The EFSA Journal (2006) 384, 1-9

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1 The EFSA Journal (2006) 384, 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 Biosaf Sc 47, a preparation of Saccharomyces cerevisiae, as a feed additive for horses (Question N o EFSA-Q ) Adopted on 12 September 2006 SUMMARY The additive Biosaf Sc 47 is composed of a concentrate of dried live yeast Saccharomyces cerevisiae (NCYC Sc 47). It is already permanently authorised for its use in cattle for fattening, dairy cows, rabbits for fattening, sows and piglets. The applicant is now asking for authorisation to use the additive in horses. According to Regulation (EC) No 1831/2003, the European Food Safety Authority (EFSA) should deliver an opinion on the efficacy and the safety of the product Biosaf Sc 47 for the target animals, consumers, users and the environment. In its assessment, the FEEDAP Panel considered that the safety of this organism for the consumer, the user and the environment had already been established and would not be changed by the proposed extension of use. Consequently, the Panel considered only the safety and the efficacy of the additive for the proposed additional target species. The four efficacy studies provided demonstrate a similar action to that reported as operating in the major ruminant species for which the product is authorised. Both the in vitro and in vivo studies showed that the supplementation with Biosaf Sc 47 at the maximum recommended level enhanced the degradation of fibre components. Consequently, the FEEDAP Panel concludes that Biosaf Sc 47 is efficacious in improving fibre digestion in horses. This product is best described as a digestibility enhancer, and this should be modified accordingly in the register entry. Biosaf Sc 47 has been shown to be safe for horses when fed with more than ten times the maximum recommended dose. No adverse effects on the gastrointestinal microbiota were seen in adult horses. The FEEDAP Panel also considers it very unlikely that this extension of use would introduce hazards for consumers of products from animals given the additive, users or for the wider environment that have not already been taken into account. Key words: Saccharomyces cerevisiae, yeast, probiotic, efficacy, safety, horses

2 Opinion on Biosaf Sc 47 for 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 Lesaffre Feed Additives 2 for authorisation of the product Biosaf Sc 47 to be used as a feed additive for horses (category: zootechnical additives) 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. 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 04 of May of The additive Biosaf Sc 47 is a preparation of Saccharomyces cerevisiae (NCYC Sc 47). This product is already permanently authorised for its use in cattle for fattening, dairy cows, rabbits for fattening, sows and piglets (E 1702). The Scientific Committee on Animal Nutrition (SCAN) issued an opinion (26 September 1997, updated 25 April 2003) on the safety of this product for the piglets, sows, beef and dairy cattle, including the safety for the user, the consumer and the environment 3, and another on the efficacy for the cattle for fattening (2 December 2002). 4 EFSA issued two opinions on the safety and efficacy of this product, one for lambs for fattening (9 February 2006), 5 and one for dairy small ruminants (12 July 2006). 6 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, consumers, users and the environment of the product Biosaf Sc 47, which is a preparation of Saccharomyces cerevisiae when used under the conditions described in Table 1. 1 OJ L 268, , p Lesaffre Feed Additives, 1 rue du Haut Touquet Marquette-Lez-Lille, France

3 Opinion on Biosaf Sc 47 for horses 3/9 Table 1. Register entry as proposed by the applicant. Additive Registration number/ec No/No Category of additive Functional group of additive Saccharomyces cerevisiae Zootechnical additives Other zootechnical additives Composition, description Preparation of Saccharomyces cerevisiae NCYC Sc 47 containing a minimum of 5 x 10 9 CFU g -1 of additive Chemical formula Description Purity criteria Method of analysis - Not appropriate Not appropriate Trade name Biosaf Sc 47 Name of the holder of authorisation Société Indurtrielle Lesaffre Conditions of use Species or category of animal Maximum Age Minimum content Maximum content CFU kg -1 of complete feedingstuffs Withdrawal period Horses - 8 x x 10 9 Not appropriate 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 Reserved exclusively to the manufacture of animals feed Production/ expiry dates and batch number printed on the commercial packaging Stable for pellets at dye temperatures up to 83 C Store in a cool and dry place Not appropriate Not appropriate Marker residue Maximum Residue Limit (MRL) Species or category of animal Target tissue(s) or food products Maximum content in tissues Not appropriate Not appropriate Not appropriate Not appropriate

4 Opinion on Biosaf Sc 47 for horses 4/9 ASSESSMENT 1. Introduction The additive Biosaf Sc 47 is composed of a concentrate of dried live yeast Saccharomyces cerevisiae (NCYC Sc 47). This product is already permanently authorised for its use in cattle for fattening, dairy cows, rabbits for fattening, sows and piglets (E 1702). The applicant is asking for an extension of use to horses of all ages. The additive Biosaf Sc 47, based on a well-known and researched strain of bakers/brewers yeast (Saccharomyces cerevisiae) has been assessed for its safety when included in feed for farmed animals. SCAN issued an opinion (26 September 1997, updated 25 April 2003) on the safety of this product for the piglets, sows, beef and dairy cattle, including the safety for the user, the consumer and the environment, and another on the efficacy for cattle for fattening (2 December 2002). The European Food Safety Authority (EFSA) issued two opinions on the safety and efficacy of this product for lambs for fattening (9 February 2006) and for dairy small ruminants (12 July 2006). 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 for the consumer, the user and the environment is established and will not be changed by the proposed extension of use. Consequently the FEEDAP Panel has considered only the safety and the efficacy of the additive for the target species, in particular regarding to the gut flora of horses, since it is known to be sensitive to disruption with consequences for the animal health. 2. Characterisation of the product. The active ingredient of the product is the yeast strain Saccharomyces cerevisiae (NCYC Sc 47). Biosaf Sc 47 is included in horse feed in order to improve digestibility. The product is a preparation of this strain containing a minimum of 5 x 10 9 CFU g -1 of the additive with a dry matter concentration of 91-94%. The additive is intended for use in feed for horses at a minimum content of 8 x 10 8 and a maximum content of 7 x 10 9 CFU kg -1 complete feedingstuff. Saccharomyces cerevisiae (NCYC Sc 47) is deposited at the National Collection of Yeast Culture, Great Britain. Quality control parameters are in place for heavy metals (cadmium, lead, arsenic and mercury) and bacterial contaminants (coliforms, Escherichia coli, Staphylococcus aureus, Salmonella and Clostridium perfringens). Saccharomyces cerevisiae (NCYC Sc 47) is characterised by pulsed field gel electrophoresis or by DNA hybridisation techniques Evaluation 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 report can be found in the Appendix. 3. Efficacy Two in vitro studies and one in vivo study were conducted between 2001 and An additional in vivo study was performed in 2006 upon a request from the FEEDAP Panel. Study 1 In study 1 the effect of Biosaf Sc 47 on dry matter disappearance (DMD) and on disappearance of fibre components was tested per week on 24 samples (500 mg per sample) of each of four forages namely alfalfa, ryegrass, grass hay and grass haylage

5 Opinion on Biosaf Sc 47 for horses 5/9 during 3 weeks (total 72 samples of each feed). The samples were placed in filter bags, which were in turn placed in jars containing 0.2% pepsin solution and incubated at 39ºC for 48h. Following pepsin digestion and rinsing the bags were returned to the jars and inoculated with one part faecal horse liquor and four parts buffer solution. Three concentration levels of the product were added to the jars: the minimum recommended dose (8 x 10 8 ), 4 x 10 9 and 8 x 10 9 CFU kg -1 feed. The jars were incubated at 39ºC for 48h. Then the bags were washed and dried overnight. A control jar without Biosaf Sc 47 was included in each weekly experiment and the results were compared with a standard, prepared using a neutral cellulose/gamanase (NCG) kit to thus provide an independent value of DMD for each feed. The disappearance of each fibre component was used to calculate the neutral detergent fibre (NDF) disappearance (DNDF) and acid detergent fibre (ADF) disappearance (ADFD) components of the different feeds. The results varied greatly for the different feeds with respect to NDF, ADF, crude protein and NCG-DMD. The results were analysed by ANOVA showing that the inoculum-corrected DMD exerted by Biosaf Sc 47 was significantly increased (P 0.001) for all four tested feeds and Biosaf Sc 47 concentrations compared to untreated feeds. Study 2 A second study was done to determine the degradation of dried and ground perennial rye grass haylage by inoculated equine faeces. Dry matter degradation and gas production using an automated gas production technique were determined in vitro. Faeces were sampled from two stabled ponies fed haylage (predominantly ryegrass) ad libitum. One pony (B) was also given 10 g Biosaf Sc 47 in 75 g of pasture mix. Pony A was given a similar pasture mix without Biosaf. Faeces were collected from pony B after a 14-day adaptation period to yeast in the diet. Faeces were collected from each pony and buffer added and the flasks were gassed with CO g (DM) samples of haylage were added to each bottle for fermentation. Biosaf Sc 47 was added to the bottles in increasing dosages. Maximum gas production, gas production rate and DMD were determined. The results were analysed by ANOVA and correlation and regression techniques to detect possible yeast-dosage related trends. It was shown that Biosaf Sc 47 significantly increased maximum gas production (P 0.01), gas production rate (P 0.05) and DMD (P 0.001). Increased levels of Biosaf Sc 47 appeared to enhance the overall feed degradation and also the rate of degradation. Study 3 An in vivo study with pregnant mares was done to examine the digestibility of feed supplemented with increasing concentrations of Biosaf Sc 47. Four warm-blooded mares were given haylage and concentrate supplemented with Biosaf Sc 47 at varying levels, 0, 7 x 10 8, 1.4 x 10 9 and 7 x 10 9 CFU kg -1 feed. The yeast content of the feed given in the trial was analysed. The horses were housed individually in boxes and allocated at random to the different diets. The trial was performed as a Latin-square design with four periods. Each period lasted 21 days, of which 14 were used for acclimatisation and the last seven for faecal collection. Total DM and concentrate intake of the horses were recorded. Faeces were collected from the floor four times daily. The digestibility of the individual feed components, DM, NDF, ADF, hemicelllulose, crude protein and fat were determined. The results were analysed by ANOVA. The supplementation of hay-concentrate with Biosaf Sc 47 at a yeast concentration of 7 x 10 9 CFU kg -1 improved the digestibility of NDF (P<0.05). Study 4 A second in vivo study was done to examine the effect of Biosaf Sc 47 on digestibility of horse feed when supplemented to a concentrate feed at a maximum level 6.5 x 10 9 CFU kg -1 feed. Five two-year old standard-bred trotters and five warm-blooded horses, 3 to 7 year-old, were used. The trial was performed as a Latin-square design experiment with

6 Opinion on Biosaf Sc 47 for horses 6/9 four periods and with five horses in each control or test group. Animals were fed hay three times a day and concentrate twice per day. The additive was top-dressed on concentrate during treatment periods. The horses were housed individually on rubber mats and allocated at random to the different diets. Each period lasted five days with a wash-out period of seven days. Total faeces and urine were collected from the horses during the 5-day test periods. The yeast content of supplemented concentrate was analysed and contained 2.3 x CFU kg -1 concentrate corresponding to 6.5 x 10 9 CFU kg -1 complete feed. Total DM, organic matter and crude fibre % of intake were analysed. The results were analysed by the GLM procedure. The supplementation of concentrate with Biosaf Sc 47 increased crude fibre digestibility from 23.7 to 28.5% (P=0.064). Conclusions on efficacy The four studies provided demonstrate a similar action to that reported as operating in the major ruminant species for which the product is authorised. Both the in vitro and in vivo studies showed that the supplementation with Biosaf Sc 47 at the maximum recommended level enhanced the degradation of fibre components. Consequently, the FEEDAP Panel concludes that Biosaf Sc 47 is efficacious in improving fibre digestion in horses. 4. Safety for the target species 4.1. Tolerance study A tolerance study was done including eight ponies of about the same weight and divided in two groups of four each. The ponies were randomly allocated to each group. Whether they were individually housed and fed is not described. A simple cross-over design was used with two 30-day feeding periods following adaptation periods of 14 days. The control group was given a dry diet ad libitum and the test group a dry diet supplemented daily with 1.7 x CFU kg -1 feed of Biosaf Sc 47 per horse, more than 10X the maximum recommended dose. Feed intake was measured daily, whereas body weight and body condition were measured weekly. Clinical examination, sampling for blood chemistry, haematology and faeces (bacteriology) were done on day 0, 30 and 60 of the trial. At day 30 two horses had elevated levels of creatin-kinase and lactate dehydrogenase and a third horse a decline of haemoglobin from day 30 to 60. These changes were not considered related to the supplementation of the feed with Biosaf Sc 47. There were no other significant changes between the groups for any of the measured parameters Effects on the microbiota of the digestive tract Four ponies were each given different diets containing different amounts of Biosaf Sc 47 for five months. One pony used as control was given non-supplemented feed. Faecal samples were examined by viable counts for yeast, other fungi, total bacterial counts, anaerobes, E. coli and coliforms. The study design was complicated and poorly described. A two-way ANOVA and the non-parametric Kruskal-Wallis rank sum test were used to analyse the data. The number of yeast cells in faeces clearly increased with increasing concentrations in the feed. Significant effects on the bacterial groups examined were not observed Conclusions on safety for the target animals Biosaf Sc 47 has been shown to be safe for horses when fed with more than ten times the maximum recommended dose. No adverse effects on the gastrointestinal microbiota were seen in adult horses.

7 Opinion on Biosaf Sc 47 for horses 7/9 5. 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 The four efficacy studies provided demonstrate a similar action to that reported as operating in the major ruminant species for which the product is authorised. Both the in vitro and in vivo studies showed that the supplementation with Biosaf Sc 47 at maximum recommended level enhanced the degradation of fibre components. Consequently, the FEEDAP Panel concludes that Biosaf Sc 47 is efficacious in improving fibre digestion in horses. This product is best described as a digestibility enhancer, and this should be modified accordingly in the register entry. Biosaf Sc 47 has been shown to be safe for horses when fed with more than ten times the maximum recommended dose. No adverse effects on the gastrointestinal microbiota were seen in adult horses. The FEEDAP Panel also considers it very unlikely that this extension of use would introduce hazards for consumers of products from animals given the additive, users or for the wider environment that have not already been taken into account. 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. 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 Biosaf Sc 47. Saccharomyces cerevisiae for horse s feed. December Submitted by Lesaffre Feed Additives. 2- Answers to the questions of EFSA. April Submitted by Lesaffre Feed Additives. 3- Answers to the question of EFSA. May Submitted by Lesaffre Feed Additives. 4- Evaluation report of the Community Reference Laboratory feed additives authorisation on the methods(s) of analysis for Biosaf Sc 47 for horses. SCIENTIFIC PANEL MEMBERS Georges Bories, Paul Brantom, Joaquim Brufau de Barberà, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël Dierick, Anders Franklin, Jürgen Gropp, Ingrid Halle, Christer Hogstrand, Lubomir Leng, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Miklos Mezes, Carlo Nebbia, Walter Rambeck, Guido Rychen, Atte von Wright and Pieter Wester

8 Opinion on Biosaf Sc 47 for horses 8/9 APPENDIX Executive Summary of the Evaluation Report of the Community Reference Laboratory Feed Additives Authorisation on the Method(s) of Analysis for Biosaf Sc 47 for horses BIOSAF Sc 47 is a zootechnical feed additive consisting of a strain of the yeast Saccharomyces cerevisiae NCYC Sc 47. The additive belongs to category 4 of zootechnical additives. The guaranteed number of viable yeast cells of the product is above 5 x 109 c.f.u./g (colony forming units / gram) additive. The current dossier for BIOSAF Sc 47 seeks an extension of its use in feeds for horses. The target level of supplementation is in the range of c.f.u./g feed. Concerning the enumeration of the active substances of BIOSAF Sc 47 (Saccharomyces cerevisiae) in the additive, the applicant proposes a pour plate method, the Standard method ISO The ISO method is considered appropriate for the purpose of routine control, as well as for official controls. Concerning the enumeration of the active substance in premixtures and feedingstuff, the applicant refers to a pour plate count method that has been published in a peer reviewed journal. This method is based on the principles of the above-mentioned ISO 7954 method and the authors of the article have validated the method in a full collaborative study applying an internationally accepted protocol for collaborative studies. Performance characteristics of this method obtained in the collaborative study were expressed in terms of relative standard deviations for repeatability (RSDr) and reproducibility (RSDR) which were around 5 % and 8 %, respectively. Both values are considered as acceptable. The limit of quantification (LOQ) of this method is 100 colony forming units (c.f.u) per gram (g) sample, i.e. well below the target application level in feedingstuffs. Alternatively, in cases where the species S. cerevisiae needs to be confirmed, a plate count method using an elective chromogenic agar was proposed, which has a limit of quantification of 1000 c.f.u. per gram (g) sample. This method was fully ring trial validated in parallel with the above mentioned pour plate method and resulted in acceptable method performance characteristics, because the RSDr was about 3 % and the RSDR was about 6%. The LOQ of this method is 1000 (c.f.u) per gram (g) sample, i.e. well below the target application level in feedingstuffs. Both methods are therefore considered appropriate for routine control and for official control purposes. Concerning the unambiguous identification of the specific strain of S. cerevisiae NCYC Sc 47 in BIOSAF Sc 47, in the additive, and if necessary in the premixture and feedingstuff, a polymerase chain reaction (PCR) method is used. This method was validated in a collaborative study which demonstrated a high level of correct identification between laboratories, and it is therefore considered suitable for routine and official control purposes. Information on the composition of ingredients other than the active agents, including impurities, physical state of the product, toxins and virulence factors, antibiotic production and resistance, stability of the additive, other physico-chemical or biological properties and incompatibilities with other feed ingredients, was provided for the dossier. However, actual methods for determining these properties were not submitted for the extension of the authorisation for this dossier. Saccharomyces cerevisiae does not belong to a taxonomic group which includes members known to be capable of production of toxins or other virulence factors. In the opinion of the CRL the methods for determination of the active substance in additives, premixtures and feedingstuff are considered fit for routine control and suitable for official control purposes. The CRL opinion is based on (i) the low LOQ relative to the target level of application which ranges between c.f.u./g of sample

9 Opinion on Biosaf Sc 47 for horses 9/9 (ii) the systematic and well performed validation studies for the quantitative microbiological enumeration method referenced in the BIOSAF Sc 47 dossier, which have acceptable performance characteristics expressed in terms of RSDr and reproducibility RSDR and (iii) the submitted identification methodology, which is a validated PCR method. On the basis of the supplied documentation, no supplementary experimental work (testing or method validation) is required by the CRL.