Scientific Opinion on the safety and efficacy of micro-organism DSM when used as a technological feed additive for pigs 1

Transcription

1 EFSA Journal 2013;11(5):3203 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of micro-organism DSM when used as a technological feed additive for pigs 1 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3 ABSTRACT European Food Safety Authority (EFSA), Parma, Italy The additive, based on a single bacterial strain (DSM 11798), is intended for use in feeds for pig to reduce the epoxide group of contaminating trichothecene mycotoxins to a less toxic metabolite. To establish an identity for the strain a phylogenetic analysis was made based on the genomic sequence of the strain and 24 genome sequences available for the family Coriobacteriaceae. The three approaches used consistently suggested an assignment to a new taxonomic unit within the family Coriobacteriaceae. The Panel accepts that a new species and/or genus may have to be established. In the interim the deposition of the bacterium in a European culture collection is sufficient to ensure that any authorisation would apply only to the additive under application. Based on the results of a tolerance study the additive is considered safe for piglets and pigs for fattening at the maximum dose proposed. The live organism is not expected to present a hazard for consumers and the negative findings in a series of genotoxicity and oral toxicity tests indicates that toxic metabolites are not produced. Since the additive is expected to act only by the biotransformation of trichothecene mycotoxins to less toxic forms, no hazards for consumers would arise from the use of the additive in animal production. The additive was nonirritant to eyes and skin and was not a skin sensitiser, but the potential for respiratory sensitization cannot be excluded. As the strain has a very limited capability to survive in aerobic conditions, its use in animal production is unlikely to introduce an environmental concern. The evidence presented in ex vivo and in vivo studies with deoxynivalenol confirms that the capacity to transform trichothecenes can be realised in pigs when the additive is incorporated into diets at a minimum dose of 1.7 x 10 8 CFU/kg complete feed. European Food Safety Authority, 2013 KEY WORDS Bacterial transformation, trichothecene mycotoxins, deoxynivalenol, safety, efficacy, piglets, pigs for fattening 1 On request from the European Commission, Question No EFSA-Q , adopted on 16 April Panel members: Gabriele Aquilina, Alex Bach, 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 Mycotoxin detoxifying agents, including Georges Bories, Andrew Chesson and Jürgen Gropp, 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), Scientific Opinion on the safety and efficacy of micro-organism DSM when used as a technological feed additive for pigs. EFSA Journal 2013;11(5):3203, 18 pp. doi: /j.efsa Available online: European Food Safety Authority, 2013

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, consumers, users and the environment and the efficacy of a bacterial product when used as a technological additive to reduce the concentration of contaminating trichothecene mycotoxins in feed. In 2005, the FEEDAP Panel published an opinion on the strain of bacterium which is the subject of this application but which was then assigned to a species of Eubacterium. As there was no data on the prevalence of the strain in the digestive tract of farm animals or humans, and thus no estimate of natural exposure, and as the tolerance studies did not provide sufficient reassurance, the Panel was unable to conclude on the safety of the strain for the target species. The applicant has now resubmitted an application for the use of this additive as a technological additive (functional group m, substances for reduction of the contamination of feed by mycotoxins) for use with pigs. In order to establish an identity for the strain under application (DSM 11798) a phylogenetic analysis was made based on the assembled and annotated genomic sequence of the strain and 24 genome sequences available for the family Coriobacteriaceae. The genome set was used to create phylogenies at three levels: (i) based on the 16S rrna gene sequence, (ii) based on a set of 58 concatenated core bacterial proteins sequences, and (iii) based on the average nucleotide identity of the whole genome sequences. All three approaches consistently place the strain in a subgroup of Coriobacteriaceae. These data suggest the assignment of the strain to a new taxonomic unit within the family Coriobacteriaceae. The Panel accepts that the taxonomic position of the active agent is unclear and may warrant establishing a new species and/or genus. In the interim the deposition of the bacterium in a European culture collection is sufficient to ensure that any authorisation would apply only to the additive under application. No resistance to antibiotics of human and veterinary clinical significance and no homologies with known virulence factors could be detected in the strain. Based on the results of a tolerance study in which piglets were exposed to a 100-fold overdose of the additive, additive is considered safe for piglets at the maximum dose proposed. This conclusion can be extended to pigs for fattening. The live organism is not expected to present a hazard for consumers and the negative findings in a series of genotoxicity and oral toxicity tests gives reassurance that toxic metabolites are not produced. As current legislation includes a guidance level for DON which already ensures animal and consumer safety and since the additive is expected to act only by the biotransformation of trichothecene mycotoxins to less toxic forms, no hazards for consumers would arise from the use of the additive in animal production at the recommended dose range. The additive was non-irritant to eyes and skin and was not a skin sensitiser. Although the additive is formulated to minimise exposure by inhalation some exposure of the respiratory tract remains possible and the potential for respiratory sensitization cannot be excluded. The active agent would appear to naturally occur in the digestive tract but, as a potentially new genus, no data on its prevalence is available. However, as the strain has a very limited capability to survive in aerobic conditions, its use in animal production is unlikely to introduce an environmental concern. The active agent has the capacity to reduce the epoxide group of trichothecenes to produce less toxic metabolites. The evidence presented in ex vivo and in vivo studies with deoxynivalenol confirms that this capacity can be realised in piglets when the additive is incorporated into diets at a minimum dose of 1.7 x 10 8 CFU/kg complete feed. The FEEDAP Panel concludes that the additive at a minimum dose of 1.7 x 10 8 CFU/kg complete feed has the capacity to biotransform trichothecenes from contaminated feed for all pigs. EFSA Journal 2013;11(5):3203 2

3 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 3 Background... 4 Terms of reference... 4 Assessment Introduction Characterisation Characterisation of the active agent Identity Antimicrobial production and antibiotic resistance Presence of virulence determinants Production and characteristics of the additive Stability and homogeneity Shelf-life Premixtures Complete feeds Homogeneity Conditions of use Analytical methods Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) Interference with the analysis of mycotoxins in feed Safety Safety for the target species Tolerance study with piglets Additional tolerance studies Conclusions on the safety for target species Safety for the consumer Exposure to the active agent Trichothecenes and metabolites Genotoxicity Oral toxicity studies Conclusions on the safety for consumers Safety for the user Effects on skin and eyes Effects on the respiratory system Conclusions on the safety for the users Safety for the environment Efficacy In vitro studies Ex vivo studies Pig intestinal sections Ussing chambers In vivo studies Short-term studies Performance studies Conclusion on efficacy Conclusions Documentation provided to EFSA References Appendix EFSA Journal 2013;11(5):3203 3

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 Biomin GmbH 5 for authorisation of the product Biomin BBSH 797, based on the microorganism DSM (Genus novus of family Coriobacteriaceae), when used as a feed additive for all animal species 6 (category: technological additive; functional group: substances for reduction of the contamination of feed by mycotoxins) 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 30 August The additive DSM (Genus novus of family Coriobacteriaceae) has not been previously authorised in the European Union (EU). The FEEDAP Panel delivered an opinion on the safety and efficacy of this product when used as a feed additive for piglets, pigs for fattening and chickens for fattening (EFSA, 2005). 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 based on the microorganism DSM (Genus novus of family Coriobacteriaceae), when used under the conditions described in Table 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, , p. 29. Biomin GmbH Industriestraße 21, A-3130 Herzogenburg, Austria. 6 During the course of the assessment, the applicant requested to limit the application to pigs. 7 EFSA Dossier reference: FAD EFSA Journal 2013;11(5):3203 4

5 Table 1: Description and conditions of use of the additive as proposed by the applicant Additive Registration number/ec No/No (if appropriate) Category(ies) of additive Functional group(s) of additive BIOMIN BBSH DSM 11798; Genus nov. (formerly Eubacterium) species nov Submission number: Technological Additives 1 m / substances for reduction of the contamination of feed by mycotoxins Composition, description Preparation of BIOMIN BBSH DSM 11798; Genus nov. (formerly Eubacterium) species nov Description Chemical Purity criteria formula (if appropriate) compliant with EU law on microbial quality, not applicable heavy metals, toxins and undesirable substances Method of analysis (if appropriate) plate count method Trade name (if appropriate) BIOMIN BBSH 797 Name of the holder of authorisation (if appropriate) Species category animal or of Maximum Age Biomin GmbH Conditions of use Minimum content Maximum content Withdrawal period CFU/kg of complete feedingstuffs (if appropriate) all pigs 1.7E E+09 none 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 store in cool, dry place (room temperature or lower) Face mask, goggles and gloves recommended. Use original container. When using do not eat, drink or smoke. After use wash hands and face. Avoid contact with eyes. Change and clean spoiled work clothing. not applicable not applicable Marker residue Maximum Residue Limit (MRL) (if appropriate) Species or category of animal Target tissue(s) or food products Maximum content in tissues EFSA Journal 2013;11(5):3203 5

6 ASSESSMENT 1. Introduction In 2005, the FEEDAP Panel published an opinion on the strain of bacterium which is the subject of this application but which was then assigned to a species of Eubacterium (EFSA, 2005). The strain was intended to be included in feed for poultry and pigs to reduce the adverse effects of any contaminating trichothecene mycotoxins. As there was no data on the prevalence of the strain in the digestive tract of farm animals or humans, and thus no estimate of natural exposure, and as the tolerance studies did not provide sufficient reassurance, the Panel was unable to conclude on the safety of the strain for the target species. The Panel acknowledged that the strain was able to reduce the epoxide group of trichothecenes to produce less toxic metabolites, but expressed some concern that reliance on the presumed efficacy of the additive could lead to unsuitable feed materials being treated and used with adverse consequences for farm animals. Since this date, the publication of guidance values (Recommendation 2006/576/EC 8 ) and the Regulation on Feed Materials (Regulation (EC) No 767/ ) restricts the use of cereals to those of merchantable quality, with levels of contaminating mycotoxins below the legal or advisory limits, removing this as a concern. The applicant has now resubmitted an application for the use of this additive as a technological additive (functional group m, substances for reduction of the contamination of feed by mycotoxins). The original application was for all animal species, but during the course of the assessment the applicant decided to limit it for use with pigs. 2. Characterisation 2.1. Characterisation of the active agent Identity The active agent was isolated from the bovine rumen and is deposited with Deutsche Stammsammlung von Mikroorganismen und Zellkulturen with the accession number DSM The strain contains no detectable plasmids and has not been subjected to any genetic modification using recombinant DNA technology (GMO-free certificate provided). 11 Its identity has proved difficult to establish. It was initially assigned on the basis of its 16S rrna gene sequence and cytosine:guanosine (CG) content to Eubacterium, then a heterogeneous group of Gram positive obligate anaerobes. However, the closest sequence similarity with known eubacteria, was 92.8%, a value which did not allow speciation and might have indicated a new genus. Subsequently, the 16S rrna gene sequence of the active strain was again compared with known 16S rrna gene sequences using the Basic Local Alignment and Search Tool (BLAST) of NCBI (National Center for Biotechnology Information). 12 The strain showed no more than 93 % 16S rrna gene sequence identity to the most closely related strain Eggerthella lenta (formerly Eubacterium lentum). In 2011, the sequencing data of the active strain was again compared with known 16S rrna gene sequences. As the sequence identity with the closest neighbour remained at 93% the applicant took the decision to assign the strain to a new genus (Gen. nov.) in the family of Coriobacteriaceae, in line with present taxonomy. Subsequently a phylogenetic analysis was made based on the assembled and annotated genomic sequence of DSM and 24 genome sequences available for the family Coriobacteriaceae and 8 Commission Recommendation of 17 August 2006 on the presence of deoxynivalenol, zearalenone, ochratoxin A, T-2 and HT-2 and fumonisins in products intended for animal feeding. OJ L 229, , p Regulation (EC) No 767/2009 of the European Parliament and of the Council of 13 July 2009 on the placing on the market and use of feed, amending European Parliament and Council Regulation (EC) No 1831/2003 and repealing Council Directive 79/373/EEC, Commission Directive 80/511/EEC, Council Directives 82/471/EEC, 83/228/EEC, 93/74/EEC, 93/113/EC and 96/25/EC and Commission Decision 2004/217/EC. OJ L 229, , p Technical dossier/section II/Annex II Technical dossier/section II/Annex II Technical dossier/section II/Annex II-01. EFSA Journal 2013;11(5):3203 6

7 two out-group genomes. The genome set was used to create phylogenies at three levels: (i) based on the 16S rrna gene sequence, (ii) based on a set of 58 concatenated core bacterial proteins sequences, and (iii) based on the average nucleotide identity of the whole genome sequences. 13 All three approaches consistently place the DSM in a subgroup of Coriobacteriaceae including the genera Eggerthella and Gordonibacter. The splits are supported with almost perfect bootstrap values for the core protein set and medium to high values for the 16S rrna gene sequences. These data also suggest the assignment of the strain DSM to a new taxonomic unit within the family Coriobacteriaceae. Since there are no known closely related strains the need for strain-specific detection is, at present, redundant. The current 16S rrna gene sequence data would be sufficient to confirm the presence of the organism. To guarantee genetic stability of the active strain, a master cell bank from the deposited culture was prepared. 14 For routine fermentation work additional working cell banks were established No genetic changes have been detected using Pulsed Field Gel Electrophoresis (PFGE) between the genetic patterns of the master and working cell banks from between 2005 and In all cases the individual restriction digestions produced identical banding patterns Antimicrobial production and antibiotic resistance The strain does not produce detectable antibiotic activity against the four indicator bacteria and the one yeast tested. 15 The strain was tested for antibiotic susceptibility using the broth dilution method. 16 The battery of antibiotics used was that recommended by EFSA (EFSA, 2008, 2012). As all minimum inhibitory concentration values for the strain fell below the corresponding cut-off defined by the FEEDAP Panel for other Gram positive strains, no further investigation is required Presence of virulence determinants The raw sequence data for strain DSM was annotated using an automated process. This identified 2738 coding sequences, none of which were identified as genes associated with adhesion, toxin, virulence, disease or defence genes. A literature search for virulence genes within the family Coriobacteriaceae also did not identify any possible targets. 17 Finally, using the approach to screen for virulence factors described by Bennedsen et al. (2011), all of the bacterial virulence gene information contained in the Virulence factor database (VFDB) 18 was converted into an artificial genome of virulence factors. This was then used as a scaffold to assemble the full genome sequencing data of the strain DSM Since no assembly to the artificial genome occurred, the bioinformatic analysis indicated that no homologies to the any of the virulence genes in the VFDB existed in the active agent Production and characteristics of the additive Details of the production process are provided in the technical dossier. The resulting additive is a grey-brown powder with a typical composition of not more than 35 % cell mass, not more than 35 % inulin and % coating agent. The minimum declared activity is CFU/g additive. Analysis of six production batches showed that this minimum content was confirmed in all cases (mean CFU, range CFU/g additive) Supplementary information/annex Phylogenetic positioning. 14 Technical dossier/section II/Annex II Technical dossier/section II/Annex II Technical dossier/section II/Annex II Supplementary information/annex Virulence genes. 18 Available online: 19 Technical dossier/section II/Annexes II EFSA Journal 2013;11(5):3203 7

8 Particle size analysis by laser diffraction of three batches of the additive showed that approximately 28 % (v/v) of particles had a diameter below 100 µm, 16 % < 50 µm and 1.5 % < 10 µm. 20 Dusting potential measured by the Stauber-Heubach method with the same three batches was measured at 0.50 g/m Batches of the additive are regularly screened for heavy metals and arsenic contamination. Limits are set for Pb (<10 mg/kg), Cd (<2 mg/kg), Hg (<0.1 mg/kg) and As (< 2 mg/kg). Analysis of three batches found all values were below the limit of quantification (LOQ) for each impurity. 22 The same three batches also were assayed for 15 of the most commonly encountered mycotoxins. Again all values were below the respective LOQs. 23 A further three batches of the additive were analysed for dioxin content which was in each case 0.07 WHO(2005) PCDD/F ng/kg dry matter. 24 The additive is routinely monitored for microbial contamination at various points in the manufacturing process and in the final product. Limits are set for Enterobacteriaceae (<10 3 CFU/g additive), and yeasts and filamentous fungi (<10 2 CFU/g additive), Escherichia coli (<10 CFU/g additive) and Salmonella (absence in 25 g additive). Data from five batches confirmed compliance with these values Stability and homogeneity Shelf-life Three batches of the additive were stored under ambient temperatures (22 ± 2 ºC) in the normal packaging used for distribution and under two controlled conditions (25 ºC/60 % relative humidity (RH) and 40 ºC/75 % RH). 26 The additive, whether stored under ambient conditions or at 25 ºC/60 % RH, showed a similar slow rate of loss of viability. In both cases, losses were < 0.5 log after 12 months. Storage at the higher temperature resulted in a significant loss of viability over the same period Premixtures The stability of the additive (three batches) in two premixtures was monitored over a six month period. 27 The first premix was a protein concentrate intended for piglet starter diets and the second a typical vitamin-mineral premix for grower/fattening pigs. In each case the additive was blended at a concentration of 1 % by weight and the premixtures stored under dry conditions at ambient temperature (22 ± 2 ºC). The loss in viability was limited over the observed storage period Complete feeds The additive (three batches) was incorporated into three complete feeds, a starter and a grower diet for pigs and a broiler diet. 28 All three were stored as mash feed under ambient conditions (22 ± 2 ºC) and monitored for three months. In addition the broiler feed was pelleted at 75 ºC and stored under the same conditions. As was seen in the premixtures there was very little loss in viability over the storage period in any of the feedstuffs monitored. However, the results with the pelleted feed did not take account of any loss due to the pelleting process. A small scale pelleting experiment (10 kg samples) was made with a poultry feed supplemented with the additive. 29 One sample was cold pelleted (40 ºC) and two further samples with steam (50 and 60 ºC) for 15 min. There was little effect of the process on the viability of the additive strain (<0.3 log loss). A very similar result was also obtained under more realistic conditions in which kg of a treated poultry feed was pelleted at 80 ºC Technical dossier/section II/Annexes II Technical dossier/section II/Annexes II Technical dossier/section II/Annexes II Technical dossier/section II/Annex II Technical dossier/section II/Annexes II Technical dossier/section II/Annexes 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 Technical dossier/section II/Annex II-64. EFSA Journal 2013;11(5):3203 8

9 Homogeneity Micro-organism DSM for pigs The results of the two studies made with a single batch of the additive showed that the distribution of the active substance incorporated in a vitamin-mineral premixture and a pelleted broiler feedingstuffs is even. 31 The colony counts of ten sub-samples of each feed demonstrated high consistency with a coefficient of variation of 6.6 % and 9.0 %, respectively Conditions of use The additive is intended for pigs with a recommended incorporation rate of to CFU/kg complete feed. Administration of the additive via premixtures is recommended Analytical methods 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 Interference with the analysis of mycotoxins in feed Specific tests were made to examine the possible effects of the additive on the routine analysis of mycotoxins. 32 In each case a sample of feed was obtained which was naturally contaminated with one or more of the more commonly occurring mycotoxins of interest. The analysed levels of contamination in the different feed samples were: aflatoxin B1 7 µg/kg and aflatoxin B2 1 µg/kg; zearalenone 54 µg/kg; deoxynivalenol µg/kg; ochratoxin A 9 µg/kg and fumonisin B µg/kg and fumonisin B2 880 µg/kg. Replicate samples (25 g) of the contaminated feed were mixed with 50 mg additive and then extracted for analysis. The presence of the additive did not affect the results obtained. 3. Safety 3.1. Safety for the target species Tolerance study with piglets A total of 120 mixed-sex weaned piglets (Piétrain (Landrace Large White)) of four weeks of age were assigned to one of three treatment groups. 33 The first group of 40 animals were fed only a basal diet without the additive, the second group received the same diet supplemented with the additive at the maximum recommended rate ( CFU/kg feed) and the third group the diet supplemented with the additive at 100 the maximum recommended rate ( CFU/kg feed). The expected microbial counts were confirmed by analysis of the feed. Animals were housed in groups of ten giving four replicates per treatment. The duration of the study was 42 days. Animals were monitored for general health and performance measured. Data was analysed by analysis of variance. There were no mortalities recorded and all animals remained in good health throughout the trial. The weight of animals at the end of the trial did not significantly differ between groups (28.8 kg vs 29.7 kg and 29.6 kg) or daily gain (470 vs 492 and 487 g/day). Feed intake also was unaffected by treatment and consequently the calculated feed to gain ratio did not significantly differ (1.63 vs 1.54 and 1.64) Additional tolerance studies Two further tolerance studies with piglets where the additive was supplied at ten-fold overdose were considered in the course of a previous application (EFSA, 2005). One of these studies was confounded by the use of DONcontaminated feed and the remaining one lacked the power to detect adverse effects. Although no specific concerns regarding safety were noted at this time, the Panel was unable to conclude on the safety of the additive for piglets. 31 Technical dossier/section II/Annex II Technical dossier/section II/Annex II Technical dossier/section III/Annex III-02. EFSA Journal 2013;11(5):3203 9

10 Conclusions on the safety for target species Micro-organism DSM for pigs The additive appeared well tolerated by piglets and is safe for this category at the maximum dose proposed. This conclusion can be extended to pigs for fattening Safety for the consumer As indicated in the previous opinion on this organism (EFSA, 2005), consumers may be exposed to the live organism or its metabolites through contamination of carcasses of animals fed the additive or to metabolites of trichothecenes in animal tissues Exposure to the active agent The active agent is a strict anaerobe with limited survivability under aerobic conditions, therefore the potential for surface contamination of carcasses is very limited. In addition, a thorough search of the virulence factor database failed to detect any homology with known bacterial virulence genes (see 2.1.3). Thus the organism itself is unlikely to prove to be hazardous to consumers, particularly when taking into account the limitations to possible exposure Trichothecenes and metabolites Microbial metabolism of trichothecenes generally involves the enzymatic reduction of the 12, 13- epoxide group to the corresponding de-epoxy metabolites (Figure 1), although other reactions can occur (Binder et al., 1998; Wu et al., 2010). The de-epoxy metabolites have been shown to be significantly less toxic than their epoxy parents in a variety of cytotoxicity tests (Swanson et al., 1987; Shima et al., 1997; Eriksen et al., 2004; Sundstol et al. 2004; Schatzmayr et al., 2006a,b) Figure 1. Reduction of the epoxide group The strain DSM was selected because of particular capacity to metabolise trichothecenes to the corresponding de-epoxy metabolites. Transformation capacity in the fermentation broth or in the resuspended final product is routinely monitored using DON as substrate and measuring the production of the single de-epoxy metabolite de-epoxydeoxynivalenol by HPLC. The in vitro transformation by strain DSM of five other Type A trichothecenes (scirentriol, T-2 triol, T-2 tetraol, T-2 toxin, HT-2 toxin) has been confirmed and the metabolites identified (Fuchs et al., 2002). As was found for DON, all gave rise to a single de-epoxy metabolite by a one or two step route. Other Group B trichothecenes similarly examined include nivalenol, fusarenon X and 3- and 15-acetyldeoxynivalenol. In these cases, deacetylation preceded de-epoxidation (Fuchs et al., 1999) Genotoxicity A Bacterial Reverse Mutation Assay was made in accordance with the OECD guideline The study included a preliminary solubility test, a preliminary range finding test, an initial mutation test (plate incorporation method) and a confirmatory mutation test (pre-incubation method). The test item was a concentrated lysate of the active agent. No mutagenic activity was detected up to the highest dose tested (5 000 µg/plate). 34 Technical dossier/section III/Annex III-19. EFSA Journal 2013;11(5):

11 The second in vitro study was a Mammalian Chromosome Aberration Test following the protocol of OECD guideline 473, again using a concentrated lysate of the active agent as test item. 35 The lysate tested up to the highest recommended concentration (5 000 µg/ml) in the presence and absence of metabolic activation system, did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells and can be considered non-clastogenic in this test system. Finally the additive was tested in the Mouse Micronucleus Test performed in accordance with the OECD guideline In the preliminary toxicity test, groups of two male and two female mice were treated with a concentrated lysate of the active agent as the test item suspended in 1 % aqueous methylcellulose at 2 000, 1 000, 500 and 250 mg/kg body weight by oral gavage. No clinical signs or marked effect on body weight were observed in these mice. Based on these observations, the highest dose level selected for the main test was mg/kg body weight. In the main test, no treatment related effects were observed on animal body weights in any treated groups. The numbers of micronuclei observed in the groups were all equal to or less than the negative control value. Thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study Oral toxicity studies A single dose acute oral toxicity study following OECD 423 was made in female rats. 37 One group of three rats was given mg of the additive/kg bw by gavage, observed for 14 days and then were subject of necropsy and macroscopic observation. No clinical or macroscopic abnormalities were observed. For confirmation, the acute oral toxicity study was repeated with a second group of three rats, with the same outcome. A sub-chronic oral toxicity study following OECD 408 was made with groups of 20 Wistar rats (ten male and ten female). 38 A control group was compared to a treatment group given , or CFU per kg body weight of the active agent for 90 days. At the end of the period animals were killed for gross pathology and histopathology. An additional treatment group given the highest dose tested were observed for a further 30 days after treatment was withdrawn before also being killed and examined. No mortalities were recorded and no adverse clinical observations made during the study. The body weight of the treated groups was comparable to the control group. Haematological and clinical chemistry values did not significantly differ between groups and no gross pathological findings were observed Conclusions on the safety for consumers The live organism is not expected to present a hazard for consumers and the negative findings in a series of genotoxicity and oral toxicity tests gives reassurance that toxic metabolites are not produced. Current legislation restricts the use of feed materials to those of merchantable quality which includes limits for most mycotoxins to a level which ensures animal and consumer safety. Since the additive is expected to act only by the reduction of trichothecene mycotoxins to less toxic forms, no hazards for consumers would arise from the use of the additive in animal nutrition at the recommended dose Safety for the user Effects on skin and eyes Dermal and eye irritation studies were made according to the protocols in OECD guidelines 404 and 405 respectively using New Zealand albino rabbits. 39 The test item was suspension of the additive although at the time the tests were made whey powder was used instead of inulin as carrier. No skin reactions were observed and no adverse effects were seen after instillation into the eye. The difference in carrier is not expected to change the outcome of these studies. 35 Technical dossier/section III/Annex III Technical dossier/section III/Annex III Technical dossier/section III/Annex III Technical dossier/section III/Annex III Technical dossier/section III/Annex III-26. EFSA Journal 2013;11(5):

12 A skin sensitisation test (maximization test) was made with guinea pigs following OECD 406. A suspension of the test substance in saline (the additive as above) was given by intradermal injection on day 1 followed by a topical challenge on day 8 and day 22. Skin responses were recorded 24 h and 48 h after patch removal. No dermal reactions were detected in this study Effects on the respiratory system The additive is a granulated coated product with a low concentration of respirable particles and a moderate dusting potential. Consequently the potential for exposure via a respiratory route is limited and any potential adverse effects further reduced by the fat coating. Nonetheless, the applicant made two acute inhalation toxicity studies both based on OECD 403. In the first of these, a group of ten Wistar rats (five male and five female) exposed only to clean air were compared with a second group of 15 rats (five female and ten male) exposed (nose only) to an aerosol of the test item for a period of four hours. 40 Rats were observed for 14 days after exposure except for five male rats from the test group which were killed after 24 h for gross pathology and microbiology. It is unclear whether the test item was the additive or the active agent alone. Item-related mild alveolar/bronchiolar neutrophilic cell infiltrate was detected in the males subjected to the necropsy at 24 hours and the active agent also could be detected. Otherwise there were no test item related findings during the 14 day observation period and none thereafter. The second study was made using the additive as test item and involved six groups of five Wistar rats (three male groups and three female groups) treated with single doses of three increasing concentrations of the test item with intratracheal delivery (two groups per treatment). 41 The dose used ranged from 0.35 g to 4.5 g/kg bw from which an LD 50 of 9.4 g/kg bw could be derived for the additive by curvilinear extrapolation. Dyspnoea and lethargy were observed in connection with the delivery of the test item but surviving animals returned to normal within two days. No other treatment related affects were seen in surviving animals but 7/30 animals had died by day 7. Despite these findings, the proteinaceous nature of the additive is taken as an indication of a potential for respiratory sensitization Conclusions on the safety for the users The additive was non-irritant to eyes and skin and was not a skin sensitiser. Although the additive is formulated to minimise exposure by inhalation some exposure of the respiratory tract remains possible and the potential for respiratory sensitization cannot be excluded Safety for the environment The active agent in the additive is a strictly anaerobic strain originally isolated from bovine rumen. It would appear to naturally occur in the digestive tract but, as a potentially new genus, no data on its prevalence is available. However, as the strain has a very limited capability to survive in aerobic conditions, its use in animal production is unlikely to introduce an environmental concern. 4. Efficacy As EFSA acknowledged in its previous opinion (EFSA, 2005), the microbial metabolism of trichothecenes generally involves the enzymatic reduction of their 12, 13-expoxide group to the corresponding de-epoxy metabolite. The de-epoxy metabolites have been shown to be less toxic than their parents in a variety of cytotoxicity tests (see ) In vitro studies A series of incubation studies with the additive and buffered solutions containing the mycotoxin deoxynivalenol (DON) confirmed that the additive was able to fully degrade DON under anaerobic conditions at 37 ºC and 40 Technical dossier/section III/Annex III Technical dossier/section III/Annex III-25. EFSA Journal 2013;11(5):

13 physiologically relevant ph values, with the de-epoxy form DOM-1 as the sole resulting metabolite. 42 These in vitro assays form part of the quality control applied to production batches of the additive. A series of parallel studies were made using rumen fluid or digesta obtained from pigs artificially contaminated with DON. 43 Incubation of rumen fluid alone showed some activity towards DON (about 25 % reduction), but addition of 10 8 CFU/mL of the active agent resulted in the total transformation of DON to DOM-1 as the sole identified metabolite. Incubation of the artificially contaminated intestinal contents of the pig, unlike rumen fluid, had little demonstrable effect on DON. However, addition of the active agent to either duodenal or ileal contents resulted in the total reduction of DON Ex vivo studies Pig intestinal sections Pig intestine was taken immediately after slaughter. 44 Sections of defined length (approximately 15 cm) were cut from the duodenum, jejunum and ileum and tied at the ends. The gut sections were then inoculated by injection with 1 ml additive suspension containing a defined cell concentration, placed in anaerobic buffer solution containing 50 mg/l DON, and incubated at 37 C under a CO 2 atmosphere. Samples for analytical determination of DON and DOM-1 were taken after a 48 h incubation period. Control batches were prepared without addition of the additive. No metabolism of DON was detected in the control sections. In the presence of the additive the greatest extent of reduction of DON to DOM-1 was seen in the duodenum with less degradation seen in the jejunum and ileum. From these results it was concluded that the intestinal environment provides conditions suitable for the biotransformation of DON to DOM-1 by the additive Ussing chambers A six-week broiler feeding trial (total 277 birds) was conducted to evaluate the effects of DONcontaminated diets on the electrophysiological parameters of the gut. 45 A control group was given feed without addition of DON, a second group was fed the same diets with 10 mg/kg DON, and a third group was fed the DON-contaminated diets additionally supplemented with CFU/kg feed. At the end of the feeding period, seven birds from each group were killed. The effect of DON on the electrophysiological parameters of the jejunum was studied using isolated gut mucosa in Ussing chambers. Basal and glucose stimulated transmural potential difference, short-circuit current (Isc) and electrical resistance were measured. The transmural potential difference did not differ (P > 0.05) among groups. The tissue resistance was significantly greater (P < 0.05) in birds receiving DON plus the additive than in the control or the DON group. Addition of D-glucose on the luminal side of the isolated mucosa increased (P < 0.05) Isc in the control group and the additive containing group, whereas it decreased (P < 0.05) in the DON group indicating that the glucose-induced Isc was altered by DON. This indicated that the DON in the diet impaired the Na + -D-glucose co-transport in the jejunum of broilers. Addition of the additive counteracted this effect In vivo studies Short-term studies Two short-term studies were made with pigs with the intention of demonstrating that the biotransformation of DON to the de-epoxy-metabolite DOM-1 occurred in vivo by analyses of serum samples. 42 Technical dossier/section IV/Annexes IV Technical dossier/section IV/Annexes IV Technical dossier/section IV/Annex IV Technical dossier/section IV/Annex IV-21. EFSA Journal 2013;11(5):

14 Control BBSH Control BBSH Control BBSH Control BBSH Control BBSH Control BBSH ng/ml Micro-organism DSM for pigs The first experiment was intended to establish optimal sampling times. 46 Four mixed sex piglets (Piétrain (Landrace Large White)) were selected on basis of body weight. Two piglets received 1.67 mg DON each by single oral administration while the remaining two piglets obtained the same amount of toxin plus CFU of the active agent. Blood samples were taken before treatment and 0.5 hours, hours and hours after test administration. In control samples taken prior to the administration of DON and in all samples from the DON plus additive group, levels of DON in serum were under the limit of detection (LOD = 8 µg/l). In the group given only DON the highest serum value was seen in the final sampling period hours after administration. No DOM-1 was detected (LOD = 4 µg/l). DOM-1 was detected only in the DON plus additive group reaching 36 µg/l. The second study involved 24 piglets of the same breed as the first study. 47 These were assigned to three groups, the first given a standard diet with low DON content (0.6 mg/kg), the second the standard diet with wheat naturally contaminated with DON (1.8 mg/kg ~ twice the guidance value of Recommendation 2006/576/EC) and the third the same diet supplemented with CFU/kg feed of the active agent via the additive. Animals were housed in six pens of four piglets, allowing two replicate pens per treatment. Animals were adapted to restricted feeding using the standard diet. Blood samples were taken on day 1 before feeding experimental diets, on day 2, 1.5 h, 4 h and 10 h after feeding, on day 3, 1.5 and 4 h after feeding and on day 4, 24 h after feeding. DOM-1 was seen only in trace amounts in serum from samples taken on day one before exposure to the test diet. In day 2 and 3 samples the highest value for DOM-1 was recorded in serum from the 1.5 h samples from the group given the additive with the concentration falling with time (Figure 2). Far lower values for DOM-1 were measured in the animals given only the DON contaminated feed and no DOM-1 could be detected in serum from control animals DON DOM-1 a a A B a B b A a b B A B b A B b A Blood collections: Figure 2: Serum DON and DOM-1 concentrations in piglets fed control diet contaminated with DON (1.8 mg/kg) or the same contaminated diet supplemented with the additive BBSH at a dose of CFU/kg feed. Blood collections were 1: after feeding a blank diet (without addition of mycotoxin or BBSH); and later after feeding diets with added DON only (Control) or with both mycotoxin and additive (BBSH) 2: 1.5 h after 1 st feeding; 3: 4 h after 1 st feeding; 4: 10 h after 1 st feeding; 5: 1.5 h after 3 rd feeding; 6: 4 h after 3 rd feeding. Different letters (a,b for DON, A,B for DOM-1) above columns within a given blood collection mean significant differences (P<0.05) Performance studies A total of six studies were provided in which the performance of piglets or pigs for fattening given feed naturally contaminated with mycotoxins were tested in the absence or presence of the additive. Five of the six trials have been previously considered and reported by EFSA (2005). In three studies the mean final bodyweight of the 46 Technical dossier/section IV/Annex IV Technical dossier/section IV/Annex IV-36. EFSA Journal 2013;11(5):

15 animals were significantly higher in the groups treated with the additive compared to their respective controls while in the remaining three trials there were no significant differences found. Current legal and advisory limits for mycotoxin contamination of feedingstuffs are set to levels which ensure that no adverse effects on the health or performance of the most sensitive livestock species occur. As a consequence feeding studies in which the only parameters measured relate to growth, feed intake and the efficiency of feed conversion are considered inappropriate. Such studies are of value only when combined with the measurement of uptake and/or excretion of specific mycotoxins and their metabolites. None of the feeding studies made with piglets and pigs submitted in support of the current application included such measures Conclusion on efficacy As previously concluded (EFSA, 2005), the active agent has the capacity to reduce the epoxide group of trichothecenes to produce less toxic metabolites. The evidence presented in ex vivo and in vivo studies with DON confirms that this capacity can be realised in piglets when the additive is incorporated into diets at a minimum dose of 1.7 x 10 8 CFU/kg complete feed. The FEEDAP Panel notes that the level of DON used in the core study with piglets exceeded the guidance value. Given the presumed mode of action and the small deviation of the DON concentration in the diet in comparison to the guidance value, the FEEDAP Panel considers that the conclusion on biotransformation can be extended to lower dietary DON levels. The FEEDAP Panel finally concludes that the additive at a minimum dose of 1.7 x 10 8 CFU/kg complete feed has the capacity to biotransform trichothecenes from contaminated feed for all pigs. CONCLUSIONS The FEEDAP Panel accepts that the taxonomic position of the active agent is unclear and may warrant establishing a new species and/or genus. In the interim the deposition of the bacterium in a European culture collection is sufficient to ensure that any authorisation would apply only to the additive under application. No resistance to antibiotics of human and veterinary clinical significance and no homologies with known virulence factors could be detected in the strain. The additive is safe for piglets at the maximum dose proposed. This conclusion can be extended to pigs for fattening. The live organism is not expected to present a hazard for consumers and the negative findings in a series of genotoxicity and oral toxicity tests gives reassurance that toxic metabolites are not produced. As current legislation includes a guidance level for DON which already ensures animal and consumer safety and since the additive is expected to act only by the biotransformation of trichothecene mycotoxins to less toxic forms, no hazards for consumers would arise from the use of the additive in animal production at the recommended dose range. The additive is non-irritant to eyes and skin and is not a skin sensitiser. Although the additive is formulated to minimise exposure by inhalation some exposure of the respiratory tract remains possible and the potential for respiratory sensitization cannot be excluded. The active agent would appear to naturally occur in the digestive tract but, as a potentially new genus, no data on its prevalence is available. However, as the strain has a very limited capability to survive in aerobic conditions, its use in animal production is unlikely to introduce an environmental concern. The active agent has the capacity to reduce the epoxide group of trichothecenes to produce less toxic metabolites. The evidence presented in ex vivo and in vivo studies with DON confirms that this capacity can be realised in piglets when the additive is incorporated into diets at a minimum dose of CFU/kg complete feed. The FEEDAP Panel concludes that the additive at a minimum dose of CFU/kg complete feed has the capacity to biotransform trichothecenes from contaminated feed for all pigs. DOCUMENTATION PROVIDED TO EFSA 1. Biomin BBSH 797. June Submitted by Biomin GmbH 2. Biomin BBSH 797. Supplementary information. January Submitted by Biomin GmbH. EFSA Journal 2013;11(5):