1 = 593 g/pig, T2 = 792 g/pig, P < 0.05; body weight gain: C = 5.9 kg, T1 = 6.6 kg, T2 = 7.7 kg, P < 0.05). Faecal scores were not significantly influenced by treatment. 4.2. Safety for weaned piglets A tolerance trial was performed with a total of 168 piglets (Landrace, four weeks of age, 8 kg body weight) randomly distributed into 12 blocks. 19 Eight of the blocks had three pens with five animals each, and four of the blocks had three pens with four animals each. Weaned piglets were fed ad libitum a pelleted diet with no additive (control) or the basal diet containing Cylactin either at the maximum recommended dose (T1 = 1.4 10 9 CFU/kg complete feed) or at a 100 overdose (T2 = 1.4 10 11 CFU/kg complete feed). All doses were confirmed by analysis. The trial lasted 42 days. Animals health and performance were monitored by measuring body weight and feed intake. Weight gain and feed efficiency were calculated. The performance parameters measured were compared among treatments using ANOVA. The pen was used as the experimental unit. Mortality was low (average 2.5 %) and was not treatment related, and no other health problems were reported. Zootechnical parameters were not significantly affected by treatment (final body weight: C = 23.2 kg, T1 = 22.6 kg, T2 = 23.8 kg; daily weight gain: C = 366 g, T1 = 352 g, T2 = 380 g; feed to gain ratio: C = 1.67, T1 = 1.70, T2 = 1.56). 4.3. Safety for sows A tolerance trial was conducted with 41 sows (Landrace, with an initial weight of 233 kg) from four consecutive batches and distributed in three treatment groups: 13 sows in the control group, 13 sows in treatment 1 and 15 sows in treatment 2. 20 The trial started after insemination and continued throughout gestation and from farrowing until the day of weaning of the offspring (at around 26 days post farrowing). After insemination, within each batch, the animals were divided into groups of three sows that were as similar as possible (considering age, parity number and body weight). The three sows in each group were randomly assigned to one of the three experimental treatments: control (no supplementation), treatment 1 (basal diet supplemented with Cylactin at 1.3 10 9 CFU/kg complete feed, 1 the maximum recommended dose) and treatment 2 (basal diet supplemented with a 100 overdose, 1.4 10 11 CFU/kg complete feed). All doses were confirmed by analysis. Fostering of piglets between sows, in order to homogenise litters, was performed within the first three days and between sows of the same treatment. During gestation, feed was restricted (adjusted to body condition of the sows) and provided manually twice a day. During lactation, feed was offered ad libitum and provided manually twice a day. Feed was presented in pelleted form. The trial lasted 151 days overall. The parameters monitored over the experimental period were body weight of sows, feed intake, number of piglets born, weight of piglets (at birth and at weekly intervals until weaning) and the interval from weaning to the conception of the sows. Data were compared among treatments using the general linear model (GLM) procedure considering the sow as the experimental unit. For statistical analysis, a randomised block design was used. The average measurements of performance for each sow were used to calculate the mean values for each treatment, and they were compared taking into account the effects of parity (classified in three categories: second parity, third to sixth parity and seventh or eighth parity) and experimental batch (four consecutive batches). 19 Technical dossier/ Section III/Annex III_4. 20 Technical dossier/section III/Annex III_2. EFSA Journal 2015;13(7):4158 9
No sows died during the course of the trial. No statistically significant differences were observed in any of the parameters measured during gestation or lactation (the number of piglets born alive was, on average, 9.3 and the number of piglets weaned was, on average, 8.7). However, numerically, the sows on the 100 overdose treatment presented the lowest weight loss during lactation (C = 538 g/day, T1 = 489 g/day, T2 = 365 g/day) and the highest initial litter weights (C = 14.6 kg, T1 = 15.04, T2 = 15.44 kg) and piglet weights at weaning (C = 7.17 kg, T1 = 7.01, T2 = 7.54 kg). 4.4. Conclusions on the safety for the target species When used at 100 times the maximum recommended dose, Cylactin did not show any adverse effect on the performance or mortality of suckling and weaned piglets and sows. Therefore, the additive is considered safe for suckling and weaned piglets, pigs for fattening and sows at the maximum proposed dose. 5. Efficacy 5.1. Efficacy for sows and suckling piglets A total of eight studies conducted in four Member States were submitted to determine the benefits for piglets when Cylactin is added to feed for sows and suckling piglets. However, one 21 was disregarded owing to insufficient reporting, which did not allow a proper assessment to be done, and a second 22 was disregarded because of a significant difference in the mean number of piglets (total born and born alive) from the control (total born, 13.4; born alive, 13.1) and treated sows (total born, 10.3; born alive, 10.2), which confounded the results on mortality and feed to gain ratio. A third study 23 was also not further considered because of high counts of E. faecium in the control feed (control = 2 10 7 vs. Cylactin group = 4.3 10 8 CFU/kg feed) and a fourth study 24 was not further considered owing to a high mortality of piglets during the lactation period (control = 23 % vs. Cylactin group = 17 %). In the first of the remaining four studies, two consecutive batches of 26 sows (breed not specified) were divided into two groups (13 sows per treatment and per batch) and fed a lactation diet with or without Cylactin (control and treatment with the additive at 0.7 10 9 CFU/kg feed), starting about two weeks before farrowing and continuing during the 21 days of lactation, covering 38 days overall. 25 Their 518 suckling piglets were divided into four experimental groups in a 2 2 factorial design as follows: sow control + control piglets (no Cylactin ) sow control + piglets with Cylactin at 1 10 9 CFU/kg feed sow with Cylactin at 0.7 10 9 CFU/kg feed + piglets control sow with Cylactin at 0.7 10 9 CFU/kg feed + piglets with Cylactin at 1 10 9 CFU/kg feed. Piglets received a creep diet over 21 days and a starter feed from weaning until 42 days of age with or without the additive. Analyses of the feed showed values of 2.7 10 9 CFU/kg feed in the case of sows and 7.7 10 9 CFU/kg feed in the case of piglets. The parameters measured per sow were weight of piglets at 48 hours and at weaning (21 days), daily feed intake, piglets weekly intake and mortality of piglets. The parameters measured in weaned piglets were initial pen body weight and, on a weekly basis, pen weekly feed intake, mortality and morbidity (Table 2). Data were analysed using ANOVA. From all parameters tested, the mortality rate of piglets was significantly reduced when both sows were treated with Cylactin at the minimum recommended dose and piglets at the maximum 21 Technical dossier/annex IV/Section IV.6. 22 Technical dossier/section IV/Annex IV.9. 23 Technical dossier/section IV/Annex IV.12. 24 Technical dossier/section IV/Annex IV.13. 25 Technical dossier/section IV/Annex IV.8. EFSA Journal 2015;13(7):4158 10
recommended dose (Table 2). No other significant effects were observed in the other parameters measured. The second study included 49 sows (PIC) divided in two groups and fed a lactation diet over 38 days (from 10 days before farrowing until the end of lactation) without Cylactin (control) and with the additive at 0.7 10 9 CFU/kg feed. 26 Cross-fostering was done in the first days within the treatment group. Their offspring (546 suckling piglets, 273 in each treatment) were distributed in two treatment groups and fed a pelleted diet over 28 days with or without Cylactin at 1 10 9 CFU/kg feed. All doses were confirmed by analysis. The parameters measured per sow were feed intake, number of piglets born alive and dead, weight of piglets at birth and at weaning, and mortality of piglets. The parameters measured in the piglets were initial body weight and, on a weekly basis, daily feed intake, mortality and morbidity (Table 2). Data were analysed using ANOVA. Piglets weight at weaning was significantly improved when Cylactin was supplemented simultaneously in lactating sows feed at the minimum recommended dose and in suckling piglets feed at the maximum recommended dose (Table 2). No other parameter was influenced by treatment. The third experiment involved 36 sows (Finnish Dutch Landrace from three consecutive batches) divided in two groups (of three pens with six sows each) according to parity and weight and fed a lactation diet over 36 days (from two weeks before parturition until weaning). 27 The control group received a basal diet and the Cylactin group received the same diet supplemented with the additive at 0.7 10 9 CFU/kg feed. Litters were equalised within treatment groups (approximately 10 piglets/litter) one day after parturition. A total of 360 suckling piglets were maintained in the trial (18 replicates/litters per group) and fed creep feed either with or without Cylactin at 1 10 9 CFU/kg feed from approximately six days of age until day 18, and at 0.7 10 9 CFU/kg feed from day 19 to the end of the trial (four weeks after weaning). All doses were confirmed by analysis. The parameters measured for the sows were individual body weight and back fat thickness after parturition and after weaning, and feed intake (on a daily basis during the 36 days). Those parameters measured for the litter were the number of piglets born alive and dead, individual body weight at birth, litter weight during the last three weeks of the suckling period, individual body weight at weaning and at the end of the experiment, and litter weights one and two weeks after weaning and at the end of experiment (Table 2). Morbidity and mortality were also monitored over the whole experimental period. Piglets faecal score was monitored (visually) on a weekly basis using a four-point scale with 0 denoting normal faeces and 3 denoting liquid faeces. Data were analysed as a randomised complete block design with the litter as the experimental unit and period and parity class as block. Data were subject to an ANOVA. Mortality during lactation was approximately 8 % and not influenced by treatment. Supplementation of Cylactin significantly improved sows feed intake during lactation, but no significant effects were seen in piglets (Table 2). The fourth trial involved 60 sows (starting in late gestation) randomly distributed in two groups and fed a lactation diet over 42 days (period between day 90 of gestation until weaning) without the additive (control) or containing Cylactin at 0.7 10 9 CFU/kg feed. Their 626 (312/314) suckling piglets were also fed a pelleted feed without the additive or with Cylactin at 1 10 9 CFU/kg until weaning (at 28 days) and then 7 10 8 CFU/kg feed until the end of the trial (at 56 days of age). 28 Health status, live weight and feed intake were monitored in sows before and after farrowing. The number of born piglets, overall losses until weaning, live weight of piglets at birth and at weaning, and as their growth rate, feed intake and feed conversion ratio during the four weeks after weaning were monitored (Table 2). All data were analysed with ANOVA. 26 Technical dossier/section IV/Annex IV.10. 27 Technical dossier/section IV/Annex IV.11. 28 Technical dossier/section IV/Annex IV.14. EFSA Journal 2015;13(7):4158 11
Supplementation of Cylactin to sows at the minimum recommended dose and to suckling piglets at the maximum recommended dose significantly improved body weight of piglets at weaning (Table 2). Table 2: Overview of the results of efficacy studies with Cylactin in sows and their offspring Trial No Cylactin (CFU/kg feed) (1) Number of sows (replicates) Piglets at birth (n) Piglet mortality at weaning (%) Piglet weight at weaning (kg) Total feed intake of sows during lactation (kg) 1 0/0 13 n.a. 4.0 b 6.7 n.a. 0/1 10 9 13 3.1 b 6.8 0.7 10 9 /0 13 1.8 ab 6.8 0.7 10 9 /1 10 9 13 0.8 a 6.9 2 0/0 25 11.6 6.3 6.9 a n.a. 0.7 10 9 /1 10 9 24 12.4 5.3 7.7 b 3 0/0 18 11.7 9.0 8.2 135 a 0.7 10 9 /1 0.7 10 9 18 11.7 7.1 8.8 145 b 4 0/0 30 13.0 6.1 6.9 a 156.1 0.7 10 9 /1 0.7 10 9 30 13.0 4.4 7.3 b 157.2 n.a.: not available. a,b: Means in a column within a given trial with different superscript letters are significantly different (P < 0.05). (1): Dose applied to diets for sows/offspring of each group. 5.2. Efficacy for weaned piglets Eight trials were performed in three Member States to demonstrate the efficacy of Cylactin in improving the performance of weaned piglets. However, two 29 were disregarded owing to insufficient reporting and two 30 were disregarded because they had durations that were too short. In all the remaining cases, weaned piglets were distributed in two or more dietary treatments, one without any supplementation and one or more with the additive supplemented at different doses, all confirmed by analysis. The first study involved 84 weaned piglets ((Pietrain (Duroc Landrace) of both sexes, 25 days of age) distributed according to sex, weight and litter origin in two groups of 14 replicates each (Table 3). 31 The diets of the two groups were based on wheat/barley/maize/soybean meal and differed by the addition of Cylactin at 3.5 10 8 CFU/kg feed. Body weight gain, feed intake and feed to gain ratio were measured for each pen at weekly intervals and until the end of the trial at day 42. In addition, health status of the piglets and faecal consistency were monitored on a daily basis (four-point scale with 1 denoting normal faeces and 4 denoting watery faeces with colour changes). Data were subjected to an ANOVA. After checking the homogeneity of the variances, means were compared by Tukey test. No animals died during the experiment. Weight gain of piglets in the Cylactin group was significantly increased compared with that of control animals. A significantly improved feed to gain ratio of animals in the Cylactin group compared with that of animals in the control group was also observed (Table 3). The second study involved 240 weaned piglets (Pietrain (Duroc Large White), 50 % females and 50 % males, 35 days of age) distributed in two groups according to sex, weight and litter origin (12 replicates each): a control group (basal diet based on wheat/soybean meal/barley) and a treated group with Cylactin at 3.5 10 8 CFU/kg feed. Body weight gain, feed intake and feed to gain ratio were measured for each pen at biweekly intervals until the end of the trial on day 56. 32 The data obtained in 29 Technical dossier/section IV/Annexes IV.6 and IV.16. 30 Technical dossier/section IV/Annexes IV.9 and IV.15. 31 Technical dossier/section IV and Supplementary information June 2014/Annexes IV.17 and 44. 32 Technical dossier/supplementary information June 2014/Annex 42. EFSA Journal 2015;13(7):4158 12
the experiment were subjected to one-way or two-way ANOVA using the pen as the experimental unit and the differences between mean values were estimated using the Duncan test. No mortality occurred during the trial. Cylactin -treated animals had a significantly higher final weight and daily weight gain and a lower feed to gain ratio than animals from the control group (Table 3). In the third study, 220 weaned piglets (Pietrain Topigs, both sexes, 28 days old) were randomly distributed in two dietary groups: control (with no supplementation) and Cylactin (with diet supplemented at 3.5 10 8 CFU/kg feed). 33 Each treatment group was replicated 11 times. Feed was in pelleted form and based on wheat/barley/soybean meal. Piglets were weighed and feed intake was recorded at the start of the study, on day 21 and at the end of the experiment on day 42. Mortality was also monitored, and weight gain and feed to gain ratio were calculated. The data were analysed by ANOVA using the pen as the experimental unit. Daily weight gain of animals in the Cylactin group was significantly higher than that of the control group (Table 3). The other parameters were not affected by the treatment. Mortality was low (1.8 %) and not treatment related. The last study was performed with 480 weaned piglets (Porkuss PIC hybrids, both sexes, approximately 26 days old) separated into two treatment groups: a control, which received a basal diet, and the Cylactin group, which received the same basal diet supplemented with the additive at 3.5 10 8 CFU/kg feed. 34 Each treatment consisted of 20 replicates per pen with 12 animals per replicate. Diets were in mash form and based on wheat/barley/soybean meal. The duration of the study was 42 days. The parameters measured were individual body weight (on days 0, 14 and 42), feed intake and daily body weight gain, and feed conversion ratio. Data were statistically analysed using the GLM procedure followed by a Bonferroni test. Feed to gain ratio of the Cylactin -treated animals was significantly lower than that of the control. The other parameters were not affected by the treatment (Table 3). Mortality was low (0.8 %) and not treatment related. Table 3: Overview of the results of efficacy studies with Cylactin in weaned piglets Trial No (duration, days) Total animals (replicates/ treatment animals/ replicate) Cylactin (CFU/kg) Initial weight (kg) Final weight (kg) Weight gain (g/day) Feed intake (g/day) Feed to gain ratio 1 (42) 84 (14 3) 0 5.9 19.6 a 327 a 539 1.64 a 3.5 10 8 5.9 21.2 b 363 b 548 1.51 b 2 (56) 240 (12 10) 0 7.5 29.7 a 398 a 860 2.16 a 3.5 10 8 7.4 31.1 b 425 b 877 2.05 b 3 (42) 220 (11 10) 0 8.5 29.0 476 a 707 1.52 3.5 10 8 8.5 30.0 500 b 732 1.49 4 (42) 480 (20 12) 0 8.1 27.9 470 753 1.61 a 3.5 10 8 8.1 27.9 471 736 1.57 b a,b: Means in a column within a given trial with different superscript letters are significantly different (P < 0.05). 33 Technical dossier/supplementary information June 2014/Annex 43. 34 Technical dossier/supplementary information June 2014/Annex 45. EFSA Journal 2015;13(7):4158 13
5.3. Efficacy for pigs for fattening Cylactin for porcine species Six studies were provided to demonstrate the efficacy of Cylactin in pigs for fattening, one in the form of a publication, four trials performed in the European Union (EU) and one performed outside the EU. However, three 35 of these were disregarded owing to insufficient reporting; in one case, the data did not specifically address the fattening period and another had an unacceptable experimental design (lack of replication). A fourth study 36 was discarded because counts of the Cylactin active agent in treated feeds were substantially lower than expected (measured levels: 6.8 10 6 CFU/kg in the starter feed and 3.6 10 6 CFU/kg and 2.4 10 6 CFU/kg in the finisher feeds) and not different from the control (measured levels: 4 10 6 CFU/kg in the grower feed and < 2.5 10 5 CFU/kg in the finisher feeds). The first acceptable experiment involved 120 cross-bred pigs (from two batches, 25 kg, both sexes) distributed to blocks according to weight and sex. 37 Within each of the two blocks, 15 pens of one pig each were randomly assigned to one of the four dietary treatment groups: control (with no supplementation), Cylactin at 3.5 10 8 CFU/kg feed, Cylactin at 1.0 10 9 CFU/kg feed and Cylactin at 1.0 10 10 CFU/kg feed. All doses were confirmed by analysis. Pigs were fed ad libitum diets prepared according to the growing phase. The trial lasted until the pigs reached the slaughter weight (approximately 112 kg) over 121 126 days. The parameters monitored were individual body weight (on days 0, 28 and 63 and at slaughter) and individual feed intake on the same days and on the day of the delivery to the slaughterhouse. These data were used to calculate weight gain and feed to gain ratio. Morbidity (incidence of diarrhoea based on visual assessment of faecal consistency on a three-point scale: normal, mild diarrhoea and watery diarrhoea) and mortality were recorded. Data were analysed using regression analysis. The chi-squared test was used to compare the data on mortality and the number of veterinary treatments between groups. The supplementation of Cylactin at 1.0 10 9 CFU/kg significantly improved the feed to gain ratio compared with all other groups (Table 4). No other parameter, including mortality, was significantly influenced by treatment. The second trial aimed at testing the effect of Cylactin from birth to slaughter. Twenty-four sows were distributed between control and treatment groups, the latter receiving diets supplemented with Cylactin at 3.5 10 8 CFU/kg feed. 38 Similarly, piglets resulting from the different litters were maintained in the same treatment groups. The 216 piglets (112 in the control group and 104 in the treated group) were allocated to 14 pens per treatment. Initial weights were not significantly different between treatments (control 21.3 kg vs. Cylactin 22.8 kg). The duration of the study was in the range 84 and 112 days (the animals were slaughtered when they reached the weight of 105 110 kg). The following parameters were checked and calculated: live weight, feed intake, average daily gain and feed to gain ratio. An ANOVA was applied to the data using the pen as the experimental unit. Feed to gain ratio was significantly improved in Cylactin -treated animals, while final body weight, daily gain and feed intake were not affected by the treatment (Table 4). However, as animals were treated with the additive from birth until the end of the fattening period, it cannot be excluded that the beneficial effect observed is due only to supplementation during the fattening period. 35 Technical dossier/section IV/Annexes IV.19, 18 and 20. 36 Technical dossier/supplementary information February 2015/Annexes 3 4. 37 Technical dossier/section IV/Annex IV.21. 38 Technical dossier/section IV and Supplementary information February 2015/Annexes IV.9 and 2. EFSA Journal 2015;13(7):4158 14
Table 4: Results of the efficacy study with Cylactin in pigs for fattening Trial Total animals (replicates/ treatment animals/ replicate) Cylactin (CFU/kg) Final weight (kg) Average daily gain (g/day) Feed intake (kg/day) Feed:gain Mortality and removal (%) 1 120 (30 1) 0 111.6 861 2.19 2.55 a 6.7 3.5 10 8 112.2 867 2.21 2.55 a 3.3 1.0 10 9 113.4 880 2.18 2.48 b 10 1.0 10 10 111.3 859 2.20 2.57 a 0 2 216 (14 7/8) 0 107.3 858 2.28 2.69 a 0 3.5 108 108.8 882 2.27 2.60 b 1 a,b: Means in a column within a given trial with different superscript letters are significantly different (P < 0.05). 5.3.1. Conclusions on the efficacy for the target species The FEEDAP Panel concludes that (i) Cylactin has the potential to improve one or more performance parameters of sows and of suckling piglets when supplemented to both sows at 7.0 10 8 CFU/kg feed and suckling piglets at 1 10 9 CFU/kg feed, (ii) Cylactin also has the potential to be efficacious in weaned piglets when supplemented at the minimum recommended dose of 3.5 10 8 CFU/kg feed and (iii) insufficient data were provided to support the efficacy of Cylactin when used in pigs for fattening. 6. Post-market monitoring 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 39 and Good Manufacturing Practice. CONCLUSIONS Results on the safety and efficacy apply to all forms of the additive when delivering the same dose. When used at 100 times the maximum recommended dose, Cylactin did not show any adverse effect on the performance or mortality of piglets and sows. Therefore, the additive is considered safe for suckling and weaned piglets, pigs for fattening and sows at the maximum proposed dose. Cylactin has the potential to be efficacious in sows and suckling piglets when supplemented to both sows and suckling piglets at 7.0 10 8 CFU/kg feed and 1 10 9 CFU/kg feed, respectively. Cylactin also has the potential to be efficacious in weaned piglets when supplemented at the minimum recommended dose of 3.5 10 8 CFU/kg feed. Insufficient data were provided to support the efficacy of Cylactin when used in pigs for fattening. DOCUMENTATION PROVIDED TO EFSA 1. E. faecium NCIMB 10415 as a gut flora stabiliser, zootechnical additive for sows in order to have benefits in piglets, piglets (suckling and weaned) and pigs for fattening. November 2010. Submitted by DSM Nutritional Products Ltd. 2. E. faecium NCIMB 10415 as a gut flora stabiliser, zootechnical additive for sows in order to have benefits in piglets, piglets (suckling and weaned) and pigs for fattening. Supplementary information. July 2012. Submitted by DSM Nutritional Products Ltd. 39 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 2015;13(7):4158 15
3. E. faecium NCIMB 10415 as a gut flora stabiliser, zootechnical additive for sows in order to have benefits in piglets, piglets (suckling and weaned) and pigs for fattening. Supplementary information. June 2014. Submitted by DSM Nutritional Products Ltd. 4. E. faecium NCIMB 10415 as a gut flora stabiliser, zootechnical additive for sows in order to have benefits in piglets, piglets (suckling and weaned) and pigs for fattening. Supplementary information. February 2015. Submitted by DSM Nutritional Products Ltd. 5. Evaluation report of the European Union Reference Laboratory for Feed Additives on the Methods(s) of Analysis for Cylactin. 6. Comments from Member States received through the ScienceNet. REFERENCES EC (European Commission), 1997, updated 2003. Opinion on the use of certain micro-organisms as additives in feedingstuffs. Available online: http://ec.europa.eu/food/fs/sc/scan/out93_en.pdf EC (European Commission), 2002, online. Opinion of the Scientific Committee on Animal Nutrition on the safety of use of product Oralin in turkeys. Available online: http://ec.europa.eu/ food/fs/sc/scan/out92_en.pdf EFSA (European Food Safety Authority), 2004a. 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 Cylactin for dogs and cats. The EFSA Journal 2004, 138, 1 7. EFSA (European Food Safety Authority), 2004b. Scientific Opinion of the Panel on Additives and Products or Substances used in Animal feed on the safety of product Oralin for dogs. The EFSA Journal 2004, 51, 1 6. EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2014a. Scientific Opinion on the safety and efficacy of Cylactin (Enterococcus faecium NCIMB 10415) as a feed additive for chickens for fattening, chickens reared for laying, minor poultry species for fattening and minor poultry species reared for laying. EFSA Journal 2014;12(11):3906, 11 pp. doi:10.2903/j.efsa.2014.3906 EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2014b. Scientific Opinion on the safety and efficacy of Oralin (Enterococcus faecium) as a feed additive for calves for rearing, piglets, chickens for fattening, turkeys for fattening and dogs. EFSA Journal 2014;12(6):3727, 19 pp. doi:10.2903/j.efsa.2014.3727 EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2014c. Scientific Opinion on the safety and efficacy of Oralin (Enterococcus faecium) when used as a feed additive for cats. EFSA Journal 2014;12(3):3602, 13 pp. doi:10.2903/j.efsa.2014.3602 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2010a. Scientific Opinion on the safety and efficacy of Cylactin (Enterococcus faecium) as a feed additive for chickens for fattening on request from the European Commission. EFSA Journal 2010;8(7):1661, 13 pp. doi:10.2903/j.efsa.2010.1661 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2010b. Statement on the assessment of the safety of Cylactin (Enterococcus faecium) used in animal nutrition. EFSA Journal 2010;8(12):1955, 14 pp. doi:10.2903/j.efsa.2010.1955 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2013a. Scientific Opinion on the safety and efficacy of Cylactin (Enterococcus faecium) as a feed additive for cats and dogs. EFSA Journal 2013;11(2):3098, 15 pp. doi:10.2903/j.efsa.2013.3098 EFSA Journal 2015;13(7):4158 16
EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2013b. Scientific Opinion on Cylactin (Enterococcus faecium) for calves, lambs and kids for rearing and for fattening. EFSA Journal 2013;11(2):3097, 14 pp. doi:10.2903/j.efsa.2013.3097 EFSA Journal 2015;13(7):4158 17
Annex Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for Cylactin 1 In the current application authorisation is sought under Article 10(2) for Enterococcus faecium NCIMB 10415, 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 as micro-encapsulated or granulated form with concentrations of Enterococcus faecium NCIMB 10415: ranging from 5 10 9 to 3.5 x 10 10 CFU/g. Specifically, authorisation is sought for the use of the feed additive for pigs for fattening, 2 piglets (suckling and weaned), sows, cats and dogs. 3 The feed additive is intended to be used either via premixtures or incorporated directly into the feedingstuffs. The Applicant proposed the following content of Enterococcus faecium NCIMB 10415 in complete feedingstuffs: - for dogs and cats a minimum content of (4.5 and 5) 10 6 CFU/kg; - for pigs and piglets a content range of (3.5 10) 10 8 CFU/kg; and - for sows a content range of (0.7 1.25) 10 9 CFU/kg. For the enumeration of Enterococcus faecium NCIMB 10415 in the feed additive, premixtures and feedingstuffs the Applicant proposed two single laboratory validated pour plate/spread plate methods. The EURL identified instead the internationally recognised ring-trial validated spread plate CEN method (EN 15788), using Bile Esculin Azide Agar, for the enumeration of Enterococcus spp. The performance characteristics of the CEN method reported after logarithmic transformation (CFU) are: a repeatability standard deviation (s r ) ranging from 0.12 to 0.2 log 10 CFU/g, a reproducibility standard deviation (s R ) ranging from 0.23 to 0.41 log 10 CFU/g; and a limit of detection (LOD) of 1 x 10 5 CFU/kg feedingstuffs. Based on the performance characteristics presented, the EURL recommends for official control, the CEN method (EN 15788) for the enumeration of Enterococcus faecium NCIMB 10415 in feed additive 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. 1 The full report is available on the EURL website: http://irmm.jrc.ec.europa.eu/sitecollectiondocuments/finrep-fad- 2010-0269+0294.pdf 2 FAD-2010-0269. 3 FAD-2010-0294. EFSA Journal 2015;13(7):4158 18