SCIENTIFIC OPINION. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3

Transcription

1 EFSA Journal 2015;13(3):4055 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of Cibenza EP150 (a preparation of Bacillus licheniformis (ATCC 53757) and its protease (EC )) as a feed additive for chickens for fattening, chickens reared for laying and minor avian species for fattening and to point of lay and ornamental birds 1 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3 ABSTRACT European Food Safety Authority (EFSA), Parma, Italy Cibenza EP150 is a feed additive containing a protease produced by fermentation of a single strain of Bacillus licheniformis. The final preparation contains the enzyme, spores of the production organism and spent medium. It is intended for use as a zootechnical feed additive for chickens for fattening, chickens reared for laying and all relevant minor avian species in the growing or fattening phases but excluding laying birds. Chickens for fattening appeared to tolerate the additive at ten times the recommended dose and consequently the additive is considered safe at the recommended dose of 500 mg/kg complete feedingstuffs. This conclusion applies to chickens reared for laying and can be extrapolated to minor avian species used for meat production and ornamental birds when the same dose is applied. Use of the additive in feed for chickens for fattening, chickens reared for laying and other avian species is considered safe for consumers. The additive is not a skin irritant but is an eye irritant. Because of the observed severe irritation in the eye and the known capacity of enzyme products to induce sensitisation, the additive has the potential to be a skin sensitiser and to induce sensitisation following respiratory exposure. The organism, a strain of a naturally-occurring soil bacterial species, and its fermentation products are considered not to present a hazard for the environment. A significant production benefit of the addition of the additive using the recommended dose was seen in chickens for fattening but only when a reduced protein diet was provided. No effects were seen when a standard poultry diet was fed. This conclusion on efficacy is taken to apply to chickens reared for laying and can be extrapolated to other avian species used for meat production when the same dose is used. Use of the additive is compatible with coccidiostats. European Food Safety Authority, 2015 KEY WORDS zootechnical additive, Bacillus licheniformis, protease, chickens for fattening, minor avian species, safety, efficacy 1 On request from the European Commission, Question No EFSA-Q , adopted on 11 March Panel members: Gabriele Aquilina, 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 Other Zootechnical Additives, including Andrew Chesson, 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 Cibenza EP150 (a preparation of Bacillus licheniformis (ATCC 53757) and its protease (EC )) as a feed additive for chickens for fattening, chickens reared for laying and minor avian species for fattening and to point of lay and ornamental birds. EFSA Journal 2015;13(3):4055, 16 pp. doi: /j.efsa Available online: European Food Safety Authority, 2015

2 SUMMARY Following a request from 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 and efficacy of the feed additive Cibenza EP150 Cibenza EP150 contains a protease produced by fermentation of a single strain of Bacillus licheniformis. The final preparation contains the specified enzyme, spores of the production organism (both of which are declared constituents) and spent medium. It is prepared in dry form and is intended for use as a zootechnical feed additive (functional group other zootechnical additives ) for chickens for fattening, chickens reared for laying and all relevant minor avian species in the growing or fattening phases (but excluding laying birds) and ornamental birds. On the basis of a tolerance trial made with chickens for fattening, birds appeared to tolerate the additive at ten-times the recommended dose and consequently the additive is considered safe at the recommended dose of 500 mg/kg complete feedingstuffs. This conclusion also applies to chickens reared for laying and can be extrapolated to other avian species used for meat production and ornamental birds when the same dose is applied. Use of the additive in feed for chickens for fattening, chickens reared for laying and other avian species used for meat production is considered safe for consumers. The additive is not a skin irritant but is an eye irritant. Because of the observed severe irritation in the eye and the known capacity of enzyme products to induce sensitisation, the additive has the potential to be a skin sensitiser and to induce sensitisation following respiratory exposure. The organism, which is a strain of a naturally-occurring soil bacterial species, and its fermentation products are considered not to present a hazard for the environment. A significant production benefit of the addition of the additive using the recommended dose (500 mg/kg complete feedingstuffs) was seen in chickens for fattening but only when a reduced protein diet was provided. No significant effects were seen when a standard poultry diet was fed. This conclusion on efficacy is taken to apply to chickens reared for laying and can be extrapolated to other avian species used for meat production and ornamental birds when the same dose is used. Use of the additive is compatible with the following coccidiostats: diclazuril, nicarbazin, decoquinate, semduramycin, lasalocid sodium, monensin sodium, robenidine hydrochloride, maduramicin ammonium, narasin and salinomycin. EFSA Journal 2015;13(3):4055 2

3 TABLE OF CONTENTS Abstract... 1 Summary... 2 Background... 4 Terms of reference... 4 Assessment Introduction Characterisation Characterisation of the production organism/active agent Identity Genetic stability Toxigenic potential Antibiotic resistance and antibiotic production Characterisation of the protease Characterisation of the additive Production process Purity Physical properties Stability and homogeneity Conditions of use Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) Safety Safety for the target species Safety for chickens for fattening Safety for chickens reared for laying Safety for minor avian species for fattening and to point of lay and ornamental birds Safety for the consumer Safety for the user Effects on eyes and skin Sensitization Safety for the environment Efficacy Efficacy for chickens for fattening Compatibility with coccidiostats when used with chickens for fattening Efficacy for chickens reared for laying Efficacy for minor avian species for fattening and to point of lay and ornamental birds Post-market monitoring Conclusions Documentation provided to EFSA References Annex A. Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Methods of Analysis for Cibenza EP EFSA Journal 2015;13(3):4055 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 Novus Europe S.A./N.V. 5 for authorisation of the product Cibenza EP150, a preparation of protease (EC ) and Bacillus licheniformis (ATCC 53757), when used as a feed additive for chickens for fattening, chickens reared for laying and minor avian species for fattening and to point of lay (category: zootechnical additives; functional group: other zootechnical additives) under the conditions mentioned in Table 1. According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the application to the European Food Safety Authority (EFSA) as an application under Article 4(1) (authorisation of a feed additive or new use of a feed additive). EFSA received directly from the applicant the technical dossier in support of this application. 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 11 September The additive Cibenza EP150 is a preparation of Bacillus licheniformis (ATCC 53757) and its protease (EC ). This product has not been previously authorised in the European Union (EU). 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 Cibenza EP150, a preparation of Bacillus licheniformis (ATCC 53757) and its protease (EC ), 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. Novus Europe S.A./N.V. Woluwe Atrium, Neerveld , 1200 Brussels, Belgium. EFSA Dossier reference: FAD EFSA Journal 2015;13(3):4055 4

5 Table 1: Description and conditions of use of the additive as proposed by the applicant Cibenza EP 150 for poultry Additive Registration number/ec No/No (if appropriate) Category(ies) of additive Functional group(s) of additive Protease, EC and Bacillus licheniformis, PWD-1 (ATCC 53757) 4dxxx Zootechnical Other (performance enhancer) Composition, description Preparation of protease and Bacillus licheniformis Description Chemical formula Protease, EC and Bacillus licheniformis, PWD-1 (ATCC 53757) Purity criteria (if appropriate) Complies with EU feed hygiene law Method of analysis (if appropriate) Validated spectrophotometric method for protease; BS EN 15784:2009 for Bacillus licheniformis Trade name (if appropriate) Name of the holder of authorisation (if appropriate) Cibenza EP150 Novus Europe S.A./N.V. Species or category of animal Chickens for fattening Maximum Age To slaughter age and weight Conditions of use Minimum content Maximum content Withdrawal Units of activity or CFU/kg of complete period feedingstuffs (if appropriate) Chickens reared for laying Minor avian species for fattening 24 weeks (point of lay) Up to slaughter age and weight U protease/kg; Bacillus licheniformis, PWD-1 (ATCC 53757), CFU/kg Not applicable Not applicable Minor avian species to point of lay weeks (depending on species) 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 original closed packaging and in a cool, dry place (<25ºC) Do not breathe dust / spray. Avoid contact with eyes, skin, or clothing. Use personal protection equipment when handling the product (goggles, masks, rubber gloves and protective clothing). As per EU feed hygiene regulation: traceability, HACCP-based quality control, formal product/service complaints procedure, and product recall capability. Final feedingstuff should contain minimum 500 mg Cibenza EP150/kg Maximum Residue Limit (MRL) (if appropriate) Species or category of Target tissue(s) or Maximum content Marker residue animal food products in tissues Not applicable Not applicable Not applicable Not applicable EFSA Journal 2015;13(3):4055 5

6 ASSESSMENT 1. Introduction Cibenza EP150 is a feed additive containing a protease produced by fermentation of a single strain of Bacillus licheniformis. The final preparation contains the specified enzyme, spores of the production organism (both of which are declared constituents) and spent medium. It is prepared in dry form and is intended for use as a zootechnical feed additive (functional group other zootechnical additives ) for chickens for fattening, chickens reared for laying and all relevant minor avian species in the growing or fattening phases to point of lay, as well as young or adult ornamental or performing birds, but excluding laying birds. It is not authorised in the EU. 2. Characterisation 2.1. Characterisation of the production organism/active agent Identity The single strain of B. licheniformis was originally isolated from a poultry waste digester in the USA and is deposited in the American Type Culture Collection (ATCC) with the accession number ATCC It has not been genetically engineered and does not harbour plasmids. Identification was confirmed by biochemical parameters and sequencing of the full 16S rrna gene and the 16S 23S internal transcribed spacer Genetic stability The potential for genetic drift was studied after ten consecutive subcultures using macrorestriction analysis followed by pulsed field gel electrophoresis. 9 For this purpose, two different enzymes, Not I and Sfi I, were used. No differences in the restriction patterns at the start and end of the experiment were observed with either enzyme. A study of this type gives no information on long-term stability which, ideally should be measured over a period of years. It can, however, indicate how many times the same working culture can be used as an inoculum before obtaining a fresh sub-culture of the reference material Toxigenic potential Application was made at the time when the guidance on the assessment of the toxigenic potential of Bacillus spp. introduced in 2011 (EFSA FEEDAP Panel, 2011) was the recommended approach. Following this approach no haemolytic activity was found when B. licheniformis was streaked onto bovine blood agar plates and incubated at 30 ºC for 72 h. 10 A PCR assay for non-ribosomal peptide synthase genes made using primers for surfactin, fengycins, plipastatins and mycosubtilin. There was no detectable amplification of surfactin or plipastatins, although in the latter case the positive control was also negative. A weak signal was found for mycosubtilin but a much stronger one was seen for fengycins Subsequent to the publication of the 2011 guidance, it was recognised that non-ribosomal peptides are commonly, if not universally, present in Bacillus species belonging to the B. subtilis group. In addition the association between the presence of such surfactin-like peptides and the occurrence of food intoxication has not been established. For this reason the 2011 guidance was reassessed and revised (EFSA FEEDAP Panel, 2014). This revised approach retained only a cytotoxicity assay made with culture filtrates without concentration. 7 Technical dossier/section II/Annex_II_2_1_2_1. 8 Technical dossier/section II/Annex_II_2_1_2_2. 9 Technical dossier/section II/Annex_II_2_1_2_2. 10 Technical dossier/section II/Annex_II_2_2_2_1 and Annex_II_2_2_2_2. EFSA Journal 2015;13(3):4055 6

7 Two cytotoxicity assays were made using Vero cells which fulfilled the revised approach as recommended by EFSA in In both assays, the effect of the culture supernatant of B. licheniformis (or a ten-fold concentrate of the supernatant) was monitored by measuring the uptake of radio-labelled leucine. In the first assay, there was a weak indication of inhibition of protein synthesis with the culture supernatant (~28%) and an almost total inhibition by the concentrated supernatant (~98%). Repetition of the Vero cell cytotoxicity assay, including a heat treatment of the test items, showed similar results for the non heat-treated supernatant. No inhibition of protein synthesis was seen in the presence of the culture filtrate although a ten-fold concentration again produced almost total inhibition. Heat-treatment reduced by about half the inhibitory effect of the concentrated supernatant. The Panel considers that the evidence provided indicate that the strain does not exhibit a toxigenic potential Antibiotic resistance and antibiotic production The strain was tested for antibiotic susceptibility using the broth dilution method and two growth media (trypticase soya broth and Mueller Hinton broth). 12 The battery of antibiotics used was that recommended by EFSA (EFSA FEEDAP Panel, 2012). As all minimum inhibitory concentration (MIC) values for the B. licheniformis strain fell below the corresponding breakpoints defined by the FEEDAP Panel regardless of the media used no further investigation is required. Two similar methods were used to detect the production of antimicrobial substances. 13 Either a filter paper impregnated with supernatant from an overnight culture of B. licheniformis or a plug of agar from an overnight plate culture was applied to a lawn culture of the test bacterial species. A zone of clearing was taken as evidence of antimicrobial activity by B. licheniformis. No activity against any of the test strains (Escherichia coli, Serratia marcescens, Bacillus cereus, Staphylococcus aureus, Streptococcus pyogenes and Bacillus circulans) was detected Characterisation of the protease The protease (EC ) in the additive derives entirely from the fermentation of B. licheniformis (ATCC 53757) Characterisation of the additive Production process The additive is produced by batch fermentation of B. licheniformis in a typical industrial medium (details provided). After fermentation the entire fermentation broth is concentrated by evaporation and spray dried. The fermentation concentrate is then blended with sufficient carrier (CaCO 3 ) to meet specifications. Full details of the quality control procedures taken during manufacture are provided. The final additive is guaranteed to contain a minimum of units 14 protease activity and CFU B. licheniformis spores/g additive. Data from five batches show compliance with the minimum specification (mean protease units/g with a cv% of 4.1 and mean spore count CFU/g with a cv% of 8.2). 15 The applicant stated that no antimicrobials are used during the fermentation process and further downstream processes for the production of Cibenza EP Purity Five batches of the additive were analysed for heavy metals, arsenic, dioxins and dioxin-like PCBs, and for the presence of six commonly encountered mycotoxins in feed (aflatoxins, fumonisin B1, 11 Technical dossier/section II/Annex_II_2_2_2_1 and Annex_II_2_2_2_2. 12 Technical dossier/section II/Annex_II_2_1_2_2. 13 Technical dossier/section II/Annex_II_2_1_2_2. 14 One azocasein unit of protease activity is defined as an increase of absorbance of 0.01 units per 15 min period as measured at 410 nm in a 1 ml volume at ph 8.0 and temperature of 37 C with azocasein as the substrate. 15 Technical dossier/section II/Annex_II_1_3_3_2. EFSA Journal 2015;13(3):4055 7

8 zearalenone, T2 toxin, deoxynivalenol and ochratoxin A). 16 Mean values for the heavy metals were cadmium, 0.38 mg/kg, lead, 0.63 mg/kg and mercury <0.01 mg/kg and for arsenic was <1 mg/kg additive. The mean value found for dioxins (dioxin-teq) was 0.18 ng/kg additive and for dioxin-like PCBs (WHO PCDD/F-TEQ) ng/kg additive. All of the mycotoxins were below the respective limits of detection. None of these values are cause for concern. A microbial examination was made with three batches of the product. 17 This showed that counts of filamentous fungi, total coliforms and E. coli were all <10 CFU/g additive, yeasts had a mean count of ~ 50 CFU/g and that no Shigella or Salmonella spp. could be detected in 25 g additive. In a separate exercise six batches were examined for the presence of B. cereus and all were found to have counts of <10 CFU/g additive Physical properties The additive is formulated as a dry brown powder with a mean particle size of around 35 µm based on the laser diffraction measurements of three batches. Approximately 20 % of particles in the additive have diameters < 10 µm and are considered respirable, with this value doubling to 40 % when the dust fraction collected during the analysis of dustiness alone is examined. Despite the low particle size, the dustiness determined on the same three batches of product were relatively low (mean 0.5 g/m 3 with a range of 0.25 to 0.64 g/m 3 ) Stability and homogeneity Shelf life Three batches of the additive were stored at 25 ºC/60% relative humidity (RH) or 40 ºC/75% RH for 24 and six months, respectively. 19 No loss of bacterial viability was observed at the lower temperature, while protease activity was reduced by approximately 10% during the 24 months storage. At the higher temperature some 30% of protease activity was lost within four months but bacterial counts were essentially unaffected. However a significant reduction was observed at 6 months (50% in protease activity, 4 5 log reduction in bacterial counts). Premixtures Three batches of the additive were incorporated into a vitamin-mineral premix and stored for a period of six months under constant conditions (25 ºC/60 % RH). 20 Samples were withdrawn at monthly intervals for the first three months and then after six months. No loss of bacterial viability was seen over the entire storage period while protease activity decreased by an average of 23 % after six months. A second study was performed with a similar design which confirmed the above results. 21 Feed Three batches of the additive were incorporated into a wheat-soybean based diet prepared in both mash and pelleted form. 22 Prepared diets were stored at 25 ºC/60 % RH for three months. Essentially no loss of bacterial viability was observed in either diet form after three months. The protease activity was similarly little affected. However, the analysis of the pelleted feed used post-pelleting values as the baseline for comparison. The process of pelleting at 65 ºC resulted in a loss of approximately half of the protease activity and a 35 % reduction in bacterial counts. 16 Technical dossier/section II/Annex_II_1_4_2. 17 Technical dossier/section II/Annex_II_1_4_3. 18 Technical dossier/section II/Annex_II_1_4_4. 19 Technical dossier/section II/Annex_II_4_1_1 and Supplementary information January 2015/Annex_II_4_1_4. 20 Technical dossier/section II/Annex_II_4_1_2. 21 Technical dossier/supplementary information January 2015/Annex_II_4_1_5. 22 Technical dossier/section II/Annex_II_4_1_3. EFSA Journal 2015;13(3):4055 8

9 Homogeneity of mixing The ability of the additive to be uniformly distributed was examined using a single batch of product mixed into a premix and a mash feed. 23 In both cases ten sub-samples were randomly collected and analysed. Protease activity was found uniformly mixed in both premix and mash feed at the expected concentration (CV of 5 % in both cases). Surprisingly, the bacterial component of the additive showed a far greater variation between sub-samples with a CV of 12.8 % in the case of the premix and 16.6 % in the case of the mash feed Conditions of use The additive is intended for use with chickens for fattening, minor avian species used for meat production, chickens reared for laying to the point of lay, minor avian species also reared to the point of lay and ornamental birds. A single dose is proposed for all applications of 500 mg additive/kg complete feed delivering a minimum of units of protease and CFU B. licheniformis 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 substance and agent in animal feed. The Executive Summary of the EURL report can be found in Annex A. 3. Safety 3.1. Safety for the target species Safety for chickens for fattening A tolerance study was made with 800 one-day-old male and female Ross 308 chickens for fattening. 24 Birds were allocated to one of four treatment groups and housed in groups of 20 birds of the same sex. A total of ten pens were used for each treatment (five male and five female pens). Birds were fed a typical maize/soybean meal poultry diet in mash form without the additive or supplemented with 500 mg, mg or mg Cibenza EP150/kg feed. This corresponded to the recommended dose and five and ten times the recommended dose (confirmed by analysis). All diets contained diclazuril as coccidiostat and phytase. The study duration was 35 days during which time birds were monitored for adverse effects and performance. Feed to gain values were calculated. Blood samples were collected at the end of the study on day 35 and analysed for limited haematology 25 and clinical chemistry parameters. 26 Faeces were collected on days 21 and 35 and a subjective assessment of litter quality made on days 13 and 27. All data was subject to an analysis of variance. Birds reached approximately 1.75 kg body weight by the end of the experiment with no significant differences between groups. Similarly average daily gain, feed intake and feed to gain ratio was not significantly affected by the additive. Mortality was approximately 4% and similar in all treatment groups. Litter quality was good for all treatments and there were no recorded cases of diarrhoea. No significant differences were observed in haematology but serum albumin content was significantly reduced in the 1 and 5 treatment groups compared to the control. This was reflected in the total protein values which were similarly significantly reduced in these groups. However, all values remained within the normal range and the highest dose group did not show the same reduction and was not significantly different from the mean control value. 23 Technical dossier/section II/Annex_II_4_1_3. 24 Technical dossier/section III/Annex III_1_1_1. 25 Total blood cell count, haemoglobin content and haematocrit value. 26 Aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase, glucose, uric acid, total protein and albumin. EFSA Journal 2015;13(3):4055 9

10 Overall birds appeared to tolerate the additive at ten-times the recommended dose and consequently the additive is considered safe for chickens for fattening at the recommended dose of 500 mg/kg complete feedingstuffs Safety for chickens reared for laying The conclusion on safety for chickens for fattening is taken to apply to chickens reared for laying when the same dose is used. Therefore, the additive is considered safe for chickens reared for laying at 500 mg/kg feed without the need for additional studies Safety for minor avian species for fattening and to point of lay and ornamental birds Since the safety of the additive for chickens for fattening has been established with a wide margin of safety and considering that the applicant proposes the use of the same dose in minor avian species, safety for minor avian species can be presumed without the need for specific studies. Therefore, Cibenza EP150 can be presumed safe for minor avian species grown for meat production or reared to the point of lay and ornamental birds at a dose of 500 mg/kg feed without the need for additional studies Safety for the consumer The additive consists of viable cells of a strain of B. licheniformis, a protease enzyme, other metabolites produced during fermentation and spent medium. B. licheniformis meets the criteria for a qualified presumption of safety (QPS) approach to safety assessment. Its identity is established and it does not carry resistance determinants to antibiotics of human clinical and veterinary importance or exhibit a toxigenic potential as judged by the current guidance. Consequently, the strain is presumed safe for the target species, consumers and the environment. The protease present will be degraded as any other protein by the target species and so no direct exposure of consumers will occur. In addition, as the enzyme derives from an organism presumed safe by the QPS approach, no other metabolites of concern produced during fermentation and included in the additive are to be expected. Use of the additive in feed for chickens for fattening, chickens reared for laying and other avian species used for meat production is considered safe for consumers Safety for the user Effects on eyes and skin GLP compliant skin 27 and eye 28 irritation tests were made following OECD guidelines 404 and 405 respectively. Limited erythema was seen in two of five rabbits at 24 h after application of the test substance (the additive), disappearing within 72 hours. Accordingly, and following the classification of Council Directive 67/548/EEC, 29 the additive was found to be non-irritant to skin. The eye irritation test was made with a single rabbit with 0.1 g of the additive instilled into one eye with the other eye serving as control. Serious signs of irritation were observed after one hour. After 24 hours and despite washing of the treated eye, because of the severity of the symptoms the animal was euthanized Sensitization Because of the observed severe irritation in the eye and the known capacity of enzyme products to induce sensitisation, no sensitisation assays were made. The applicant acknowledges that the additive has the potential to be a skin sensitiser and, because of the low particle size, also to induce 27 Technical dossier/section III/Annex III_3_1_2_1. 28 Technical dossier/section III/Annex III_3_1_2_2. 29 Council Directive 67/548/EEC of 27 June 1967 on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances. OJ L 196, , p. 1. EFSA Journal 2015;13(3):

11 sensitisation by respiratory exposure. The recommendations made by the applicant for handling the product, including the use of goggles and a respirator, reflect the seriousness of the hazards identified Safety for the environment On the basis of the QPS assessment, the organism which is a strain of a naturally-occurring soil bacterial species, and its fermentation products are considered not to present a hazard for the environment. 4. Efficacy 4.1. Efficacy for chickens for fattening A total of five efficacy trials are described, three made in the USA and two in different EU Member States. Throughout the applicant uses the term positive control to describe a standard poultry diet without the inclusion of the additive and negative control for a diet with deliberately reduced total protein and amino acid content without the additive. All of the studies took essentially the same form in which male only Cobb broiler chicks were assigned to treatments which always included a positive control, one or two negative (reduced protein) controls and the negative control diet supplemented with the additive at 500 mg/kg diet and in two studies additionally at 250 mg/kg diet. In only the two European studies was the effect of the additive tested using a standard diet which fully met requirements for protein. Basal diets were similar in composition and were based on maize/soybean meal fed initially in crumb and latterly in pelleted form. The duration of the studies was 42 days (studies 2 5) or 45 days (study 1). The three studies made in the USA tested essentially the ability of the additive to restore performance when diets deliberately formulated with reduced protein content were used. The extent of the reduction depended on the stage of growth but was in the order of 5 6 % when measured as crude protein (starter feed for control: %). In the first study chicks were assigned to one of four treatments (nine pens of 50 birds/treatment. In the second were allocated to one of three treatments (eight pens of 45 birds/treatment) and in the third 800 to one of four treatments (ten pens of 20 birds/treatment). Performance characteristics were measured and the data subjected to statistical analysis by analysis of variance using the pen as the experimental unit. The results of these three studies are summarised in Table 2. In all three studies, as would be expected, there was a trend towards a reduction in performance in birds fed the reduced protein diet compared to the positive control. There was evidence that this reduction could be reversed by the addition of the additive. This only reached significance for average weight gain in one study but the feed to gain ratio showed significant benefits associated with addition of the additive in all studies. Systems for intensive poultry production are very similar in the USA and Europe and, consequently, the results obtained are likely to be relevant to the European situation. EFSA Journal 2015;13(3):

12 Table 2: Summary of the results of the three studies made in the USA on the effects of Cibenza EP150 on the performance of chickens for fattening Treatment Average weight gain (kg/bird) Average feed intake (kg/bird) Feed:gain Mortality (%) Study 1 (1) Positive control 3.01 b a 2.7 Negative control 2.90 a b 5.3 Negative control mg/kg additive 2.99 b a 5.6 Negative control mg/kg additive 3.03 b a 4.9 Study 2 (2) Positive control ab 6.7 Negative control b 9.4 Negative control mg/kg additive a 7.3 Study 3 (3) Positive control ab 3.5 ab Negative control a 9.0 ab Negative control mg/kg additive b 10.0 a Negative control mg/kg additive ab 3.0 b a, b: Means in a column within the same trial with different superscript letters are significantly different (P 0.05). (1): Technical dossier/section IV/Annex IV_3_1. (2): Technical dossier/section IV/Annex IV_3_2. (3): Technical dossier/section IV/Annex IV_3_3. The remaining two studies also included a reduced protein diet with and without the additive as in the American studies, but also included a test group in which the additive was added to the positive control (starter feed: 21%). In the first of the European studies (study 4) 719 Ross 308 broiler chicks (males and females) were allocated to one of four treatment groups (ten pens of 18 birds/treatment) and in the second Ross 308 male only broiler chicks were allocated to one of six treatments (ten pens of 35 birds/treatment). Diets were based on maize/soybean meal and fed in pelleted form. As with the previous studies, the reduced protein diets in study 4 contained approximately 5% less crude protein than the diets prepared as positive controls while in study 5 two levels of depletion were used (5% and 7.5%). Performance characteristics were measured and the data analysed by analysis of variance. Results for these studies are shown in Table 3. Table 3: Summary of the results of the two studies made in Europe on the effects of Cibenza EP150 on the performance of chickens for fattening Treatment Average weight gain (kg/bird) Average feed intake (kg/bird) Feed:gain Mortality (%) Study 4 (1) Positive control Negative control Positive control mg/kg additive Negative control mg/kg additive Study 5 (2) Positive control 2.85 bc Negative control 1 (- 5% protein) 2.84 ab Negative control 2 (-7.5% protein) 2.82 a Positive control mg/kg additive 2.88 c Negative control mg/kg additive 2.87 c Negative control mg/kg additive 2.86 bc a,b,c: Means in a column within the same trial with different superscript letters are significantly different (P 0.05). (1): Technical dossier/section IV/Annex IV_3_4. (2): Technical dossier/section IV/Annex IV_3_5. EFSA Journal 2015;13(3):

13 In study 4 no significant differences were observed. Small but significant differences in average weight gain were observed between positive and negative control groups in study 5 and the loss in performance was restored by the addition of the additive. This was not reflected in the feed to gain values. Overall a significant benefit of the addition of the additive was only seen when a protein depleted diet was provided (studies 1 and 5 for average weight gain and studies 1 3 for feed to gain ratio). The applicant provided a meta-analysis of the five trials based on results for the positive and negative controls and the negative control supplemented with 500 mg/kg feed (the recommended dose) which confirmed and strengthened this conclusion. 30 An average weight gain of 2.64 kg in the positive control was reduced to 2.58 kg in the negative control and restored to 2.64 kg by the additive (P = ). Similarly a feed to gain ratio of 1.76 was increased to 1.79 for the negative control and returned to 1.76 in the presence of the additive (P = ). Average feed intake was not affected. The Panel concludes that evidence has been provided that the additive has the potential to be efficacious in chickens for fattening at the recommended dose (500 mg/kg complete feedingstuffs) but only when a reduced protein diet was provided. No significant effects were seen when a standard poultry diet was fed Compatibility with coccidiostats when used with chickens for fattening In vitro studies involving exposure of the B. licheniformis strain to two-fold serial dilutions of a range of coccidiostats showed that the MIC values for diclazuril, nicarbazin, decoquinate and semduramycin were all equal to or greater than four times the maximum permitted use level. 31 Consequently compatibility with these coccidiostats can be assumed. For other coccidiostats an in vivo trial was made to compare differential counts of spores and vegetative cells in chickens given the additive in the presence or absence of coccidiostats (or an organic acid preparation). 32 A total of 330 male Ross 308 chicks were assigned to one of nine treatment groups comprising a negative control without the additive, a positive control with the additive and seven test groups in which both the additive and test substance was included in the feed. Six replicate cages were used for each treatment housing 4 9 birds per cage. The tested coccidiostats were included at the maximum permitted concentration and the additive at twice the recommended dose. The concentration of B. licheniformis in feed was confirmed by analysis. After 37 days birds were killed and the gut contents recovered. The number of birds killed was adjusted to ensure a sample of sufficient volume for analysis was obtained from each cage. Microbial counts were made with and without heat treatment and the results analysed by ANOVA using the Dunnett test for paired comparisons with the positive control. Bacterial counts were not significantly reduced in the presence of lasalocid sodium, monensin sodium, robenidine hydrochloride, maduramicin ammonium, narasin or salinomycin in comparison with the positive control (the additive alone). A mixture of formic and propionic acids and their ammonium salts included at mg/kg feed also did not significantly affect numbers of B. licheniformis. However, although compatibility was demonstrated for this organic acid preparation, the result should not be extrapolated to other organic acids and their mixtures Efficacy for chickens reared for laying The conclusion on efficacy for chickens for fattening is taken to apply to chickens reared for laying when the same dose (500 mg/kg feed) is used. 30 Technical dossier/section IV/Annex IV_3_6. 31 Technical dossier/section II/Annex_II_2_1_2_2 and Annex_II_2_1_2_3. 32 Technical dossier/section II/Annex_II_4_4_1. EFSA Journal 2015;13(3):

14 4.4. Efficacy for minor avian species for fattening and to point of lay and ornamental birds Since the applicant proposes the use of the same dose in minor avian species and as the mechanism of action of the additive can be reasonably assumed to be the same, the conclusion on efficacy for chickens for fattening can be extrapolated to minor avian species grown for meat production or reared to the point of lay at a dose of 500 mg/kg feed without the need for additional studies. 5. 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 Regulation 33 and Good Manufacturing Practice. CONCLUSIONS Chickens for fattening appeared to tolerate the additive at ten-times the recommended dose and consequently the additive is considered safe at the recommended dose of 500 mg/kg complete feedingstuffs. This conclusion also applies to chickens reared for laying and can be extrapolated to other avian species used for meat production and ornamental birds when the same dose is applied. Use of the additive in feed for chickens for fattening, chickens reared for laying and other avian species used for meat production is considered safe for consumers. The additive is not a skin irritant but is an eye irritant. The additive has the potential to be a skin sensitiser and to induce sensitisation following respiratory exposure. The organism, which is a strain of a naturally-occurring soil bacterial species, and its fermentation products are considered not to present a hazard for the environment. A significant production benefit of the addition of the additive using the recommended dose (500 mg/kg complete feedingstuffs) was seen in chickens for fattening but only when a reduced protein diet was provided. No significant effects were seen when a standard poultry diet was fed. This conclusion on efficacy is taken to apply to chickens reared for laying and can be extrapolated to other avian species used for meat production and ornamental birds when the same dose is used. Use of the additive is compatible with the following coccidiostats: diclazuril, nicarbazin, decoquinate, semduramycin, lasalocid sodium, monensin sodium, robenidine hydrochloride, maduramicin ammonium, narasin and salinomycin. DOCUMENTATION PROVIDED TO EFSA 1. Cibenza EP150 (Protease, EC and Bacillus licheniformis, ATCC 53757) Zootechnical additive for chickens for fattening, chickens reared for laying and relevant minor avian species. April Submitted by Novus Europe S.A./N.V. 2. Cibenza EP150 (Protease, EC and Bacillus licheniformis, ATCC 53757) Zootechnical additive for chickens for fattening, chickens reared for laying and relevant minor avian species. Supplementary information February Submitted by Novus Europe S.A./N.V. 3. Evaluation report of the European Union Reference Laboratory for Feed Additives on the Methods(s) of Analysis for Cibenza EP Comments from Member States received through the ScienceNet. 33 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, , p. 1. EFSA Journal 2015;13(3):

15 REFERENCES EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), Technical Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition. EFSA Journal 2011;9(11):2445, 13 pp. doi: /j.efsa EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. EFSA Journal 2012;10(6):2740, 10 pp. doi: /j.efsa EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition. EFSA Journal 2014;12(5):3665, 10 pp. doi: /j.efsa EFSA Journal 2015;13(3):

16 Annex A. Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Methods of Analysis for Cibenza EP150 In the current application authorisation is sought under article 4(1) for Cibenza EP150 under the category/functional group 1(d) zootechnical additives / other zootechnical additives, according to the classification system of Annex I of Regulation (EC) No 1831/2003. The authorisation is sought for the use of the feed additive for chickens for fattening, chickens reared for laying, and minor avian species (ducks, geese, pigeons and other game birds) for meat production. Cibenza EP150 is a dry preparation containing two active substances: a protease (EC ) and viable spores of non-genetically modified Bacillus licheniformis (strain PWD-1, ATCC 53757). The Applicant stated the following minimum activity/concentration in the product: 6x105 U/g for protease and 1x109 Colony Forming Units (CFU)/g for Bacillus licheniformis. The Applicant defined the protease activity units (U) as the amount of protease that liberates 1 micromole of para-nitroaniline (pna) from the Succinyl-Ala-Ala- Pro-Phe-pNA (C30H36N6O9) substrate per minute at ph 8.0 and 37 C. Cibenza EP150 is intended to be used in premixtures and feedingstuffs, with the minimum activity/concentration in feedingstuffs of: 3x105 U/kg for protease and 5x108 CFU/kg for Bacillus licheniformis, respectively. For the identification and characterisation of the strain Bacillus licheniformis ATCC the Applicant studied biochemical parameters and applied DNA sequencing, together with Pulsed Field Gel Electrophoresis (PFGE). The EURL recommends for official control PFGE, the generally recognised standard methodology for microbial identification that is currently being evaluated by the CEN Technical Committee 327 to become European Standards. For the enumeration of spores of Bacillus licheniformis ATCC in feed additive, premixtures and feedingstuffs, the Applicant submitted the ring-trial validated CEN spread plate method (EN 15784). However, this method is not applicable to mineral feeds composed mainly of minerals and containing at least 40 % crude ash. During the review process one of the National Reference Laboratories identified the ring-trial validated VDLUFA method for the enumeration in mineral feed. Based on the performance characteristics available, the EURL recommends for official control the EN and VDLUFA methods. For the quantification of the protease activity in the feed additive, premixtures and feedingstuffs the Applicant submitted a single-laboratory validated and further verified colorimetric methods based on the quantification of para-nitroaniline (pna) released by the action of protease on the synthetic peptide substrate (Succinyl-Ala-Ala-Pro-Phe-pNA). Based on the satisfactory experimental evidence provided the EURL recommends for official control the colorimetric methods submitted. 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. EFSA Journal 2015;13(3):