SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of InteSwine (Saccharomyces cerevisiae) as a feed additive for weaned piglets 1,2 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 3,4 European Food Safety Authority (EFSA), Parma, Italy ABSTRACT InteSwine consists of 10 10 CFU/g of a strain of Saccharomyces cerevisiae and is intended to be used as a feed additive for weaned piglets at a dose range of 6 x 10 9 to 4 x 10 10 CFU/kg feed. Saccharomyces is considered by the EFSA to be suitable for the QPS approach to safety assessment. In the view of the FEEDAP Panel, the identity of the production strain is established as Saccharomyces cerevisiae and therefore safety for the target species, consumer and the environment can be presumed. The additive was shown not to be irritant to skin and eyes. As the product InteSwine does not contain particles smaller than 100 µm, it is not expected to present an inhalation hazard for workers. Three trials performed on weaned piglets showed that growth performance was significantly improved in each of the trials and feed conversion in two studies. Since the studies were made with different additive concentrations, the FEEDAP Panel concludes that InteSwine at the highest dose tested (4 x 10 10 CFU/kg feed) has the potential to improve growth performance of weaned piglets. European Food Safety Authority, 2011 KEY WORDS Zootechnical additive, gut flora stabiliser, Saccharomyces cerevisiae, InteSwine, weaned piglets, efficacy, QPS 1 On request form the European Commission, Question No EFSA-Q-2010-00992, adopted on 11 May 2011. 2 This scientific opinion has been edited following the provisions of Article 8(6) and Article 18 of Regulation (EC) No 1831/2003. The modified sections are indicated in the text. 3 Panel members: Gabriele Aquilina, Georges Bories, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël Albert Dierick, Mikolaj Antoni Gralak, Jürgen Gropp, Ingrid Halle, Christer Hogstrand, Reinhard Kroker, Lubomir Leng, Secundino López Puente, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Giovanna Martelli, Miklós Mézes, Derek Renshaw, Maria Saarela, Kristen Sejrsen and Johannes Westendorf. Correspondence: FEEDAP@efsa.europa.eu 4 Acknowledgement: The Panel wishes to thank the members of the Working Group on Micro-organisms, including Atte von Wright, Joaquim Brufau and Guido Rychen, 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 InteSwine (Saccharomyces cerevisiae) as a feed additive for weaned piglets. EFSA Journal 2011;9(5):2173. [10 pp.] doi:10.2903/j.efsa.2011.2173. Available online: www.efsa.europa.eu/efsajournal European Food Safety Authority, 2011
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 the product InteSwine consisting of a minimum of 10 10 CFU/g of viable cells of a strain of Saccharomyces cerevisiae. The product is intended to be used as a feed additive for weaned piglets at a dose range of 6 x 10 9 to 4 x 10 10 CFU/kg feed. Saccharomyces cerevisiae is a species considered by the EFSA to be suitable for the QPS approach to safety assessment. In the view of the FEEDAP Panel, the identity of the production strain is established as S. cerevisiae and therefore safety for the target species, consumer and the environment can be presumed. The additive was shown not to be irritant to skin and eyes. As the product InteSwine does not contain particles smaller than 100 µm, it is not expected to present an inhalation hazard for workers. Three trials performed on weaned piglets showed that growth performance was significantly improved in each of the trials and feed conversion in two studies. Since the studies were made with different additive concentrations, the FEEDAP Panel concludes that InteSwine at the highest dose tested (4 x 10 10 CFU/kg feed) has the potential to improve growth performance of weaned piglets. 2
TABLE OF CONTENTY Table of contents... 3 Background... 4 Terms of reference... 4 Assessment... 6 1. Introduction... 6 2. Characterisation... 6 2.1. Characterisation of the active agent... 6 2.2. Characterisation of the product... 6 2.3. Stability and homogeneity... 6 2.4. Conditions of use... 7 2.5. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL)7 3. Safety... 7 4. Efficacy... 8 5. Post-market monitoring... 8 Conclusions... 8 Documentation provided to EFSA... 9 References... 9 Appendix... 10 3
BACKGROUND Regulation (EC) No 1831/2003 5 establishes the rules governing the European Union authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lays down that any person seeking authorisation for a feed additive or for a new use of a feed additive shall submit an application in accordance with Article 7. The European Commission received a request from the company RM Associates Ltd 6 for authorisation of the product InteSwine, Saccharomyces cerevisiae, when used as a feed additive for weaned piglets (category: zootechnical additives; 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 4(1) (authorisation of a feed additive or new use of a feed additive). EFSA received directly from the applicant the technical dossier in support of this application. 7 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 21 October 2010. The additive InteSwine is a microbiological feed additive containing cells of the yeast Saccharomyces cerevisiae NCYC R-625. This product has not been previously authorised in the European Union. 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 animal(s), consumer, user and the environment and the efficacy of the product InteSwine (Saccharomyces cerevisiae), when used under the conditions described in Table 1. 5 6 7 OJ L 268, 18.10.2003, p. 29. RM Associates Ltd on behalf of Integro Gida ve Ticaret AS; 34349 Gayrettepe, Istanbul, Turkey EFSA Dossier reference: FAD-2010-0051. 4
Table 1: Description and conditions of use of the additive as proposed by the applicant Additive Registration number/ec No/No Categoryof additive Functional group(s) of additive Saccharomyces cerevisiae Zootechnical additives Gut flora stabiliser Description Chemical Purity criteria Method of analysis Composition, description formula Saccharomyces cerevisiae N/A N/A N/A Trade name Name of the holder of authorisation InteSwine Integro Gida SAN. Ve TIC. A.S Conditions of use Species or category of animal Maximum Age Minimum content Maximum content Withdrawal period CFU/kg of complete feedingstuffs piglets 35 kg 6 x 10 9 4 x 10 10 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 None None None None Maximum Residue Limit (MRL) Species or category of Target tissue(s) or Maximum content in Marker residue animal food products tissues None None None None 5
ASSESSMENT 1. Introduction The microbial additive InteSwine is a preparation of viable cells of the yeast Saccharomyces cerevisiae. The applicant has requested an authorisation under the category of zootechnical additives (functional group: gut flora stabilisers) for use in diets for weaned piglets. This product has not been previously authorised in the European Union. 2. Characterisation 2.1. Characterisation of the active agent The active agent of InteSwine is Saccharomyces cerevisiae strain NCYC R-625. The strain has been deposited at the UK National Collection of Yeast Culture with accession number R-625. 8 It has been identified based on morphological and phenotypic properties, and PFGE fingerprinting. It has not been subject to any genetic modification. The genetic stability of the strain (five samples) between January 2009 and December 2010 has been shown with PCR amplification of sigma sequences according to Fernandez-Espinar et al. (2001). 9 2.2. Characterisation of the product 10 InteSwine is produced by batch fermentation in a growth medium based on molasses. After centrifugation the yeast cell mass is washed, concentrated and further dried until microgranules of yeast are obtained, with about 95 % dry weight. The minimum concentration of yeast in the final product is 10 10 CFU/g. Batch to batch variation was measured in ten samples of five production batches of InteSwine. 11 The mean value was 10 10 CFU/g with a CV of 18 %, indicating a low batch to batch variation. Action limits are set for microbial contaminants (mesophilic microbiota, coliforms, Escherichia coli, Staphyloccoccus aureus, Salmonella, Clostridium perfringens spores) and chemical impurities (Cd, Pb, As, Hg). 12 Analytical results from five batches confirm that observed levels are well below the action limits, and where applicable within the maximum concentrations set by Directive 2002/32/EC. Particle size distribution was measured by sieving. 13 Results indicate that the product InteSwine does not contain inhalable (<100 µm) and respirable (<10 µm) particles. 2.3. Stability and homogeneity Five batches of InteSwine were stored in their original packaging at an average temperature of 22 o C and RH 68 % for 12 months. 14 The product was stable under these conditions (average reduction in viability < 0.1 Log-values). The stability of the product was also shown at 30 o C, RH 75 % for 12 months (average reduction in viability < 0.1 Log-values). 15 Three batches of InteSwine were chosen at random and were added each to the three lots of a vitamin/mineral premixture (nine replicates in total). 16 The stability was examined over a period of six months under ambient conditions with an average temperature of 22 C. Results indicated that the 8 Technical dossier/section II 9 Technical dossier/supplementary information Mar 11/Annex 4 10 This section has been edited following the provisions of Article 8(6) and Article 18 of Regulation (EC) No 1831/2003. 11 Technical dossier/supplementary information Mar 11/Annex 1 12 Technical dossier/supplementary information Mar 11/Annex 2 13 Technical dossier/supplementary information Mar 11/Annex 3 14 Technical dossier/section II/Reference 2 15 Technical dossier/section II/Reference 3 16 Technical dossier/supplementary information Mar 11/Annexes 5_1-5_2 6
product is stable for at least six months in a piglet premixture (start 8.9 x 10 11 CFU/kg, reducing to 7.6 x 10 11 CFU/kg). The stability of the additive was examined in typical piglet feeds supplemented with InteSwine at 5 x 10 10 CFU/kg (three additive batches in three different feeds). 17 Ten subsamples of each of the three feeds were analysed for the stability over a period of three months under ambient conditions and with an average temperature of 22 C. The stability of InteSwine was also evaluated in pelleted feeds (the mash feeds used above were pelleted at 85 C). 18 Ten subsamples were analysed for the stability over a period of three months at ambient conditions and at an average temperature of 22 C. Results of these studies (table 2) show that the product is stable for three months in compound mash feed, during pelleting and during storage of pelleted feed. Table. 2 Stability of InteSwine in piglet feeds Duration of storage Day 0 Mash feed (CFU/kg) 3 months Mash feed (CFU/kg) Feed before pelleting (CFU/kg) 3 months after pelleting (CFU/kg) Average 5.2 x 10 10 5.0 x 10 10 5.1 x 10 10 4.9 x 10 10 Standard 1.3 x 10 10 1.0 x 10 10 0.9 x 10 10 0.8 x 10 10 deviation CV (%) 26 % 20 % 18 % 15 % The feeds used in the stability trial were also used to evaluate the homogenous distribution of the additive in piglet feed. 19 In 30 subsamples of mash feed (10 sub-samples of three batches), a CV of 19 % was found. In 30 subsamples of pelleted feeds (as above), the CV was of 17 %. 2.4. Conditions of use The product is intended for use in complete feed for weaned piglets at a minimum content of 6 x 10 9 and a maximum content of 4 x 10 10 CFU/kg feedstuffs. 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 Saccharomyces cerevisiae is considered by the EFSA to be suitable for the QPS approach to safety assessment (EFSA, 2010). In the view of the FEEDAP Panel, the identity of the production strain is established as S. cerevisiae and therefore safety for the target species, consumer and the environment is presumed. An acute dermal irritation study according to OECD 404 and an acute eye irritation study according to OECD 405 were conducted in New Zealand Rabbits. 20 The product caused transient erythema in the acute dermal irritation study, disappearing after 24 hours and no eye irritation. Consequently, the test sample was evaluated as non-irritant. 20 No study on skin sensitisation was provided. 17 Technical dossier/supplementary information Mar 11/Annexes 5_3-5_8 18 Technical dossier/supplementary information Mar 11/Annexes 5_9-5_11 19 Technical dossier/supplementary information Mar 11/Annexes 6_1-6_2 20 Technical dossier/supplementary information Mar 11/Annex 7 7
As the product InteSwine does not contain particles smaller than 100 µm, it is not expected to present an inhalation hazard for workers. 4. Efficacy Three trials were performed in two different Member States. The studies had an essentially similar design with weaned animals of both sexes (cross-breeds with initial weights of 7.4-8.5 kg) allocated to two groups of the same size, one serving as the control and the other receiving the same basal diet (in mash form, except for trial 2) supplemented with the additive. In each trial, doses of InteSwine were confirmed by analyses. All trials were of 42 days duration. Data were analysed as a block design using the GLM procedure (feed intake measured at the pen level, weight gain measured individually, the experimental unit was the pen). Table 3 shows further aspects of the experimental designs and the resulting zootechnical performances of the animals. Table 3. Zootechnical performances of weaned piglets supplemented with InteSwine Trial number Total number of piglets (Replicates/treatment X Animals/replicate) 1 21 216 (12 X 9) 2 22 240 (12 X 10) 3 23 156 Dose (CFU/kg feed) Final weight (kg) 0 6 x 10 9 19.1 20.2 (6 X 13) a,b For a given trial, treatment means differ significantly P< 0.05 ADG: average daily gain ADG (kg) Feed to gain 0.40 a 1.89 a 0.43 b 1.69 b 0 21.6 a 0.32 a 1.47 4.5 x 10 10 22.3 b 0.34 b 1.44 0 19.0 a 0.26 a 1.76 a 1.2 x 10 10 20.1 b 0.29 b 1.50 b Growth performance was significantly improved in each of the trials and feed conversion in two studies. Feed intake was not significantly affected. However, the studies were made with doses ranging from the lowest recommended dose to the highest. The FEEDAP Panel concludes that InteSwine has the potential to improve growth performance of piglets at a dose of 4 x 10 10 CFU/kg feed. 5. 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 24 and Good Manufacturing Practice. CONCLUSIONS The active agent has been identified as a strain of Saccharomyces cerevisiae and consequently meets the qualifications required by the QPS approach. It can thus be presumed safe for the target species, consumers and the environment. 21 Technical dossier/section IV/Annexes IV_Reference_9 and 10 22 Technical dossier/section IV/Annexes IV_Reference_11 and 12 23 Technical dossier/section IV/Annexes IV_Reference_13 and 14 24 OJ L 35, 8.2.2005, p. 1. 8
The additive is not irritant to skin and eyes. As the product InteSwine does not contain particles smaller than 100 µm, it is not expected to present an inhalation hazard for workers. InteSwine has the potential to improve growth performance of weaned piglets at a dose of 4 x 10 10 CFU/kg feed. DOCUMENTATION PROVIDED TO EFSA 1. InteSwine for piglets. June 2010. Submitted by RM Associates Ltd. 2. InteSwine for piglets. Supplementary information. March 2011. Submitted by RM Associates Ltd. 3. Evaluation report of the European Union Reference Laboratory for Feed Additives on the methods(s) of analysis for InteSwine. 4. Comments from Member States received through the ScienceNet. REFERENCES EFSA (European Food Safety Authority), 2010. EFSA Panel on Biological Hazards (BIOHAZ); Scientific Opinion on the maintenance of the list of QPS biological agents intentionally added to food and feed (2010 update. EFSA Journal 2010;8(12):1944. [56 pp.] Fernández-Espinar MT, López V, Ramón D, Bartra E and Querol A (2001). Study of the authenticity of commercial wine yeast strains by molecular techniques. International Journal of Food Microbiology 70 (2001) 1 10 9
APPENDIX Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for InteSwine In the current application authorisation is sought under article 4(1) for product InteSwine under the category 'zootechnical additives', functional group 4(b) 'gut flora stabilisers', according to Annex I of Regulation (EC) No 1831/2003. The active component of InteSwine is a pure culture of the strain Saccharomyces cerevisiae NCYC R-625, with a minimum concentration of 9 x 10 10 Colony Forming Units (CFU)/g. Specifically, the authorisation is sought for the use of inteswine for weaned piglets. The feed additive is intended to be mixed to complete feedingstuffs at a dose ranging from 6 x 10 9 to 4.5 x 10 10 CFU/kg of Saccharomyces cerevisiae NCYC R-625. For the enumeration of Saccharomyces cerevisiae NCYC R-625 in feed additive, premixtures and feedingstuffs the Applicant proposes the ring trial validated CEN pour plate method for the enumeration of yeast probiotic strains (EN 15789), using yeast extract dextrose chloramphenicol agar (CGYE). The performance characteristics of the EN 15789 method reported after logarithmic transformation (CFU) are: - a repeatability standard deviation (s r ) ranging from 0.17 to 0.36 log 10 CFU/g, - a reproducibility standard deviation (s R ) ranging from 0.55 to 0.60 log 10 CFU/g; and - a limit of detection (LOD) of 1 x 10 5 CFU/kg, well below the minimum dose proposed by the applicant (6x10 9 CFU/kg of feedingstuffs). Based on these performance characteristics the EURL recommends, for official control, the CEN method EN 15789 for the enumeration of Saccharomyces cerevisiae NCYC R-625 in feed additives, premixtures and feedingstuffs. Molecular methods were used by the Applicant for identification of the active agent. The EURL recommends for official control Polymerase Chain Reaction (PCR), a generally recognised standard methodology for identification of yeasts. 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. 10