The EFSA Journal (2006) 385, 1-9

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1 The EFSA Journal (2006) 385, 1-9 Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on the safety and efficacy of the product Levucell SC20/Levucell SC10ME, a preparation of Saccharomyces cerevisiae, as a feed additive for leisure horses (Question N o EFSA-Q ) Adopted on 12 September 2006 SUMMARY The microbial feed additive Levucell SC20/Levucell SC10ME, a product consisting of dried cells of Saccharomyces cerevisiae, is authorised without time limit for its use in dairy cows and cattle for fattening. The applicant is now seeking authorisation of the product to be used as a feed additive for leisure horses. The Scientific Committee on Animal Nutrition issued an opinion on the safety of this product for beef and dairy cattle, including the safety for the consumer, the user and the environment. In one efficacy study, the differences in weight gain, despite showing significant effects (P < 0.05) in favour of the Levucell-treated group, are not convincing because of the low weight gain of the control and the housing-treatment interaction. Although the difference in body weight gain during the experiment may reflect better fibre digestion, this was not directly demonstrated. Consequently, the FEEDAP Panel, in the absence of evidence of a similar mode of action, cannot conclude on the efficacy of the product in leisure horses. Thirty times the recommended dose of Levucell SC20 in the 30 day tolerance experiment did not produce any adverse effects in adult horses. Microbiological analysis of the faecal microbiota of horses given Levucell SC20 did not show any significant differences compared to control animals other than the expected increase in yeast numbers. Consequently, the FEEDAP Panel considers Levucell SC in either formulation (SC20 and SC10ME) safe for horses. The FEEDAP Panel also considers it unlikely that this extension of use would introduce hazards for the consumers, users or for the wider environment that have not already been considered. Key words: Saccharomyces cerevisiae, leisure horses, safety, efficacy

2 Opinion on Levucell SC for leisure horses 2/9 BACKGROUND Regulation (EC) No 1831/ establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lies down that any person seeking an authorisation for a feed additive or for a new use of a feed additive shall submit an application in accordance with Article 7. The European Commission received a request from the company Lallemand SAS 2 for authorisation of the product Levucell SC20/Levucell SC10ME to be used as a feed additive for horses (category: zootechnical additives) under the conditions mentioned in Table 1. The application, initially made for horses, was subsequently restricted to leisure horses with the acceptance of the European Commission. According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the application to the European Food Safety Authority (EFSA) as an application under Article 4.1 (authorisation of a feed additive or new use of a feed additive). EFSA received directly from the applicant the technical dossier in support of this application. According to Article 8 of that Regulation, EFSA, after verifying the particulars and documents submitted by the applicant, shall undertake an assessment in order to determine whether the feed additive complies with the conditions laid down in Article 5. The particulars and documents in support of the application were considered valid by EFSA as of 7 of December of The additive Levucell SC20/Levucell SC10ME is a preparation of Saccharomyces cerevisiae (CNCM I-1077). This product has been authorised without a time limit for use in dairy cows and cattle for fattening (E 1711). The Scientific Committee on Animal Nutrition (SCAN) issued an opinion (26 September 1997, updated 25 April 2003) on the safety of this product for beef and dairy cattle, including the safety for the user, the consumer and the environment. 3 The FEEDAP Panel of EFSA has issued an opinion (15 June 2006) on the safety and efficacy of this product for dairy goats and dairy ewes. 4 TERMS OF REFERENCE According to Article 8 of Regulation (EC) No 1831/2003 EFSA shall determine whether the feed additive complies with the conditions laid down in Article 5. Therefore, EFSA shall deliver an opinion on the efficacy and the safety for the target animals, user and consumer and the environment of the product Levucell SC20/Levucell SC10ME, which is a preparation of Saccharomyces cerevisiae (CNCM I-1077) when used under the conditions described in Table 1. 1 OJ L 268, , p Lallemand SAS, 19, rue des briquetiers, Blagnac, France

3 Opinion on Levucell SC for leisure horses 3/9 Table 1. Register entry as proposed by the applicant. Additive Saccharomyces cerevisiae CNCM I-1077 Registration number/ec No/No Category of additive 7 Zootechnical additives Functional group of additive Other zootechnical additives Composition, description Non coated form : 80% revivable dried yeast cells 14 % non revivable yeast cells 5% sorbitane monostearte (E491) 5% residual water Coated form: 50% non coated form 50% fatty acids Chemical formula - Description Purity criteria Se < 1 ppm As < 5 ppm Cd < 1 ppm Hg < 0,1 ppm Pb < 5 ppm Aflatoxins < 1 µg kg -1 Zearalenone < 20 µg kg -1 Ochratoxine < 10 µg kg -1 Coliforms < 10 g -1 Pathogenic Staph. < 100 g -1 SRA < 10 g -1 C. perfringens < 10 g -1 Salmonella 0 Method of analysis Active dry yeast cell count: Sabouraud medium + chloramphenicol Biochemical stability: API 20 C gallery Genetic purity: PCR method Trade name Name of the holder of authorisation LEVUCELL SC20 / LEVUCELL SC10ME LALLEMAND SAS Conditions of use Species or category of animal Maximum Age Minimum content Maximum content CFU kg -1 of complete feedingstuffs Withdrawal period Leisure horses x x 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 Use the coated form for any inclusion through a pelleted feed If the product is handled or mixed in confined atmosphere, it is recommended to use safety glasses and masks for mixing if the mixers are not equipped with exhaust systems Not applicable Do not overpass 50ºC with Levucell SC20 and 80ºC with Levucell SC10ME Maximum Residue Limit (MRL) Marker residue Species or category of Target tissue(s) or Maximum content

4 Opinion on Levucell SC for leisure horses 4/9 animal food products in tissues Not applicable - - -

5 Opinion on Levucell SC for leisure horses 5/9 ASSESSMENT 1. Introduction The microbial feed additive Levucell SC20/Levucell SC10ME, a product consisting of dried cells of Saccharomyces cerevisiae CNCM I-1077, is authorised for its use in dairy cows and cattle for fattening. The current dossier contains data supporting a request for an extension of use of the additive Levucell SC20/Levucell SC10ME to be used as a feed additive for leisure horses. The Scientific Committee on Animal Nutrition (SCAN) issued an opinion (26 September 1997, updated 25 April 2003) on the safety of this product for beef and dairy cattle, including the safety for the user, the consumer and the environment. 5 The FEEDAP Panel is not aware of any new or additional information that would lead it to revise this opinion. Given the ubiquitous nature of the organism, its long history of safe use as human food or in food production, the FEEDAP Panel is of the opinion that the safety of this organism (and its growth medium) for the consumer, the user and the environment is established and that no risk assessment in these respect is necessary. Consequently, the FEEDAP Panel has considered only the safety and the efficacy of the additive Levucell SC20/Levucell SC10ME for the target species. 2. Characterisation of the product The active ingredient of the additive Levucell SC20/Levucell SC10ME is the yeast Saccharomyces cerevisiae CNCM I The additive Levucell SC20 is composed of approximately 80% revivable Saccharomyces cerevisiae I-1077 dried yeast cells and approximately 14% non-viable Saccharomyces cerevisiae I-1077 yeast cells which lost their viability during the drying process. This feed additive is available under two forms: - Levucell SC20, with a guaranteed minimal concentration of viable yeast cells in the additive of 20 x 10 9 CFU g -1 - Levucell SC10ME, coated formula, with a guaranteed minimal concentration of viable yeast cells in the additive of 10 x 10 9 CFU g -1 The yeast strain Saccharomyces cerevisiae CNCM I-1077 was deposited at Collection Nationale de Cultures De Microorganismes (CNCM) of Institut Pasteur, Paris, France. The biochemical and physiological characteristics of this strain have been described and the strain has been characterized by means of genetic techniques, such as genomic DNA genetic fingerprints using Restriction Fragment Length Polymorphism (RFLP), genomic DNA karyotype by pulsed field electrophoresis, mitochondrial DNA polymorphism using RFLP and repeated yeast sequence PCR. The product has been found free from heavy metals (cadmium, lead, arsenic and mercury) and bacterial contaminants (mesophilic aerobic bacteria, coliforms, staphylococci coagulase positive, sulphite reducing anaerobic bacteria, Clostridium perfringens and Salmonella). Analyses of aflatoxins (B1, B2, G1 and G2), zearalenone and ochratoxin demonstrated that no mycotoxin was found above the limit of detection Intended use of the product The product is intended for use in feed for leisure horses at a dose of 3 x 10 9 CFU kg -1 of complete feedingstuffs. 5

6 Opinion on Levucell SC for leisure horses 6/9 3. Assessment of the analytical methods by the Community Reference Laboratory (CRL) EFSA has verified the CRL report as it relates to the methods used for the control of the active agent in animal feeds. The Executive Summary of the CRL can be found in the Appendix. 4. Efficacy One study was performed in the UK to assess the efficacy of Levucell SC20 on weight gain in yearling horses (initial weight 194 ± 46 kg). The experiment was aimed to assess the hypothesis that Levucell supplementation increases fibre utilization. The animals (23 ponies) were randomly assigned to control (11 ponies) or treatment groups (12 ponies), and kept in four pens (two on each side of the barn), with mares and geldings mixed. Animals were fed an isocaloric diet composed by old meadow hay ad libitum, plus 0.75 kg per head of hard feed per day consisting of soaked sugar beet pulp and a low energy coarse mix. Animals of the treatment group were fed with 1.5 x CFU animal -1 day -1 through 25 g of premix. Microbiological analysis of feed demonstrated that horses received a calculated dose of 2.6 x 10 9 CFU kg -1 of feed. Treatments and measurements lasted for a total of 19 weeks. Weight and body condition score of the ponies were monitored throughout the trial. Animals on one side of the barn showed a significant difference in weight gain between test and control group, while those on the other side did not (18.8 vs kg, NS; 26.0 vs kg P=0.014, for treatment and control for the west and east side respectively). When the data was combined, this difference remained significant (P <0.05). The differences in weight gain, despite showing significant effects in favour of the Levucell-treated group are not convincing because of the low weight gain of the control animals and the housing-treatment interaction. The FEEDAP Panel considers that the effect of S. cerevisiae in a hindgut fermenter, such as the horse, is likely to be similar to that seen in ruminants, where there is evidence of an effect on fibre digestion. Although there was a small but significant difference in body weight gain during the experiment, which may reflect better fibre digestion, this was not directly demonstrated. Consequently, the FEEDAP Panel, in the absence of evidence of a similar mode of action, cannot conclude on the efficacy of the product. 5. Safety for the target species 5.1. Tolerance study on horses A tolerance test was performed with eight horses, divided in two homogeneous groups of four individuals. The experiment was a cross-over Latin square design (two groups, two treatments), the trial comprising three consecutive periods: a first period of 30 days for measurements (Period 1), a wash out period of 30 days, and a second treatment period of 30 days for measurements (Period 2). During the measurement periods, one group was fed the control diet, the other one the control diet supplemented with 9.1 x CFU kg -1 of standardised complete feed of Levucell SC20, corresponding at more than 30 times the maximum recommended dose. Dry matter intake, refusals if any (hay and/or pelleted feed) and horse attitude (agitated, lively, illness) were recorded daily. A clinical examination was carried out weekly, recording heart rate, respiratory frequency, rectal temperature, feet temperature, assessment of gut sounds, colour of the mucous membranes, filling time of blood capillaries, aspect of droppings in the box (consistency and colour) and live body weight. At day 0 and 30 of each period, blood parameters (haematological and biochemical) were measured.

7 Opinion on Levucell SC for leisure horses 7/9 Horses fed with 30 times the maximum recommended dose did not shown any negative effects in terms of zootechnical or clinical parameters. No significant differences were observed in blood parameters, other than γ-gt, but all observations remained within the normal values Effects on faecal microbiota The influence on the intestinal microbiota was investigated in a study on four horses. The experiment was a cross-over Latin square design (two groups, two treatments), the trial comprising three consecutive periods: a first period of 14 days for adaptation, a second treatment period of seven days for measurements and a period of wash out of 21 days. Animals were artificially stressed by transportation for two hours on lorry at the end of each adaptation period. During the adaptation and measurement periods, one group was fed the control diet, the other one the control diet supplemented with 2 x CFU animal -1 day -1 of Levucell SC20. Faecal samples were collected the day before the transportation and four days later. Total anaerobes, lactate utilizing bacteria, lactobacilli, enterococci, and yeast were enumerated using standard plating techniques. Volatile fatty acid content of faeces was determined. Supplementation with Levucell SC20 significantly increased yeast numbers, while no major changes in the counts of analysed bacterial groups were observed. The low number of animals and the individual fluctuations prevented any conclusion to be drawn on the effect of Levucell SC supplementation on volatile fatty acid profile. 6. Post-market monitoring No risks associated with the use of the product are foreseen, and therefore the FEEDAP Panel does not see the need for specific requirements for a post-market monitoring plan. CONCLUSIONS AND RECOMMENDATIONS CONCLUSIONS In one efficacy study, the differences in weight gain, despite showing significant effects (P <0.05) in favour of the Levucell-treated group, are not convincing because of the low weight gain of the control and the housing-treatment interaction. Although the difference in body weight gain during the experiment may reflect better fibre digestion, this was not directly demonstrated. Consequently, the FEEDAP Panel, in the absence of evidence of a similar mode of action, cannot conclude on the efficacy of the product in leisure horses. Thirty times the recommended dose of Levucell SC20 in the 30 day tolerance experiment did not produce any adverse effects in adult horses. Microbiological analysis of the faecal microbiota of horses given Levucell did not show any significant differences compared to control animals other than the expected increase in yeast numbers. Consequently, the FEEDAP Panel considers Levucell SC in either formulation (SC20 and SC10ME) safe for horses. The FEEDAP Panel also considers it unlikely that this extension of use would introduce hazards for the consumers, users or for the wider environment that have not already been considered. RECOMMENDATION The current requirement for defining dose in terms of complete feedingstuffs may provide only an approximation in the case of functional ruminants and other grazing animals, including horses.

8 Opinion on Levucell SC for leisure horses 8/9 Additives are usually supplied in the form of complementary feed or as a top dressing. Unless this forms part of a total mixed ration, the remainder of the feed is given ad libitum. Consequently an unambiguous definition of target dose could be given in terms of CFU head -1 day -1 and this should be provided under other provisions in the Register entry. DOCUMENTATION PROVIDED TO EFSA 1- Dossier on Levucell SC. Saccharomyces cerevisiae CNCM I-1077 EC Nº 7. Extension of use for horses. July Submitted by Lallemand SAS. 2- Complementary particulars for Levucell SC (Saccharomyces cerevisae CNCM I-1077) for horses. November Submitted by Lallemand SAS. 3- Additional information. EFSA evaluation of Levucell SC (Saccharomyces cerevisiae CNCM I-1077) for horses. May Submitted by Lallemand SAS. 4- Evaluation report of the Community Reference Laboratory feed additives authorisation on the methods(s) of analysis for Levucell SC for horses. 5- Letter from the European Commission dated 4 July 2006 on the change of animal category of application on Levucell SC for horses. 6- Comments from the Member States received via the EFSAnet. SCIENTIFIC PANEL MEMBERS Georges Bories, Paul Brantom, Joaquim Brufau de Barberà, Andrew Chesson, Pier Sandro Cocconcelli, Noël Dierick, Anders Franklin, Jürgen Gropp, Anne-Katrine Lundebye Haldorsen, Ingrid Halle, Christer Hogstrand, Joop de Knecht, Lubomir Leng, Alberto Mantovani, Miklos Mezes, Carlo Nebbia, Walter Rambeck, Guido Rychen, Atte von Wright and Pieter Wester

9 Opinion on Levucell SC for leisure horses 9/9 APPENDIX Executive Summary of the Evaluation Report of the Community Reference Laboratory Feed Additives Authorisation on the Method(s) of Analysis for Levucell SC for horses. In the current application authorisation is sought for LEVUCELL SC under the category zootechnical additives, according to Annex I of Regulation (EC) No 1831/2003. Specifically, authorisation is sought to use LEVUCELL SC in its two forms LEVUCELL SC 20 and LEVUCELL SC 10ME for horses. LEVUCELL SC 20 is a light brown powdery uncoated product whereby LEVUCELL SC 10ME is micro-encapsulated. The feed additive is proposed for use in premixtures and feedingstuffs for horses at a concentration of 3 x 10 9 colony forming units (c.f.u.) per kg complete feedingstuff. For the determination of the active agent (Saccharomyces cerevisiae CNCM I-1077) in the feed additive LEVUCELL SC, a pour plate method for enumeration and a polymerase chain reaction (PCR) method for identification are proposed which are considered appropriate for the intended purpose. For the determination of the active agent S. cerevisiae CNCM I-1077 in premixtures and feedingstuffs, the same methods as for the feed additive are proposed. The method s performance characteristics of the enumeration method include relative standard deviations for repeatability (RSDr) and between-laboratory reproducibility (RSDR) of around 5 % and 8 %, respectively [System. Appl. Microbiol. 2003, 26, ]. The limit of quantification (LOQ) of the method is 10 5 c.f.u./kg. These performance characteristics are considered acceptable. The PCR method for strain identification was ring trial validated and performed appropriately [System. Appl. Microbiol. 2004, 27, ]. Both methods are considered suitable for official control for the field of application that is sought. Official and/or standard methods are proposed by the applicant for the determination of impurities (heavy metals, mycotoxins, microbiological quality) in the feed additive. The methods are therefore considered suitable for official control purposes. On the basis of the supplied documentation, no supplementary experimental work (testing or method validation) is required by the CRL.

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