EFSA Journal 2015;13(1):3970 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of Bacillus subtilis PB6 (Bacillus subtilis) as a feed additive for laying hens and minor poultry species for laying 1 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3 ABSTRACT European Food Safety Authority (EFSA), Parma, Italy Bacillus subtilis PB6 is the trade name for a feed additive based on viable spores of a strain of Bacillus subtilis. This species is considered by EFSA to be suitable for the qualified presumption of safety approach to safety assessment. This approach requires the identity of the active agent to be established and the absence of toxigenic potential and resistance to antibiotics of human or veterinary clinical significance to be demonstrated. No evidence of toxigenic potential or of resistance to antibiotics of human and veterinary importance was found as judged by the current guidelines. Consequently, the strain of B. subtilis in the additive is presumed safe for target animals, consumers and the environment. This conclusion covers the use of the additive in feed for laying hens and minor poultry species for laying. Use of the additive in feed for laying hens and minor poultry species will not introduce hazards for users not already considered. All four trials made with laying hens showed some evidence of a beneficial effect of the additive on egg production when used at a minimum dose of 1 10 8 colony-forming units (CFU)/kg complete feed, by increasing laying rate or average egg weight. The FEEDAP Panel concludes that Bacillus subtilis PB6 shows some potential to be efficacious in laying hens. This conclusion on efficacy for laying hens can be extended to minor poultry species raised for laying when the additive is used at the same minimum dose. European Food Safety Authority, 2015 KEY WORDS zootechnical additive, Bacillus subtilis PB6, QPS, efficacy, laying hens, minor poultry species for laying 1 On request from the European Commission, Question No EFSA-Q-2014-00288, adopted on 11 December 2014. 2 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 Micro-organisms, including Andrew Chesson, Pier Sandro Cocconcelli and Ingrid Halle, 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), 2015. Scientific Opinion on the safety and efficacy of Bacillus subtilis PB6 (Bacillus subtilis) as a feed additive for laying hens and minor poultry species for laying. EFSA Journal 2015;13(1):3970, 10 pp. doi:10.2903/j.efsa.2015.3970 Available online: www.efsa.europa.eu/efsajournal European Food Safety Authority, 2015
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 and efficacy of the product Bacillus subtilis PB6 as feed additive for laying hens and minor poultry species for laying. Bacillus subtilis PB6 is the trade name for a feed additive based on viable spores of a strain of Bacillus subtilis. The product is intended for use in feed for laying hens and minor poultry species for laying at a dose of 1 10 8 CFU/kg complete feedingstuffs. B. subtilis is considered by EFSA to be suitable for the qualified presumption of safety (QPS) approach to establishing safety for the target species, consumers and the environment. This approach requires the identity of the active agent to be established and the absence of toxigenic potential and susceptibility to a selected range of antibiotics to be demonstrated. No evidence of a toxigenic potential or resistance to antibiotics of human and veterinary importance was found as judged by the current guidelines. Consequently, the strain of B. subtilis in the additive is presumed safe for target animals, consumers and the environment. This conclusion covers the use of the additive in feed for laying hens and minor poultry species for laying. Use of the additive in feed for laying hens and minor poultry species will not introduce hazards for users not already considered. All four trials made with laying hens showed some evidence of a beneficial effect of the additive on egg production when used at a minimum dose of 1 10 8 CFU/kg complete feed, by increasing laying rate or average egg weight. The FEEDAP Panel concludes that Bacillus subtilis PB6 shows some potential to be efficacious in laying hens. This conclusion on efficacy for laying hens can be extended to minor poultry species raised for laying when the additive is used at the same minimum dose. EFSA Journal 2015;13(1):3970 2
TABLE OF CONTENTS Abstract... 1 Summary... 2 Background... 4 Terms of reference... 4 Assessment... 6 1. Introduction... 6 2. Characterisation... 6 2.1. Characterisation of the active agent... 6 2.2. Characterisation of the additive... 7 2.3. Conditions of use... 7 2.4. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) 7 3. Safety... 7 4. Efficacy... 7 4.1. Efficacy for laying hens... 7 4.2. Efficacy for minor poultry species for laying... 8 5. Post-market monitoring... 9 Conclusions... 9 Documentation provided to EFSA... 9 References... 9 EFSA Journal 2015;13(1):3970 3
BACKGROUND Regulation (EC) No 1831/2003 4 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 Kemin Europa N.V. 5 for authorisation of the product Bacillus subtilis PB6, to be used as a feed additive for laying hens and other minor species for laying (category: zootechnical additive; functional group: gut flora stabiliser) 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 4 June 2014. The additive Bacillus subtilis PB6 is a microbial preparation of Bacillus subtilis (ATCC PTA-6737). This product is authorised for use chickens for fattening, 7 chickens reared for laying, ducks for fattening, quails, pheasants, partridges, guinea fowl, pigeons, geese for fattening, ostriches, 8 and turkeys for fattening and reared for breeding. 9 EFSA issued one opinion on the safety and efficacy of Bacillus subtilis PB6 for chickens for fattening (EFSA FEEDAP Panel, 2009), another for chickens reared for laying, ducks for fattening, quails, pheasants, partridges, guinea fowl, pigeons, geese for fattening and ostriches (EFSA FEEDAP Panel, 2011), one for weaned piglets and weaned minor porcine species (EFSA FEEDAP Panel, 2012a) and one for turkeys for fattening and turkeys reared for breeding (EFSA FEEDAP Panel, 2013). An opinion on the compatibility of Bacillus subtilis PB6 with coccidiostats was also issued in 2010 (EFSA FEEDAP Panel, 2010). 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 Bacillus subtilis PB6 (Bacillus subtilis ATCC PTA-6737), when used under the conditions described in Table 1. 4 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. 5 Kemin Europa N.V., Toekomstlaan 42, 2200 Herentals, Belgium. 6 EFSA Dossier reference: FAD-2014-0005. 7 Commission Regulation (EU) No 107/2010 of 8 February 2010 concerning the authorisation of Bacillus subtilis ATCC PTA-6737 as a feed additive for chickens for fattening (holder of authorisation Kemin Europa NV). OJ L 36, 9.2.2010, p. 1. 8 Commission Implementing Regulation (EU) No 885/2011 of 5 September 2011 concerning the authorisation of Bacillus subtilis (ATCC PTA-6737) as a feed additive for chickens reared for laying, ducks for fattening, quails, pheasants, partridges, guinea fowl, pigeons, geese for fattening and ostriches (holder of authorisation Kemin Europa N.V.). OJ L 229, 6.9.2011, p. 3. 9 Commission Implementing Regulation (EU) No 787/2013 of 16 August 2013 concerning the authorisation of a preparation of Bacillus subtilis (ATCC PTA-6737) as a feed additive for turkeys for fattening and turkeys reared for breeding (holder of authorisation Kemin Europa N.V.). OJ L 220, 17.8.2013, p. 15. EFSA Journal 2015;13(1):3970 4
Table 1: Description and conditions of use of the additive as proposed by the applicant Additive Registration number/ec No/No Category(-ies) of additive Functional group(s) of additive Bacillus subtilis ATCC PTA-6737 4b1823 Zootechnical additive Gut flora stabilisers Composition, description Preparation of Bacillus subtilis ATCC PTA-6737 containing a minimum of 1 10 10 CFU/g additive Characterisation of the active substance: Spores of Bacillus subtilis ATCC PTA-6737 Chemical formula N.A. Description Purity criteria N.A. Mehtods of analysis Analytical method: Enumeration: spread plate method using tryptone soya agar with pre heattreatment of feed samples. Identification: pulsed-field gel electrophoresis (PFGE) method. Trade name Name of the holder of authorisation Bacillus subtilis PB6 Kemin Europa NV Conditions of use Species or category of animal Layinh hens and other minor laying poultry birds Maximum Age Minimum content Maximum content CFU/kg of complete feedingstuffs Withdrawal period N.A. 1 x 10 8 N.A. N.A. Other provisions and additional requirements for the labelling Specific conditions or restrictions for use Specific conditions or restrictions for handling Post-market monitoring Specific conditions for use in complementary feedingstuffs Store in a dry place at room temperature for max. 3 years. Stable for pelleting up to 90 C N.A. N.A. May be used in feed containing the permitted coccidiostats: lasalocid, maduramycin, monensin, narasin, salinomycin, decoquinate, robenidine, diclazuril and narasin/nicarbazin Marker residue Maximum Residue Limit (MRL) Species or category of animal Marker residue Species or category of animal N.A. N.A. N.A. N.A. EFSA Journal 2015;13(1):3970 5
ASSESSMENT 1. Introduction Bacillus subtilis PB6 is the trade name for a feed additive based on viable spores of a single strain of B. subtilis at a minimum concentration of 1 10 10 CFU/g. EFSA has previously assessed the additive when used in feed for chickens for fattening (EFSA, 2009), chickens reared for laying and minor poultry species (EFSA FEEDAP Panel, 2011), weaned piglets and weaned minor porcine species (EFSA FEEDAP Panel, 2012a) and turkeys for fattening and turkeys reared for breeding (EFSA FEEDAP Panel, 2013). The compatibility of the Bacillus subtilis PB6 strain with coccidiostats has also been assessed (EFSA FEEDAP Panel, 2010). The species B. subtilis is considered by EFSA to be suitable for the qualified presumption of safety (QPS) approach to establishing safety for the target species, consumers and the environment (EFSA, 2007; EFSA BIOHAZ Panel, 2013). This approach requires the identity of the active agent to be established and the absence of toxigenic potential and susceptibility to a selected range of antibiotics to be demonstrated. EFSA considered these issues in its opinion on the safety and efficacy of Bacillus subtilis PB6 as a feed additive for chickens for fattening (EFSA FEEDAP Panel, 2009) following the provisions of the guidance applicable at the time and concluded that the strain could be presumed safe for the target species, consumers and the environment. Subsequently, EFSA has introduced new guidance on the determination of antibiotic susceptibility (EFSA FEEDAP Panel, 2012b) and assessing the toxigenic potential of Bacillus species (EFSA FEEDAP Panel, 2014). Consequently, these elements of the previous assessment are reconsidered in this opinion. In the course of the 2009 assessment for use with chickens for fattening, safety for users was also considered. The applicant is now requesting the authorisation of the additive in diets for Bacillus subtilis PB6 for laying hens and minor laying species. In the view of the FEEDAP Panel, use in these additional poultry species will not introduce hazards for users of the additive not already considered. 2. Characterisation 2.1. Characterisation of the active agent The strain is deposited in the American Type Culture Collection with the accession number ATCC PTA-6737. 10 Its identity was clearly established by phenotypic and molecular methods, genetic stability confirmed and the means of distinguishing the product strain from closely related strains demonstrated. 11 The battery of antibiotics currently recommended (EFSA FEEDAP Panel, 2012b) for the testing of antibiotic susceptibility amongst species of Bacillus were all included in the recommended list applicable at the time of the 2009 submission. 12 The results presented in the application for chickens for fattening are still valid and, as the cut-off values are the same, the conclusion on the susceptibility of the strain to all tested antibiotics remains unchanged. The technical guidance on the assessment of the toxigenic potential of Bacillus species introduced in 2014 contains a substantial revision applicable to B. subtilis (EFSA FEEDAP Panel, 2014). The emphasis of the assessment changed from the detection of B. cereus-like toxins in the former guidance to the detection of cyclic lipopeptides able to cause measurable cell cytotoxicity. It recommends the use of a single cytoxicity assay, preferably carried out with Vero cells and using an established method, such as inhibition of radio-labelled leucine uptake, as evidence of toxicity. The data set provided for the 2009 application included the results of such an assay using Vero cells and a 10-fold 10 Technical dossier/section II/Annex II.13. 11 Technical dossier/section II/Annexes II.12-16. 12 Technical dossier/section II/Annex II.19. EFSA Journal 2015;13(1):3970 6
concentrated culture supernatant of strain B. subtilis PTA-6737. 13 The presence of the supernatant did not inhibit leucine uptake, unlike the positive control, which fully inhibited uptake. Consequently, these data are considered to fulfil the requirements of the present guidance and allow to conclude that the strain is not toxigenic. 2.2. Characterisation of the additive The additive which is the subject of the present application has the same formulation and method of manufacture as that considered in previous applications. Thus, the data pertaining to impurities, physical properties and shelf life still apply. Although no specific data on stability in feed for laying hens or ability to mix in such feed were provided, the data on stability in premixes, mash and pelleted feed for chickens for fattening are considered sufficient given the similarity in feed formulation. 2.3. Conditions of use The product is intended for use in feed for laying hens and minor poultry species for laying at a minimum dose of 1 10 8 CFU/kg complete feedingstuffs. 2.4. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) The EURL considered that the conclusions and recommendations reached in the previous assessment are valid and applicable for the current application. 14 3. Safety No evidence of a toxigenic potential or resistance to antibiotics of human and veterinary importance was found as judged by the current requirements. Thus, the conclusion reached in the previous opinion on chickens for fattening (EFSA FEEDAP Panel, 2009) and in subsequent opinions that the strain B. subtilis ATCC PTA-6737 is presumed safe for target animals, consumers and the environment, is still considered to apply. This conclusion automatically covers the use of the additive in feed for laying hens and minor poultry species for laying. In the view of the FEEDAP Panel, use in these additional poultry species will not introduce hazards for users of the additive not already considered. 4. Efficacy 4.1. Efficacy for laying hens Four trials, each of 168 days duration, were carried out in laying hens in three Member States. Birds at the start of the trials were between 18 and 21 weeks of age and the breeds used were Hyline (trial 1) or Lohmann brown (trials 2, 3 and 4). Birds were allocated either to a control group given only the basal diet (based on wheat/soybean in trial 1, wheat/corn/soybean in trials 2 and 3 and corn/soybean in study 4) or to one or two experimental groups in which the basal diet was supplemented with the additive at 1 10 8 CFU/kg feed (all four trials) and 1 10 7 CFU/kg feed (trials 1 and 2). All doses were confirmed by analysis. Each treatment was replicated as shown in Table 2. Individual or pen body weights were recorded at the start and end of each study and birds were routinely monitored for general health status. Feed intake was recorded and used to calculate the feed to egg ratio. The number of eggs produced, percentage laying rate, mean egg weight and daily or total egg mass determined. In trial 2 the incidence of cracked and soft-shelled eggs and in trials 3 and 4 egg shell stability were also recorded. All data were subject to an analysis of variance using the pen as experimental unit. A summary of the results for the whole period studied is shown in Table 2. 13 Technical dossier/section II/Annex II.18. 14 The full report is available on the EURL website: https://ec.europa.eu/jrc/sites/default/files/finrep-fad-2008-0039.pdf EFSA Journal 2015;13(1):3970 7
Mortality was low or absent in all of the studies (trial 1: 2.8, 3.7 and 1.9 %; trial 2: 4.6, 2.1 and 3.5 %; trial 3: 0.7 % and 0 %; trial 4: 0 %) and no health problems requiring intervention were reported. In all four studies birds gained weight but the final body weight and daily weight gain were affected only by treatment in study 4, in which the final body weight of the treated group (2.00 kg) was significantly higher (P < 0.05) than that of the control group (1.95 kg). Table 2: Summary of the performance results of the trials made with laying hens Trial No Total number of animals Treatment (CFU/kg Laying rate (%) Average egg weight Egg mass or daily egg Feed intake Feed to egg ratio No of replicates per treatment No of birds per replicate feed) (g) mass (g) (a) (g/bird) 1 (b) 324 0 91.1 63.5 4700 126.0 2.26 36 3 1 10 7 90.5 62.9 * 4603 125.5 2.28 1 10 8 92.4* 62.7* 4751 125.4 2.24 2 (c) 432 0 90.8 62.6 57.1 113.2 2.02 8 18 1 10 7 93.2* 63.0 58.9* 114.6 1.97 1 10 8 94.0* 63.4 59.8* 114.1 1.93* 3 (d) 324 0 85.9 56.3 49.4 111.4 2.75 9 18 1 10 8 85.2 56.8* 49.5 111.9 2.74 4 (e) 288 0 92.2 61.3 57.0 118.1 2.19 9 16 1 10 8 92.2 61.8* 57.5 119.1* 2.18 (a): Average egg mass per phase (28 days) and replicate in trial 1 and daily egg mass in trials 2, 3 and 4. (b): Technical dossier/section IV/Annexes IV.27-28. (c): Technical dossier/section IV/Annexes IV.29-31. (d): Technical dossier/section IV/Annexes IV.32-34. (e): Technical dossier/section IV/Annexes IV.35-36. *Significantly different from the control value (P < 0.05). All four trials showed some evidence of a beneficial effect on egg production. Laying rate was significantly improved in two of the four trials at the recommended dose of 1 10 8 CFU/kg feed. Average egg weight was also significantly increased in two studies, but significantly reduced in a third (trial 1). In this trial egg mass remained constant and the fall in egg weight was probably related to the increase observed in laying rate. In the two trials in which egg weight was increased, there was no difference in laying rate. Feed intake was not affected by treatment in any of the trials, except for trial 4. Feed to egg ratio was improved in one trial (trial 2), remaining essentially constant in the other three. Egg quality was considered in three of the four trials (trials 2, 3 and 4), although different measures were applied. Shell deformation was measured in trials 2 and 3 and found to be marginally greater in the treated group given the recommended dose of the additive than in the control group in trial 2 but not significantly different in trial 3. However, the incidence of cracked eggs was significantly reduced in the treated group in trial 2 (2.4 % vs. 1.5 %; P < 0.05) but was unaffected in trial 3. In trial 4 shell stability, as measured by a plate pressure test, was significantly improved in the treated group compared with the control group, and shell thickness was also significantly increased (0.36 vs. 0.37 mm; P < 0.05). Therefore, the FEEDAP Panel concludes that Bacillus subtilis PB6 shows some potential to be efficacious in laying hens. 4.2. Efficacy for minor poultry species for laying The dose proposed for use with minor egg-laying poultry species is the same as that demonstrated to be effective in a physiologically similar major species (laying hen) and it can be reasonably assumed that the mode of action is the same. Consequently, the conclusion on efficacy for laying hens can be extended to minor poultry species raised for laying. EFSA Journal 2015;13(1):3970 8
5. Post-market monitoring Bacillus subtilis PB6 for laying hens and minor laying species 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 15 and Good Manufacturing Practice. CONCLUSIONS No evidence of a toxigenic potential or resistance to antibiotics of human and veterinary importance was found as judged by the current guidance documents. The conclusion reached in the opinion on chickens for fattening and subsequent opinions that the strain B. subtilis ATCC PTA-6737 is presumed safe for target animals, consumers and the environment is still considered to apply. This conclusion automatically covers the use of the additive in feed for laying hens and minor poultry species for laying. Use of the additive in feed for laying hens and minor poultry species will not introduce hazards for users not already considered. All four trials made with laying hens showed some evidence of a beneficial effect of the additive on egg production when used at a minimum dose of 1 10 8 CFU/kg complete feed, by increasing laying rate or average egg weight. The FEEDAP Panel concludes that Bacillus subtilis PB6 shows some potential to be efficacious in laying hens. This conclusion on efficacy for laying hens can be extended to minor poultry species raised for laying when the additive is used at the same minimum dose. DOCUMENTATION PROVIDED TO EFSA 1. Bacillus subtilis ATCC PTA-6737. New use: Laying hens and other minor laying poultry birds. January 2014. Submitted by Kemin Europa N.V. 2. Comments from Member States received through the ScienceNet. 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. The EFSA Journal 2007, 587, 1 16. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2009. Scientific Opinion on the safety and efficacy of Bacillus subtilis PB6 (Bacillus subtilis) as a feed additive for chickens for fattening. EFSA Journal 2009;7(9):1314, 14 pp. doi:10.2903/j.efsa.2009.1314 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2010. Scientific Opinion on the on the compatibility of Bacillus subtilis PB6 with coccidiostats in chickens for fattening. EFSA Journal 2010;8(10):1863, 6 pp. doi:10.2903/j.efsa.2010.1863 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2011. Scientific Opinion on the safety and efficacy of Bacillus subtilis PB6 for chickens reared for laying, ducks for fattening, quails, pheasants, partridges, guinea fowl, pigeons, geese for fattening and ostriches. EFSA Journal 2011;9(3):2114, 8 pp. doi:10.2903/j.efsa.2011.2114 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2012a. Scientific Opinion on the safety and efficacy of Bacillus subtilis PB6 as a feed additive for weaned piglets and weaned minor porcine species. EFSA Journal 2012;10(5):2671, 8 pp. doi:10.2903/j.efsa.2012.2671 15 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. 1. EFSA Journal 2015;13(1):3970 9
EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP); 2012b. 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 Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2013. Scientific Opinion on the safety and efficacy of Bacillus subtilis PB6 (Bacillus subtilis) as a feed additive for turkeys for fattening and turkeys reared for breeding. EFSA Journal 2013;11(4):3176, 10 pp. doi:10.2903/j.efsa.2013.3176 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), 2014. Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition. EFSA Journal 2014;12(5):3665, 10 pp. doi:10.2903/j.efsa.2014.3665 EFSA Journal 2015;13(1):3970 10