Scientific Opinion on the safety and efficacy of Lactobacillus buchneri (DSM 22963) as a silage additive for all species 1

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1 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of Lactobacillus buchneri (DSM 22963) as a silage additive for all species 1 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3 ABSTRACT European Food Safety Authority (EFSA), Parma, Italy Lactobacillus buchneri is a technological additive intended to improve the ensiling process at a proposed dose of 1.0 x 10 8 CFU/kg fresh material. The bacterial species L. buchneri is considered by EFSA to be suitable for the Qualified Presumption of Safety approach. As the identity of the strain has been clearly established and as no antibiotic resistance was detected, the use of the strain in the production of silage is considered safe for livestock species, consumers of products from animals fed the treated silage and for the environment. Evidence of a lack of irritancy was provided for one formulation of the additive. It is unlikely that given the nature of the alternative food grade excipients, different results would be obtained for other formulations containing this strain of L. buchneri. However, the proteinaceous nature of the active agent means its potential to be a skin/respiratory sensitizer cannot be excluded. Five studies with laboratory-scale silos are described, each lasting at least 90 days, made using samples of forage covering a range of dry matter content (20 to 71 %) with differing water-soluble carbohydrate content. In each case, replicate silos containing treated forage were compared to identical silos containing the same untreated forage and, after opening, the period before deterioration became evident was measured. The results showed that the additive containing this specific strain of L. buchneri has the potential to improve the production of silage from all forages by increasing acetic acid production resulting in an extended aerobic stability of the treated silage. European Food Safety Authority, 2011 KEY WORDS Technological additive, silage additive, Lactobacillus buchneri, QPS, safety, efficacy 1 On request from the European Commission, Question No EFSA-Q , adopted on 7 April 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 Lopez 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 3 Acknowledgement: The Panel wishes to thank the members of the Working Group on Silage 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 Lactobacillus buchneri DSM as a silage additive for all species. EFSA Journal 2011;9(4):2138. [11 pp.] doi: /j.efsa Available online: European Food Safety Authority, 2011

2 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 for the target animals, consumer, user and for the environment and on the efficacy of a product based on a single strain of Lactobacillus buchneri, when used as a technological additive intended to improve the ensiling process at a proposed dose of 1.0 x 10 8 CFU/kg fresh material. The bacterial species L. buchneri is considered by the European Food Safety Authority to be suitable for the Qualified Presumption of Safety approach. Therefore, it does not require any specific demonstration of safety other than confirming the absence of any determinants of resistance to antibiotics of human and veterinary clinical significance and the safety for the user. As the identity of the strain has been clearly established and as no antibiotic resistance was detected, the use of the strain in the production of silage is considered safe for livestock species, consumers of products from animals fed the treated silage and for the environment. Once an active agent has been authorised as a silage additive, different formulations can be placed on the market with reference to that authorisation. The applicant listed several cryoprotectants and carriers which would allow multiple formulations of the additive to be produced and consequently, not all forms can be directly tested for user safety. However, for assessing the safety for the user of the additive, the active agent is the principal concern provided that other components do not introduce concerns. Evidence of a lack of irritancy was provided for one formulation of the additive. It is unlikely that given the nature of the alternative food grade excipients, different results would be obtained for other formulations containing L. buchneri DSM Given the proteinaceous nature of the active agent, its potential to be a skin/respiratory sensitizer cannot be excluded. Five studies with laboratory-scale silos are described, each lasting at least 90 days, made using samples of forage covering a range of dry matter content (20 to 71 %) with differing water-soluble carbohydrate content. In each case, replicate silos containing treated forage were compared to identical silos containing the same untreated forage and, after opening, the period before deterioration became evident was measured. The results showed that the additive containing L. buchneri DSM has the potential to improve the production of silage by increasing acetic acid production resulting in an extended aerobic stability of the treated silage. 2

3 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 3 Background... 4 Terms of reference... 4 Assessment Introduction Characterisation Identity and properties of the active agent Production and characteristics of the additive Stability Conditions of use Evaluation of the analytical methods by the European Union Reference Laboratory (EURL)7 3. Safety Efficacy... 8 Conclusions... 9 Documentation provided to EFSA References Appendix

4 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 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 Lactosan GmbH&Co.KG 5 for reevaluation of the product Lactobacillus buchneri DSM 22963, to be used as a feed additive for all animal species (category: technological additive; functional group: silage additive) 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. 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 15 December The additive Lactobacillus buchneri DSM 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 animals, consumer, user and the environment and the efficacy of the product Lactobacillus buchneri DSM 22963, when used under the conditions described in Table 1. 4 OJ L 268, , p.29 5 Lactosan GmbH&Co.KG. Industriestrasse West 5, 8605 Kapfenberg, Austria 6 EFSA Dossier reference: FAD

5 Table 1: Description and conditions of use of the additive as proposed by the applicant Additive Lactobacillus buchneri DSM Registration number/ec No/No (if appropriate) Category(ies) of additive Functional group(s) of additive - Technological Silage additive Composition, description Lactobacillus buchneri DSM Activity: min. 5 x10 11 CFU/g Description Chemical Purity criteria formula (if appropriate) Impurities: Fungi < 100 CFU/g Clostridia < 10 CFU/g Enterobacteria <10 CFU/g Salmonella not detectable in 25 g Method of analysis (if appropriate) Quantification of lactic acid bacteria (according ISO 15787:2009) Trade name (if appropriate) Name of the holder of authorisation (if appropriate) Not applicable Not applicable Species or category of animal All species and animal categories Maximum Age Conditions of use Minimum content Maximum content CFU/kg of complete feedingstuffs The min. dose is CFU/g fresh matter, corresponding to 0.2 g of the additive Lactobacillus buchneri DSM per ton forage. Withdrawal period (if appropriate) Specific conditions or restrictions for use (if appropriate) Specific conditions or restrictions for handling (if appropriate) Post-market monitoring (if appropriate) Specific conditions for use in complementary feedingstuffs (if appropriate) Other provisions and additional requirements for the labelling The directions for use must indicate storage temperature, shelf-life Maximum Residue Limit (MRL) (if appropriate) Species or category of Target tissue(s) or Maximum content in Marker residue animal food products tissues 5

6 ASSESSMENT 1. Introduction Six genera of lactic acid producing bacteria are commonly associated with forage species and collectively contribute to the natural ensiling process. The present additive is based on a preparation of a single strain of one of those six genera, Lactobacillus buchneri, and is intended to be added to forages to promote ensiling (technological additive, functional group: silage additive) for the eventual use of the silage in any animal species. The species L. buchneri is considered by EFSA to be suitable for the Qualified Presumption of Safety (QPS) approach to safety assessment (EFSA, 2007, 2010). This approach requires the identity of the strain to be conclusively established and evidence that the strain does not show acquired resistance to antibiotics of human and veterinary importance. 2. Characterisation 2.1. Identity and properties of the active agent The strain of L. buchneri was isolated from silage and is deposited with the Deutsche Sammlung von Mikroorganisms und Zellkulturen (DSMZ) with the accession number DSM It has not been genetically modified. Strain identity was established by its phenotypic properties (API) and by the full 16S rrna gene sequence which by comparison with sequences recorded in databases was unambiguously identified as L. buchneri. Strain-specific detection is based on the use of pulsed field gel electrophoresis after cleavage with a number of restriction enzymes used individually (ApaI, SfiI, AscI and SmaI). Genetic stability was established using a finger-printing method based on RAPD-PCR amplification products. Using this method the master culture is routinely compared with working cultures used to inoculate fermentation batches. No differences in the resultant patterns have been observed to date. The strain was tested for antibiotic susceptibility using two-fold broth dilutions. The battery of antibiotics tested was that recommended by EFSA (EFSA, 2008) excluding vancomycin which is not required for this species. 8 As all minimum inhibitory concentration values for the L. buchneri strain were equal to or fell below the corresponding breakpoints defined by the FEEDAP Panel no further investigation is required Production and characteristics of the additive The active agent is grown in a sterilised medium typical of those used for lactic acid bacteria and then separated from the growth medium by centrifugation. Cryoprotectants are added and the cell mix is freeze-dried and ground. The ground powder is then blended with sufficient carrier to meet the minimum specified concentration of 5 x CFU/g additive. The resultant additive consists of % cells and solids from the fermentation and % excipients. Material safety data sheets are provided for all cryoprotectants (ascorbic acid, lactose, mannitol, monosodium glutamate, sodium citrate or whey powder) and carrier materials (glucose, maltodextrin or whey powder) and all are of food grade and do not introduce safety concerns. 9 Data on five production batches showed that the minimum specification was exceeded in all cases (mean 6.4 x CFU/g additive). 10 A single batch of the additive (excipients unknown) was examined for particle size distribution by laser diffraction and for dusting potential using a Heubach dustometer. The mean particle size was ~123 µm with approximately 20 % by weight of the additive consisting of particles with diameters 7 Technical dossier/section II/Annex II Technical dossier/section II/Annex II Technical dossier/section II/Annexes II.3-4 to II Technical dossier/section II/Annex II.1-2 6

7 below 50 µm and ~2 % below 5 µm. 11 However, the dusting potential of 0.78 g/m 3 is considered moderate. 12 The additive is routinely monitored for microbial contamination at various points in the manufacturing process and in the final product. Limits are set for Enterobacteriace and yeasts and filamentous fungi (<10 3 CFU/g additive), Escherichia coli (<10 CFU/g additive) and Salmonella spp. (absence in 25 g additive). Data from five batches confirmed compliance with the value for yeasts and fungi and Enterobacteriace 13 but absence of Salmonella and E. coli was shown only for a single batch. 14 Given the nature of the fermentation medium and the food grade excipients, the probability of contamination with heavy metals or mycotoxins is considered to be low and consequently not included in routine monitoring. One batch of the additive was, however, sent for analysis to confirm this position. Aflatoxins B1, B2, G1, and G2, zearalenone and deoxynivalenol, the metals Pb, Hg and Cd and arsenic could not be detected Stability The additive is described as hydroscopic and must be stored in packaging which protects against moisture. When stored in the original packaging, although losses were recorded, the additive met the minimum specification after two months storage at 40 ºC, one year at 25 ºC and two years at 4 ºC. 16 This data is based on three batches of the additive. Although the precise formulation of the additive used for these studies was not given, the stability data is likely to apply to any combination of the excipients listed in Section 2.2. As the additive is intended to be distributed by the spraying of an aqueous suspension, the short-term stability in water was measured. 17 This showed that bacterial numbers were maintained for at least two days at 20 ºC and seven days if refrigerated (4 ºC) Conditions of use The additive is intended for use with forages at ensiling at a proposed minimum dose of 1.0 x 10 8 CFU/kg fresh materials for the improvement of the aerobic stability in material of different botanical origin with dry matter content ranging from %. It is recommended that the additive is suspended in an appropriate amount of water and applied to the forage using a spraying device 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 The species, L. buchneri, is considered by EFSA to be suitable for the QPS approach to safety. Therefore, it does not require any specific demonstration of safety other than confirming the absence of any determinants of resistance to antibiotics of human and veterinary clinical significance and the safety for the user. In the view of the FEEDAP Panel, the antibiotic resistance qualification has been met and the identity of the production strain established. Accordingly, no further assessment of safety for the target species, consumers of products from animals fed treated silage or the environment is required. 11 Technical dossier/section III/Annex III Technical dossier/section III/Annex III Technical dossier/section II/Annex II Technical dossier/section II/Annex II Technical dossier/section II/Annex II Technical dossier/section II/Annex II Technical dossier/section II/Annex II.4-2 7

8 Dermal and eye irritation studies were made in rabbits following the relevant OECD protocols. 18 No cutaneous reactions and only mild and reversible effects on the conjunctiva were observed. Consequently, the form of the additive tested can be considered as non-irritant to skin and eyes. Skin sensitisation was not tested but its absence was assumed by the applicant as no adverse effects have been reported for workers exposed to the product in the manufacturing plant. Although users at the farm level are exposed to the additive only for a short period of time when preparing the aqueous suspension, given the proteinaceous nature of the active agent, its potential to be a skin/respiratory sensitizer cannot be excluded. Once an active agent has been authorised as a silage additive, different formulations can be placed on the market with reference to that authorisation. The applicant listed several cryoprotectants and carriers which would allow multiple formulations of the additive to be produced and consequently, not all forms can be directly tested for user safety. However, for assessing the safety for the user of the additive, the active agent is the principal concern provided that other components do not introduce safety issues. For this specific product, all excipients used are food grade, and their use in the additive would not introduce an additional risk to their conventional use. 4. Efficacy A total of five laboratory experiments made with five different forage samples, each lasting at least 90 days (90 96 days), were conducted. Each of the five studies used 6.5 L mini silos and, in one case, 1.0 L silos for intermediate samples. All had the capacity to vent gas. In each case, the contents of three replicate silos were sprayed with the additive at 1.0 x 10 8 CFU/kg forage dissolved in 10 ml water (dose was not confirmed by analysis of the applied suspension). Forage for the three control silos were sprayed with an equal volume of water without the additive. Ambient temperature was controlled at 20 ± 2 ºC. The five studies involved a range of forages of differing botanical origin and water-soluble carbohydrate (WSC) content primarily selected to show a wide range of dry matter content (see Table 2). The samples generally represented material easy to ensile (experiments 1 and 3, 4 and probably 5). Only one forage sample considered moderately difficult to ensile (experiment 2) and no samples of difficult to ensile material as defined in Regulation (EC) No 429/2008 were included (see Table 2). Table 2: Characteristics of the forage samples used in the ensiling experiments Study Test material Dry matter content WSC content 1 (% fresh material) (% fresh material) 1 19 Grass 3rd cut Lucerne 3rd cut Whole plant maize silage Ground corn cob Ground corn cob WSC: water-soluble carbohydrate Replicate silos were opened at the end of the experiment and at intermediate intervals in all experiments (between 2 5 days and at 14 days). The content of silos from the end of the experiments were analysed for dry matter content, ph, lactic and volatile fatty acids (VFA) concentration, ethanol, 18 Technical dossier/section III/Annexes III.3-3 and III Technical dossier/section IV/Annex IV.1 20 Technical dossier/section IV/Annex IV.2 21 Technical dossier/section IV/Annex IV.3 22 Technical dossier/section IV/Annex IV.4 23 Technical dossier/section IV/Annex IV.5 8

9 ammonia and total nitrogen. A reduced set of analyses were performed on the intermediate samples (ph and lactic acid). Aerobic stability was also followed by the measurement of the change of temperature after opening. A rise of 3 ºC above ambient was taken as indicating a loss of aerobic stability. Data were examined by the one-sided non-parametric Wilcoxon Kruskal-Wallis test (rank sum followed by chi-square approximation). There was a significant improvement in aerobic stability in all treated samples regardless of dry matter or WSC content. This was accompanied by a significant increase in total VFA and acetic acid concentrations compared to controls as would be expected of an obligate heterofermentative strain (Table 3). Table 3: Summary of the analysis of ensiled material recovered at the end of the experiments and the aerobic stability after opening of the silo Study Dose (CFU/kg forage) 0 Dry matter loss (%) Lactic acid ph (% ensiled material) * Acetic acid (% ensiled material) Ethanol (% ensiled material) Aerobic stability (days) x * 0.3 * 2.1 * 0.4 * 7.5 * x * 5.0 * 0.0 * 3.0 * 0.1 * 13.8 * x * 4.3 * 0.1 * 2.1 * 0.4 * 4.0 * x * * 0.4 * 0.3 * 9.3 * x * 1.7 * * * : Significantly different from the control value at P < 0.05 Due to the heterolactic fermentation pathway, by the end of the experiments the lactic acid content was lower in the treated materials compared to controls (significant in all five studies). Evidently, this reduction in lactic acid content occurred later in the ensiling process as at day 14 (and earlier) lactic acid concentrations were higher in all of the treated samples. There was also limited evidence of some increase in protein degradation as the ammonia nitrogen concentration was higher in two experiments in treated samples compared to controls. CONCLUSIONS As the identity of the strain, Lactobacillus buchneri DSM has been established and no antibiotic resistance detected, following the QPS approach the use of this strain in the production of silage is considered safe for target species, consumers of products from animals fed treated silage and for the environment. Evidence of a lack of irritancy was provided for one formulation of the additive. It is unlikely that, given the nature of the alternative food grade excipients, different results would be obtained for other formulations containing L. buchneri DSM Due to the proteinaceous nature of the active agent, its potential to be a skin/respiratory sensitizer cannot be excluded. The additive containing L. buchneri DSM has the potential to improve the production of silage by increasing acetic acid production resulting in an extended aerobic stability of the treated silage. This was demonstrated in forages with dry matter contents varying from %. 9

10 DOCUMENTATION PROVIDED TO EFSA 1. Lactobacillus buchneri DSM October Submitted by Lactosan GmbH&Co.KG. 2. Evaluation report of the European Union Reference Laboratory for Feed Additives on the methods of analysis for Lactobacillus buchneri DSM Comments from Member States received through the ScienceNet. REFERENCES EFSA (European Food Safety Authority), Opinion of the Scientific Committee on a request from EFSA on the introduction of a Qualified Presumption of Safety (QPS) approach for assessment of selected microorganisms referred to EFSA. The EFSA Journal, 587, 1-16 EFSA (European Food Safety Authority), Technical guidance prepared by the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) on the update of the criteria used in the assessment of bacterial resistance to antibiotics of human or veterinary importance. The EFSA Journal, 732, EFSA (European Food Safety Authority), 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; 8(12):

11 APPENDIX Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Methods of Analysis for Lactobacillus buchneri (DSM 22963) for all animal species In the current application authorisation is sought for the microbial feed additive Lactobacillus buchneri DSM under the category 'technological additives', functional group 'silage additives' according to Annex I of Regulation (EC) No 1831/2003. Specifically, authorisation is sought for the feed additive to be placed on the market in the form of powder, containing a minimum concentration of 5x10 11 CFU/g of Lactobacillus buchneri DSM The intended use of the current application is for all animal species. It is proposed to be mixed into silage with water providing a minimum concentration of 1x10 5 CFU/g fresh matter ensilage. For enumeration of Lactobacillus buchneri DSM in feed additive, the Applicant proposed a spread plate method based on the ring-trial validated CEN method (EN 15787). The performance characteristics reported after logarithmic transformation of measured values (CFU) are: - a standard deviation for repeatability (S r ) of 0.24 log 10 CFU/g; - a standard deviation for reproducibility (S R ) ranging from 0.29 to 0.38 log 10 CFU/g; and - a limit of detection (LOD) of 10 5 CFU/kg of feedingstuffs. Based on the performances characteristics of the method the EURL recommends for official control the EN for the determination of Lactobacillus buchneri DSM in the feed additive per se. The Applicant did not provide any experimental method or data for the determination of Lactobacillus buchneri DSM in silage. Furthermore, the method available is not able to determine the content of Lactobacillus buchneri DSM added to silage. Therefore the EURL cannot evaluate nor recommend any method for official control to determine Lactobacillus buchneri DSM in silage. Molecular methods were used by the Applicant for identification of the active agent. The EURL recommends 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. 11

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