pro 202 gt bacillus amyloliquefaciens pta 6507 bacillus amyloliquefaciens nrrl b 50013 and bacillus amyloliquefaciens nrrl b 50104 for chickens for fattening chickens reared for laying and minor poultry species for

SCIENTIFIC OPINION ADOPTED: 24 May 2016 doi: 10.2903/j.efsa.2016.4505 Safety and efficacy of Enviva® PRO 202 GT (Bacillus amyloliquefaciens PTA-6507, Bacillus amyloliquefaciens NRRL B-50013 and Bacillus amyloliquefaciens NRRL B-50104) for chickens for fattening, chickens reared for laying and minor poultry species for fattening and to point of lay EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) Abstract The additive Enviva® PRO 202 GT is a preparation containing viable spores of three strains of Bacillus amyloliquefaciens The additive is intended for use in feed for chickens for fattening, chickens reared for laying and minor poultry species for fattening and to point of lay at a minimum dose of 30 mg/kg feed, which corresponds to 7.5 107 CFU/kg complete feedingstuffs B amyloliquefaciens is considered by EFSA to be suitable for the qualified presumption of safety approach to establishing safety As the identity of the active agents was established and the lack of toxigenic potential and of resistance to antibiotics of human or veterinary clinical significance demonstrated, the additive is presumed safe for the target species, consumers and the environment Enviva® PRO 202 GT is non-irritant to skin and eyes and is not a dermal sensitiser Given the proteinaceous nature of the additive, there is a potential for respiratory sensitisation, although the risk is low due the low dusting potential Data from three trials provide evidence that the inclusion of Enviva® PRO 202 GT at the recommended dose has the potential to improve the performance of chickens for fattening This conclusion extends to the additive when used with chickens reared for laying and is extrapolated to minor avian species for fattening and to point of lay when used at the same dose The three active agents are compatible with narasin/nicarbazin, maduramicin ammonium, lasalocid A sodium, salinomycin sodium, monensin sodium, diclazuril, robenidine hydrochloride, decoquinate and semduramycin sodium and nicarbazin © 2016 European Food Safety Authority EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority Keywords: zootechnical additive, Enviva® PRO 202 GT, Bacillus amyloliquefaciens, safety, efficacy, QPS, chickens for fattening Requestor: European Commission Question number: EFSA-Q-2015-00179 Correspondence: feedap@efsa.europa.eu www.efsa.europa.eu/efsajournal EFSA Journal 2016;14(6):4505 Enviva® PRO 202 GT for poultry Panel members: Gabriele Aquilina, Giovanna Azimonti, Vasileios Bampidis, Maria de Lourdes Bastos, €rgen Gropp, Boris Georges Bories, Andrew Chesson, Pier Sandro Cocconcelli, Gerhard Flachowsky, Ju pez Puente, Marta Lo pez-Alonso, Alberto Mantovani, Baltasar Kolar, Maryline Kouba, Secundino Lo Mayo, Fernando Ramos, Guido Rychen, Maria Saarela, Roberto Edoardo Villa, Robert John Wallace and Pieter Wester Acknowledgements: The Panel wishes to thank the members of the Working Group on Microorganisms for the preparatory work on this scientific opinion The Panel also wishes to thank the members of the previous WG on Microorganisms 2012–2015, including Ingrid Halle Note: This scientific opinion has been amended following the adoption of the decision of the Commission on confidentiality claims submitted by the applicant, in accordance with Article 8(6) and Article 18 of Regulation (EC) No 1831/2003 The modified sections are indicated in the text Suggested citation: EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2016 Scientific opinion on the safety and efficacy of Enviva® PRO 202 GT (Bacillus amyloliquefaciens PTA-6507, Bacillus amyloliquefaciens NRRL B-50013 and Bacillus amyloliquefaciens NRRL B-50104) for chickens for fattening, chickens reared for laying and minor poultry species for fattening and to point of lay EFSA Journal 2016;14(6):4505, 12 pp doi:10.2903/j.efsa.2016.4505 ISSN: 1831-4732 © 2016 European Food Safety Authority EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority This is an open access article under the terms of the Creative Commons Attribution-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited and no modifications or adaptations are made The EFSA Journal is a publication of the European Food Safety Authority, an agency of the European Union www.efsa.europa.eu/efsajournal EFSA Journal 2016;14(6):4505 Enviva® PRO 202 GT for poultry Table of Contents Abstract Introduction 1.1 Background and Terms of Reference 1.2 Additional information Data and methodologies 2.1 Data 2.2 Methodologies Assessment 3.1 Characterisation 3.1.1 Characterisation of the active agents 3.1.2 Manufacturing and characterisation of the additive 3.1.3 Stability and homogeneity 3.1.4 Conditions of use 3.2 Safety 3.2.1 Safety for the target species, consumers and environment 3.2.2 Safety for the user 3.2.2.1 Effects on eyes and skin 3.2.2.2 Effects on the respiratory system 3.3 Efficacy 3.3.1 Efficacy for chickens for fattening 3.3.1.1 Conclusions on efficacy for chickens for fattening 3.3.2 Efficacy for chickens reared for laying and minor poultry species for fattening and to point of lay 3.3.3 Compatibility with coccidiostats 3.3.3.1 Conclusions on compatibility with coccidiostats 3.4 Post-market monitoring Conclusions Documentation provided to EFSA References Abbreviations Annex A – Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for Enviva® PRO 202 GT www.efsa.europa.eu/efsajournal 4 4 4 5 5 6 6 7 7 8 10 10 10 10 10 11 12 EFSA Journal 2016;14(6):4505 Enviva® PRO 202 GT for poultry Introduction 1.1 Background and Terms of Reference Regulation (EC) No 1831/20031 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 The European Commission received a request from Danisco (UK) Ltd.2 for authorisation of the product Enviva® PRO 202 GT (Bacillus amyloliquefaciens PTA-6507, Bacillus amyloliquefaciens NRRL B-50013 and Bacillus amyloliquefaciens NRRL B-50104), when used as a feed additive for chickens for fattening, chickens reared for laying and minor poultry species for fattening and to point of lay (category: Zootechnical additives; functional group: gut flora stabilisers) According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded to the European Food Safety Authority (EFSA) 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 dossiers in support of these applications The particulars and documents in support of the application were considered valid by EFSA as of 18 May 2015 According to Article of Regulation (EC) No 1831/2003, 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 EFSA shall deliver an opinion on the safety for the target animals, consumer, user and the environment and on the efficacy of the product Enviva® PRO 202 GT (Bacillus amyloliquefaciens PTA-6507, Bacillus amyloliquefaciens NRRL B-50013 and Bacillus amyloliquefaciens NRRL B-50104), when used under the proposed conditions of use (see Section 3.1.4) 1.2 Additional information ® Enviva PRO 202 GT is a preparation containing viable spores of the strains of B amyloliquefaciens (PTA-6507, NRRL B-50013 and NRRL B-50104) This additive has never been authorised in the European Union The species B amyloliquefaciens is considered by EFSA to be suitable for the qualified presumption of safety (QPS) approach to safety assessment (EFSA, 2007; EFSA BIOHAZ Panel 2013) This approach requires the identity of the strain to be conclusively established, evidence that the strain is not toxigenic and that it does not show resistance to antibiotics of human and veterinary importance Data and methodologies 2.1 Data The present assessment is based on data submitted by the applicant in the form of a technical dossier3 in support of the authorisation request for the use of Enviva® PRO 202 GT as a feed additive The technical dossier was prepared following the provisions of Article of Regulation (EC) No 1831/2003, Regulation (EC) No 429/20084 and the applicable EFSA guidance documents EFSA has verified the European Union Reference Laboratory (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 Annex A 2.2 Methodologies The approach followed by the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP Panel) to assess the safety and efficacy of Enviva® PRO 202 GT is in line with the principles laid down in Regulation (EC) No 429/2008 and the relevant guidance documents: Guidance 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, 18.10.2003, p 29 Danisco (UK) Ltd (trading as Danisco Animal Nutrition), PO Box 777, Marlborough, Wiltshire SN8 1XN, UK FEED dossier reference: FAD-2015-0008 Commission Regulation (EC) No 429/2008 of 25 April 2008 on detailed rules for the implementation of Regulation (EC) No 1831/2003 of the European Parliament and of the Council as regards the preparation and the presentation of applications and the assessment and the authorisation of feed additives OJ L 133, 22.5.2008, p www.efsa.europa.eu/efsajournal EFSA Journal 2016;14(6):4505 Enviva® PRO 202 GT for poultry on zootechnical additives (EFSA FEEDAP Panel, 2012a), Technical guidance: Tolerance and efficacy studies in target animals (EFSA FEEDAP Panel, 2011), Guidance on studies concerning the safety of use of the additive for users/workers (EFSA FEEDAP Panel, 2012b), Technical Guidance: Extrapolation of data from major species to minor species regarding the assessment of additives for use in animal nutrition (EFSA, 2008a), Technical guidance – update of the criteria used in the assessment of bacterial resistance to antibiotics of human or veterinary importance (EFSA FEEDAP Panel, 2012c), Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition (EFSA FEEDAP Panel, 2014), Technical guidance on the compatibility of zootechnical microbial additives with other additives showing antimicrobial activity (EFSA, 2008b) Assessment Enviva® PRO 202 GT is a preparation of viable spores of three strains of B amyloliquefaciens It is intended to be authorised as a zootechnical additive (functional group: gut flora stabilisers) for chickens for fattening, chickens reared for laying and minor poultry species for fattening and to point of lay 3.1 Characterisation 3.1.1 Characterisation of the active agents5 Two of the strains were isolated from turkey litter (PTA-6507 and NRRL B-50104) and the third (NRRL B-50013) is of unknown origin One is deposited with American Type Culture Collection (ATCC) with the accession number PTA-6507 and the other two in the United States Department of Agriculture Agricultural Research Culture Collection (NRRL) with the accession numbers NRRL B-50104 and NRRL B-50013.6 None of the strains has been genetically modified.7 The technical dossier contains the necessary information to demonstrate that the product strains belong to the species Bacillus amyloliquefaciens.8,9 The genetic stability was demonstrated by the comparison of cultures from 2004 and 2011 using PFGE patterns.10 Cytotoxicity tests were performed using Vero cells and the supernatants from each individual strain according to the Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition (EFSA FEEDAP Panel, 2014).11 No cytotoxic effects were detected The three active agents were tested for antibiotic susceptibility using broth micro-dilution techniques according to the technical guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance (EFSA FEEDAP Panel, 2012c).12 All minimum inhibitory concentration (MIC) values fell below the corresponding cut-off values defined by the FEEDAP Panel, and no further investigation is required 3.1.2 Manufacturing and characterisation of the additive5 The manufacturing process, and the qualitative and quantitative composition of the additive are detailed in the dossier, as well as the batch to batch variation These are based on five batches and on individual and total counts, confirming compliance with specifications.13,14,15 Specifications for chemical impurities are set for arsenic (< 15 mg/kg), lead (< 20 mg/kg), mercury (< 0.3 mg/kg), cadmium (< mg/kg), aflatoxin B1 (< 0.02 mg/kg), dioxins and PCBs (< 1.5 ng WHO-PCDD/F-PCB-TEQ/kg) Analysis of three batches showed that the product complies with the specifications and legal limits when these exist.16 Screening of mycotoxins showed the following values: deoxynivalenol and T2 toxin (< 10 lg/kg), aflatoxin B1, B2, G1 and G2 (< 0.5 lg/kg), total 10 11 12 13 14 15 16 This section has been amended following the provisions of Article 8(6) and Article 18 of Regulation (EC) No 1831/2003 Technical dossier/Section II/ Annex II.14 Technical dossier/Section II Technical dossier/Section II/ Annex II.13 Technical dossier/Section II and Supplementary information January 2016/Annexes II.15 and S1 Technical dossier/Section II/ Annex II.16 Technical dossier/Section II/ Annex II.17 Technical dossier/Section II/ Annex II.19 Technical dossier/Supplementary information January 2016 Technical dossier/Section II/Annex II.S2 Technical dossier/Supplementary information January 2016/Annex S2 Technical dossier/Section II/ Annexes II.3-5 www.efsa.europa.eu/efsajournal EFSA Journal 2016;14(6):4505 Enviva® PRO 202 GT for poultry aflatoxin (< lg/kg), ochratoxin A (< 20 lg/kg) and zearalenone (< lg/kg) Microbial quality is also monitored during the manufacturing process Tests of three batches showed levels of microbial contaminants that did not give rise to concerns (coliforms < 10 CFU/g, Escherichia coli < 10 CFU/g, Enterobacteriaceae < 10 CFU/g, Staphylococcus aureus < 20 CFU/g, Salmonella sp (absence in 25 g), Listeria sp (absence in 25 g) and Bacillus cereus < 10 CFU/g.17 The additive appears as a granular dry product Analysis of three batches by laser diffraction showed that on average ~2% (v/v) of the product consisted of particles with a diameter < 100 lm and a negligible fraction consisted of particles with a diameter < 50 lm.18 The dusting potential of the same three batches was tested following the BS EN 15051 method.18 The average dust concentration was 0.48 g/m3 3.1.3 Stability and homogeneity All the data refer to the total counts and not to counts of the single strains Being spores, the FEEDAP Panel does not expect relevant differences among the three strains The stability of three batches of the additive packed in sealed bags was monitored at 22°C and 37°C.19 Losses were lower than 0.5 log when stored at least 24 months up to 37°C Three batches of Enviva® PRO 202 GT mixed with a vitamin/mineral premix for poultry were stored for months at 22°C and 37°C in sealed foil bags.20 Microbial counts after this period showed that the product is stable over months at both temperatures (loss < 0.5 log) A single study was conducted to test the stability of the additive in mash poultry feed and its capacity of homogeneous mixing One batch of the additive was divided into three sub-batches and mixed with mash feed.21 Ten sub-samples were taken from each mixture for bacilli count determination The coefficient of variation found was 3%, demonstrating homogeneous mixing Three samples from each mixture were then taken and stored for months at 22°C and 37°C Bacterial counts remained unaffected (loss < 0.5 log) To test stability of Enviva® PRO 202 GT during feed pelleting processing, one batch of the additive was mixed with mash feed and subjected to pelleting at 95°C for 30 s.22 Pelleting did not affect viable counts of bacilli in feed (loss < 0.5 log) To test stability in poultry pelleted feed, a batch of the additive was mixed with mash feed and the mixture was subjected to a pelleting process (at 95°C for 30 s).23 The feed was then stored for months at 22°C and 37°C Bacterial counts remained stable during storage (loss < 0.5 log) 3.1.4 Conditions of use The product is intended for use in feed for chickens for fattening, chickens reared for laying and minor poultry species for fattening and to point of lay at a minimum dose of 7.5 107 CFU/kg complete feedingstuffs The applicant claims that the additive can be used in poultry feed in conjunction with coccidiostats 3.2 Safety 3.2.1 Safety for the target species, consumers and environment In the view of the FEEDAP Panel, the identity of the active agents is established as B amyloliquefaciens and the toxigenic potential and antibiotic resistance qualifications have been met Therefore, the additive is presumed safe for the target species, consumer of products derived from animals fed the additive and the environment 17 18 19 20 21 22 23 Technical Technical Technical Technical Technical Technical Technical dossier/Section II/ Annexes II.3-5 and dossier/Supplementary information January 2016/Annex S3 dossier/Section II/ Annex II.24 dossier/Section II/Annex II.25 dossier/Section II/Annex II.26 dossier/Section II/Annex II.28 dossier/Section II/Annex II.27 www.efsa.europa.eu/efsajournal EFSA Journal 2016;14(6):4505 Enviva® PRO 202 GT for poultry 3.2.2 Safety for the user 3.2.2.1 Effects on eyes and skin A skin irritation study compliant with OECD Guideline No 404 was carried out using Enviva® PRO 202 GT.24 No evidence of skin irritation was noted during the study and there were no signs of erythema/eschar or oedema Thus, Enviva® PRO 202 GT is considered not irritant to skin The eye irritancy potential of Enviva® PRO 202 GT was investigated according to OECD Guideline No 405.25 Moderate conjunctival irritation was noted in both treated eyes h after treatment with minimal conjunctival irritation noted at the 24-h observation but these signs disappeared within days Enviva® PRO 202 GT is considered not irritant to the eye A local lymph node assay was performed to assess the skin sensitisation potential of Enviva® PRO 202 GT in the CBA/Ca strain mouse following OECD Guideline 429.24 The test material was considered to be a non-sensitiser under the tested conditions 3.2.2.2 Effects on the respiratory system Respiratory toxicity was not investigated Given the proteinaceous nature of the additive, there is a potential for respiratory sensitisation, although the risk is low due the low dusting potential Conclusions on safety for the user Enviva® PRO 202 GT is non-irritant to skin and eyes, and is not a dermal sensitiser Given the proteinaceous nature of the additive, there is a potential for respiratory sensitisation, although the risk is low due the low dusting potential 3.3 Efficacy 3.3.1 Efficacy for chickens for fattening Three studies were conducted in two Member States to assess the effects of the supplementation of Enviva® PRO 202 GT to chickens for fattening In all cases, doses were confirmed by analysis of feed.13,27 The design of the studies is presented in Table and the results in Table The three trials involved 1-day-old chickens (Ross 308, 50% males and 50% females) randomly distributed in two experimental groups, one receiving the basal diets in mash form not supplemented and a second receiving the basal diets in mash form supplemented with the additive at the minimum recommended dose (7.5 107 CFU/kg complete feed) Study included a third treatment group receiving the additive at a higher dose Birds were fed ad libitum and had free access to water Observations included body weight and feed intake per pen at start, 21 and 35 days (and 42 days in trial 1) From these, weight gain and feed to gain ratio were calculated, and morbidity and mortality of birds were monitored during the whole experimental period Data were analysed using analysis of variance (ANOVA) as a completely randomised design with pen as the experimental unit Mortality was not influenced by treatment in any of the three studies Chickens supplemented with Enviva® PRO 202 GT at the minimum recommended dose showed a lower feed intake and a better feed to gain ratio in study 1, a better feed to gain ratio in study and a greater daily weight gain compared to control animals in study 24 25 26 27 Technical dossier/Section III/Annex III.1 Technical dossier/Section III/Annex III.2 Technical dossier/Section III/Annex III.3 Based on total bacilli counts and identification of the Enviva® PRO 202 GT strains by molecular methods www.efsa.europa.eu/efsajournal EFSA Journal 2016;14(6):4505 Enviva® PRO 202 GT for poultry Table 1: Trial no Details of the study design for the trials performed in chickens for fattening Basal diets (main ingredients) Total number of animals No replicates per treatment no birds per replicate Duration (days) Starter Finisher 1(a) 572 13 22 42 Maize/wheat/soybean Wheat/barley/maize/soybean 2(b) 858 13 22 35 Wheat/soybean Wheat/barley/maize/rye/soybean 3(c) 800 10 40 35 Wheat/soybean Wheat/barley/soybean (a): Technical dossier/Section IV/Annex IV.1.1 (b): Technical dossier/Section IV/Annex IV.2.1 (c): Technical dossier/Section IV/Annex IV.3.1 Table 2: Summary of the overall performance results of the three trials made with chickens for fattening Enviva® PRO 202 GT (CFU/kg feed) Feed intake (g/day) Final weight (kg) Average daily gain (g/day) Feed:gain (g/g) Mortality(1) (%) 105.5b 2.54 59.7 1.77b 4.5 a 2.54 1.98 59.7 55.4 1.72a 1.78b 7.7 5.9 Trial no 7.5 10 102.9 98.7 7.5 107 1.5 108 96.3 97.2 2.00 2.01 56.0 56.3 1.72a 1.73a 5.6 7.3 96.5 2.07 57.8a 1.67 1.5 98.6 2.14 60.0b 1.64 2.0 7.5 10 CFU: colony forming unit a,b : Means in a column within a trial with different superscript letters are significantly different (p ≤ 0.05) (1): Including culls in trials and 3.3.1.1 Conclusions on efficacy for chickens for fattening Data from three trials provide evidence that the inclusion of Enviva® PRO 202 GT at the recommended dose (7.5 107 CFU/kg complete feed) has the potential to improve the performance of chickens for fattening 3.3.2 Efficacy for chickens reared for laying and minor poultry species for fattening and to point of lay The dose proposed for use with chickens reared for laying and minor poultry species for fattening and to point of lay is the same as that demonstrated effective in a physiologically similar major species (chickens for fattening) and it can be reasonably assumed that the mode of action is the same Consequently, the conclusion on efficacy for chickens for fattening can be extended to chickens reared for laying and extrapolated to minor poultry species for fattening and to point of lay without a need for specific studies 3.3.3 Compatibility with coccidiostats5 An in vitro study was conducted to establish compatibility of the active agents with a series of coccidiostats (robenidine hydrochloride, salinomycin sodium, maduramicin ammonium, lasalocid A sodium, monensin sodium, diclazuril, nicarbazin and narasin).28 However, only the MIC for diclazuril and nicarbazin was four times greater than the maximum authorised dose in feed and compatibility can be presumed For the remaining coccidiostats, demonstration of in vivo compatibility would be required An in vivo study was conducted to establish compatibility of B amyloliquefaciens PTA-6507, B amyloliquefaciens NRRL B-50104 and B amyloliquefaciens NRRL B-50013 with narasin/nicarbazin, 28 Technical dossier/Section II/Annex II.29 www.efsa.europa.eu/efsajournal EFSA Journal 2016;14(6):4505 Enviva® PRO 202 GT for poultry maduramicin ammonium, lasalocid A sodium, salinomycin sodium, monensin sodium, diclazuril, robenidine hydrochloride, decoquinate and semduramicin sodium.29 The study involved 2,288 1-dayold chickens (Ross 708, both sexes) randomly allocated to 11 treatments, each replicated eight times and with 26 birds (13 males and 13 females) per replicate The treatments were: • • • • • • • • • • • T1: Negative control (no Enviva® PRO 202 GT – no coccidiostat) T2: Positive control (Enviva® PRO 202 GT at 107 CFU/kg feed – no coccidiostat) T3: Enviva® PRO 202 GT at 107 CFU/kg feed + narasin/nicarbazin at 100 mg/kg feed T4: Enviva® PRO 202 GT at 107 CFU/kg feed + maduramicin ammonium at mg/kg feed T5: Enviva® PRO 202 GT at 107 CFU/kg feed + lasalocid A sodium at 125 mg/kg feed T6: Enviva® PRO 202 GT at 107 CFU/kg feed + salinomycin sodium at 70 mg/kg feed T7: Enviva® PRO 202 GT at 107 CFU/kg feed + monensin sodium at 125 mg/kg feed T8: Enviva® PRO 202 GT at 107 CFU/kg feed + diclazuril at mg/kg feed T9: Enviva® PRO 202 GT at 107 CFU/kg feed + robenidine hydrochloride at 36 mg/kg feed T10: Enviva® PRO 202 GT at 107 CFU/kg feed + decoquinate at 40 mg/kg feed T11: Enviva® PRO 202 GT at 107 CFU/kg feed + semduramicin sodium at 25 mg/kg feed The trial lasted 35 days during which birds were followed for weight gain, feed to gain ratio and mortality On days 28 and 29, eight birds randomly selected from each treatment group were killed and ileal digesta samples collected and analysed for presumptive bacilli using the CEN method (EN 15784), with and without heat treatment in order to differentiate between the vegetative cells and spores The results are given in Table In addition, Bacillus morphology was observed and 60 colonies for treatment were isolated and identified at strain level using the approach described in the dossier Means of bacilli counts were compared using Dunnett multiple comparison test The technical guidance on the compatibility of zootechnical microbial additives with other additives showing antimicrobial activity (EFSA, 2008b) requires for products composed of multiple microbial strains that each strain be individually enumerated In this case, however, only total counts were provided followed by confirmation of the presence of the single strains by PFGE and SNP techniques As all three strains of the products represent B amyloliquefaciens, their differential plate counting was not possible Considering the technical difficulties, and the fact that the additive is composed of three strains in a 1:1:1 ratio, the approach was considered acceptable by the Panel Table 3: Ileal Bacillus amyloliquefaciens counts from chickens for fattening treated with the additive and different coccidiostats Treatment Log CFU B amyloliquefaciens/g ileal content À Heat treatment + Heat treatment Negative control 2.96 4.05 Positive control (PC) PC + narasin/nicarbazin 5.53 5.31 4.69 4.75 PC + maduramicin ammonium PC + lasalocid sodium 5.18 5.31 4.66 4.63 PC + salinomycin sodium PC + monensin sodium 5.07 5.19 4.71 4.46 PC + diclazuril PC + robenidine hydrochloride 5.42 5.40 4.76 4.67 PC + decoquinate 5.31 4.77 PC + semduramicin sodium 5.54 4.85 CFU: colony-forming unit The three B amyloliquefaciens strains composing the additive were identified in all ileal samples from treated birds There were no significant differences between the positive control and any of the treated groups in ileal Bacillus counts, and they were not affected by the heat treatment 29 Technical dossier/Section II and Supplementary information January 2016/Annex II.30 www.efsa.europa.eu/efsajournal EFSA Journal 2016;14(6):4505 Enviva® PRO 202 GT for poultry 3.3.3.1 Conclusions on compatibility with coccidiostats The compatibility of B amyloliquefaciens PTA-6507, B amyloliquefaciens NRRL B-50104 and B amyloliquefaciens NRRL B-50013 with narasin/nicarbazin, maduramicin ammonium, lasalocid A sodium, salinomycin sodium, monensin sodium, diclazuril, robenidine hydrochloride, decoquinate and semduramicin sodium and nicarbazin is demonstrated 3.4 Post-market monitoring The FEEDAP Panel considers that there is no need for specific requirements for a post-market monitoring plan other than those established in the Feed Hygiene Regulation30 and good manufacturing practice Conclusions The identity of the three strains as B amyloliquefaciens was established and the lack of toxigenic potential and of resistance to antibiotics of human or veterinary clinical significance demonstrated Consequently, the strains, following the QPS approach to safety assessment, are presumed safe for chickens for fattening, consumers of products from these animals fed the additive and for the environment This conclusion extends to the additive when used with chickens reared for laying and is extrapolated to minor avian species for fattening and to point of lay Enviva® PRO 202 GT is non-irritant to skin and eyes, and is not a dermal sensitiser Given the proteinaceous nature of the additive, there is a potential for respiratory sensitisation, although the risk is low due the low dusting potential Data from three trials provide evidence that the inclusion of Enviva® PRO 202 GT at the recommended dose of 7.5 107 CFU/kg complete feed has the potential to improve the performance of chickens for fattening This conclusion extends to the additive when used with chickens reared for laying and is extrapolated to minor avian species for fattening and to point of lay when used at the same dose The three active agents are compatible with narasin/nicarbazin, maduramicin ammonium, lasalocid A sodium, salinomycin sodium, monensin sodium, diclazuril, robenidine hydrochloride, decoquinate and semduramycin sodium and nicarbazin Documentation provided to EFSA 1) Enviva® PRO 202 GT (Bacillus amyloliquefaciens) for chickens for fattening, chickens reared for laying and minor poultry species for fattening and to point of laying March 2015 Submitted by Danisco (UK) Ltd 2) Enviva® PRO 202 GT (Bacillus amyloliquefaciens) for chickens for fattening, chickens reared for laying and minor poultry species for fattening and to point of laying January 2016 Submitted by Danisco (UK) Ltd 3) Evaluation report of the European Union Reference Laboratory for Feed Additives on the Methods(s) of Analysis for Enviva® PRO 202 GT 4) Comments from Member States References EFSA (European Food Safety Authority), 2007 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 EFSA Journal 2007;5(12):587, 16 pp doi:10.2903/j.efsa.2007.587 EFSA (European Food Safety Authority), 2008a Technical Guidance: Extrapolation of data from major species to minor species regarding the assessment of additives for use in animal nutrition EFSA Journal 2008;6(9):803, pp doi:10.2903/j.efsa.2008.803 EFSA (European Food Safety Authority), 2008b Technical Guidance – Compatibility of zootechnical microbial additives with other additives showing antimicrobial activity EFSA Journal 2008;6(3):658, pp doi:10.2903/ j.efsa.2008.658 30 Regulation (EC) No 183/2005 of the European Parliament and of the Council of 12 January 2005 laying down requirements for feed hygiene OJ L 35, 8.2.2005, p www.efsa.europa.eu/efsajournal 10 EFSA Journal 2016;14(6):4505 Enviva® PRO 202 GT for poultry EFSA BIOHAZ Panel (EFSA Panel on Biological Hazards), 2013 Scientific Opinion on the maintenance of the list of QPS biological agents intentionally added to food and feed (2013 update) EFSA Journal 2013;11(11):3449 108 pp doi:10.2903/j.efsa.2013.3449 EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2011 Technical Guidance: Tolerance and efficacy studies in target animals EFSA Journal 2011;9(5):2175, 15 pp doi:10.2903/ j.efsa.2011.2175 EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2012a Guidance for the preparation of dossiers for zootechnical additives EFSA Journal 2012;10(1):2536, 19 pp doi:10.2903/ j.efsa.2012.2536 EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2012b Guidance on studies concerning the safety of use of the additive for users/workers EFSA Journal 2012;10(1):2539, pp doi:10.2903/j.efsa.2012.2539 EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2012c Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance EFSA Journal 2012;10(6):2740, 10 pp doi:10.2903/j.efsa.2012.2740 EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2014 Guidance on the assessment of toxigenic potential of Bacillus species used in animal nutrition EFSA Journal 2014; 12(5):3665, 10 pp doi:10.2903/j.efsa.2014.3665 Abbreviations ANOVA ATCC CFU EURL FEEDAP Panel MIC PCB PCDD PCR PFGE QPS SNP TEQ WHO analysis of variance American Type Culture Collection colony-forming unit European Union Reference Laboratory EFSA Panel on Additives and Products or Substances used in Animal Feed minimum inhibitory concentration polychlorinated biphenyls polychlorinated dibenzo-para-dioxins polymerase chain reaction pulsed-field gel electrophoresis qualified presumption of safety single-nucleotide polymorphism toxic equivalent factor World Health Organization www.efsa.europa.eu/efsajournal 11 EFSA Journal 2016;14(6):4505 Enviva® PRO 202 GT for poultry Annex A – Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for Enviva® PRO 202 GT In the current application authorisation is sought under Article 4(1) for Enviva® PRO 202 GT under the category/functional group 4(b) ‘zootechnical additives’/‘gut flora stabilisers’, according to Annex I of Regulation (EC) No 1831/2003 Specifically, authorisation is sought for the use of the feed additive for chickens for fattening, chickens reared for laying, minor poultry species for fattening and minor poultry species reared to point of lay According to the Applicant, the feed additive contains as active substances viable spores of the three nongenetically modified strains Bacillus amyloliquefaciens BS 15A-P4 (PTA-6507), Bacillus amyloliquefaciens LSSAO1 (NRRL B-50104) and Bacillus amyloliquefaciens BS 2084 (NRRL B-50013) The feed additive is to be marketed as granular dry product containing a minimum concentration of total active substances of 2.5 109 colony forming unit (CFU)/g The feed additive is intended to be used directly in feedingstuffs or through premixtures at a minimum dose of total active substances of 7.5 107 CFU/kg complete feedingstuffs For the identification and characterisation of Bacillus amyloliquefaciens BS 15A-P4 (PTA-6507), Bacillus amyloliquefaciens LSSAO1 (NRRL B-50104) and Bacillus amyloliquefaciens BS 2084 (NRRL B-50013), the EURL recommends for official control Pulsed Field Gel Electrophoresis (PFGE), a generally recognised standard methodology for genetic identification This standard methodology for microbial identification is currently being evaluated by the CEN Technical Committee 327 to become a European Standard For the enumeration of all the Bacillus amyloliquefaciens strains (BS 15A-P4, LSSAO1 and BS 2084) in feed additive, premixtures and feedingstuffs the Applicant submitted the ring-trial validated spread plate method EN 15784 which was already evaluated by EURL in the frame of a previous Bacillus amyloliquefaciens dossier Based on the performance characteristics available, the EURL recommends for official control this CEN method (EN 15784) for the enumeration of Bacillus amyloliquefaciens (BS 15A-P4, LSSAO1 and BS 2084) in the feed additive, premixtures and feedingstuffs 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 The full report is available on the EURL website: https://ec.europa.eu/jrc/sites/default/files/finrep_fad_2015_0008_enviva_ pro202gt.pdf www.efsa.europa.eu/efsajournal 12 EFSA Journal 2016;14(6):4505 ... the product Enviva® PRO 202 GT (Bacillus amyloliquefaciens PTA- 6507, Bacillus amyloliquefaciens NRRL B- 50013 and Bacillus amyloliquefaciens NRRL B- 50104) , when used as a feed additive for chickens. .. amyloliquefaciens NRRL B- 50013 and Bacillus amyloliquefaciens NRRL B- 50104) for chickens for fattening, chickens reared for laying and minor poultry species for fattening and to point of lay EFSA... reared for laying and minor poultry species for fattening and to point of laying March 2015 Submitted by Danisco (UK) Ltd 2) Enviva® PRO 202 GT (Bacillus amyloliquefaciens) for chickens for fattening,