The EFSA Journal (2005) 288, 1-7

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1 The EFSA Journal (2005) 288, 1-7 Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on the modification of terms of authorisation of the micro-organism preparation of Bacillus cereus var. toyoi (NCIMB 40112/CNCM I-1012) (Toyocerin ) authorised as a feed additive in accordance with Council Directive 70/524/EEC (Question N o EFSA-Q ) Adopted on 30 November 2005 SUMMARY The European Food Safety Authority received a request to assess the compatibility of the micro-organism product Toyocerin with the coccidiostats diclazuril, narasinnicarbazin and maduramicin ammonium when mixed in chicken feed. An in vitro study has been performed, in which the viability of the micro-organism (Bacillus cereus var. toyoi) was assessed in experimental chicken feeds during three months of storage in the presence of diclazuril (1 mg kg -1 ), narasin-nicarbazin (100 mg kg -1 ), and maduramicin (5 mg kg -1 ). None of the coccidiostats affected the stability of the product in feed in comparison to the control. In an animal study the same experimental feeds that were used in the stability test were fed to 40 female one-day old broiler chickens for 28 days. Each experimental group (including the control) consisted of ten animals (five replicates). The faeces were analysed for B. cereus var. toyoi content on day 14 and at the end of the experiment. There were no statistically significant differences either between the time points or treatments. Nothing in the data provided suggests that an incompatibility exists between B. cereus var. toyoi (Toyocerin ) and the tested coccidiostats diclazuril, narasin-nicarbazin and maduramicin ammonium, either on storage or when fed to chickens for fattening. Keywords: Bacillus cereus var. toyoi, coccidiostat, diclazuril, narasin-nicarbazin, maduramicin ammonium, compatibility, chickens for fattening

2 Opinion on Toyocerin 2/7 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 lies down that any person seeking an 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 ASAHI VET SA 2 to modify the Annex entry of Directive 70/524/EEC to allow the use of the product Toyocerin in feed for chickens for fattening containing the coccidiostats diclazuril, narasin-nicarbazin and maduramicin ammonium under the conditions set out 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 dossier in support of this application. 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 04 of May of The additive Toyocerin is a preparation of Bacillus cereus var. toyoi (NCIMB 40112/CNCM I-1012). This product is already permanently authorised for its use in piglets up to 4 months, pigs for fattening and sows (E 1701), and was provisionally authorised (No 1) for chickens for fattening, laying hens, calves, cattle for fattening, breeding does and rabbits for fattening until 7 October 2004, including the authorisation for its use in combination with monensin sodium, lasalocid sodium, salinomycin sodium, decoquinate, robenidine, narasin and halofuginone. It is currently undergoing assessment for permanent authorisation for cattle for fattening, chickens for fattening and rabbits for fattening. The Scientific Committee on Animal Nutrition (SCAN) issued an opinion on the use of Toyocerin on toxin production and resistance to antibiotics on 5 December On 7 May 2004, EFSA issued an opinion on the efficacy of this product in feeds for pigs for fattening 4. 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. Therefore, EFSA shall deliver an opinion on the safety for the target animals, human user and consumer and the environment and on the efficacy of the micro-organism preparation of Bacillus cereus var. toyoi (NCIMB 40112/CNCM I-1012) (Toyocerin), when used under the conditions described in Table 1, taking into account the background and the information submitted by the applicant. Taking into consideration that the product is already permanently authorised for its use in piglets up to 4 months, pigs for fattening and sows and currently undergoing evaluation for permanent approval for chickens for fattening, the current assessment shall focus on the impact of the mixing of the micro-organism product Toyocerin with the coccidiostats diclazuril, narasin-nicarbazin and maduramicin ammonium in feed for chickens for fattening, when used under the conditions set out in Table 1. 1 OJ L 268, , p Asahi Vet, S.A. Avinguda de La Llana, 123. Polígono Industrial La Llana Ap. correos 283. E Rubí (Barcelona), Spain. Company name changed to RUBINUM, S.A. on 1 st April

3 Opinion on Toyocerin 3/7 Table 1. Proposal for inclusion of Toyocerin in the annex of Directive 70/524/EEC, as proposed by the applicant (proposed modifications in bold). EC No E 1701 Additive Bacillus cereus var. toyoi NCIMB 40112/CNCM I-1012 [Toyocerin] Chemical formula, description Preparation of Bacillus cereus var. toyoi containing a minimum of 1x10 10 CFU g -1 additive Species or category of animal Chickens for fattening Maximum age Minimum content Maximum content CFU kg -1 of complete feedingstuff x x 10 9 Other provisions In the directions for use of the additive and premixture indicate the storage temperature, storage life and stability to pelleting. May be used in compound feed containing the permitted coccidiostats: monensin sodium, lasolacid sodium, salinomycin sodium, decoquinate, robenidine, narasin, halofuginone, diclazuil, narasin/nicarbazin, maduramicin ammonium Period of authorisation

4 Opinion on Toyocerin 4/7 ASSESSMENT 1- Introduction Toyocerin is marketed as two commercial preparations, as feed additive (concentration of B. cereus var. toyoi CFU g -1 ) and as a premixture (concentration of B. cereus var. toyoi 10 9 CFU g -1 ). The recommended dosage range for chickens for fattening is x 10 9 CFU kg -1 feedingstuffs. 2- Compatibility with the coccidiostats diclazuril, narasin-nicarbazin and maduramicin ammonium Experimental feeds Four batches of an experimental chicken feed were prepared all with an inclusion of 1 g Toyocerin premix per kg. The inclusion rates of the coccidiostats in the respective test batches were: 1 mg diclazuril kg -1, 100 mg narasin-nicarbazin kg -1 and 5 mg maduramicin ammonium kg -1. The prepared feed batches were divided into two lots that were either sent to the company for storage trial or used immediately for animal testing. Analysed concentrations of the coccidiostats in the feeds were not presented. The effects of coccidiostats on the storage stability of Toyocerin The storage lasted for three months, after which the numbers of B. cereus var. toyoi in various feeds were determined. No information has been given about the storage conditions (type of package, temperature, humidity, etc.). The results were statistically analysed using analysis of variance and Dunnets's test. The results of the storage trial are presented in Table 2. Table 2. Numbers of B. cereus var. toyoi in feeds supplemented with coccidiostats Counts of B. cereus var. toyoi in feed (10 9 CFU kg -1 ) Coccidiostat 0 1 month 2 month 3 month None Diclazuril Narasin-Nicarbazin Maduramicin ammonium No statistically significant differences were observed between the treatments, and the stability of Toyocerin was not affected by the coccidiostats during the three-month storage. The recovery of B. cereus var. toyoi in faeces of chickens fed with coccidiostat containing feeds The experiment was performed using 40 female day old Ross 308 broiler chickens, which were distributed into 20 pens (two animals per pen). The chickens were fed for 28 days with the experimental feeds containing Toyocerin either alone (control) or together with the tested coccidiostat. The faecal samples (five replicates from two chickens per treatment) were collected after 14 days and at the end of the trial and analysed for the B. cereus var. toyoi counts. The results were statistically analysed using analysis of variance and Dunnets's test. The faecal counts of B. cereus var. toyoi are presented in Table 3.

5 Opinion on Toyocerin 5/7 Table 3. The faecal counts of B. cereus var. toyoi of chicken fed with feeds containing coccidiostats Faecal counts of B. cereus var. toyoi (log10 CFU g -1 ) Coccidiostat 14 days 28 days None Diclazuril Narasin-Nicarbazin Maduramicin ammonium There were no statistically significant differences either between the time points or treatments. Thus the coccidiostats did not have observable effects on the total counts of B. cereus var. toyoi in faeces. The effects of Toyocerin and coccidiostats on animal performance In connection with the compatibility trial, the chicken weight development was also followed by recording the initial and final weight and calculating the average daily weight gain and the daily feed consumption. The results were statistically analysed using analysis of variance and Duncan's multiple range tests (treatment means). The results are expressed in Table 4. Table 4. Performance of chickens fed diets with Toyocerin and coccidiostats (0-28d) Coccidiostat Initial weight (g) Final weight (g) Average daily gain (g) Average daily feed intake (g) Feed conversion Control ab ab Diclazuril ab ab Narasin-Nicarbazin b a Maduramicin ammonium a b a, b: Values in a column with different superscripts are statistically different (P 0.05) The design of the experiment does not allow any assessment of the efficacy of Toyocerin. 3- Assessment of the control methods by the Community Reference Laboratory (CRL) EFSA has verified the CRL report as it relates to the methods used for the control of the active agent in animal feeds. The Executive Summary of the CRL can be found in Annex A. CONCLUSIONS Nothing in the data provided suggests that an incompatibility exists between B. cereus var. toyoi (Toyocerin ) and the tested coccidiostats diclazuril, narasin-nicarbazin and maduramicin ammonium. DOCUMENTATION PROVIDED TO EFSA 1. Dossier on Compatibility of Toyocerin with coccidiostats in chicken for fattening. January Submitted by Asahi Vet, S.A. 2. Complementary information to provide the particulars requested by EFSA on in order to consider as valid and start the assessment of our application reference no. FAD submitted to EU Commission on and received by EFSA on March Submitted by Asahi Vet, S.A.

6 Opinion on Toyocerin 6/7 3. Evaluation report of the Community Reference Laboratory feed additives authorisation on the methods(s) of analysis for Toyocerin. SCIENTIFIC PANEL MEMBERS Arturo Anadón, Margarita Arboix Arzo, Georges Bories, Paul Brantom, Joaquim Brufau de Barberà, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël Dierick, Gerhard Flachowsky, Anders Franklin, Jürgen Gropp, Ingrid Halle, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Kimmo Peltonen, Guido Rychen, Pascal Sanders, Amadeu Soares, Pieter Wester and Wilhelm Windisch ACKNOWLEDGEMENTS The Scientific Panel on Additives and Products or Substances used in Animal Feed wishes to thank Professor Atte von Wright for the contribution to the draft opinion.

7 Opinion on Toyocerin 7/7 ANNEX A. Executive Summary of the Evaluation Report of the Community Reference Laboratory Feed Additives Authorisation on the Method(s) of Analysis for Toyocerin In the current application authorisation is sought for the use of Toyocerin which is a preparation of viable spores of a strain of Bacillus cereus var. toyoi (NCIMB 40112/CNCM I-1012), to be used in feed for chickens for fattening containing the permitted coccidiostats diclazuril, narasin-nicabazin and maduramicin ammonium. Concerning the determination of the active agent of Toyocerin (Bacillus cereus var. toyoi) in the additive, in premixtures and feedingstuffs, the applicant proposed a pour plate count method to enumerate viable counts of the preparation. The method is quantitative and uses a nutrient agar medium (NAM) for analysis of the additive and premixture, and NAM with the addition of antibiotics for feedingstuffs. Within-laboratory validation data were submitted by the applicant regarding the method for determination of the active substance in the additive, in premixtures and in feedingstuffs. Relative standard deviations of repeatability (RSDr) and within-laboratory reproducibility (RSDR) for the performance of the methods in the additive, the premixture and feedingstuffs were appropriate. The limit of quantification (LOQ) for the enumeration method is 5 x 10 3 colony forming units (c.f.u.) per gram of sample. Taking into account the target level of application which ranges between 10 6 c.f.u./g and c.f.u./g of sample (additive, premixture or feedingstuffs) and the acceptable performance characteristics of RSD values, in the opinion of the CRL the proposed methods are fit for the purpose of routine control of the presence of the active agent in the feed additive Toyocerin, in premixtures and in feedingstuffs. The applicant provided data confirming equivalent performance of the method of analysis when enumerating viable spores of the active agent in Toyocerin with and without the presence of three coccidiostats (diclazuril, narasin/nicarbacin and maduramicin ammonium) in broiler feed. In summary, it is considered that the proposed methods for enumeration of viable Bacillus cereus var. toyoi spores as colony forming units in the additive, in premixtures and in feedingstuffs fulfil the requirements for routine control also in the presence of the coccidiostats (diclazuril, narasin/nicarbacin and maduramicin ammonium) at levels typically applied in broiler feed. However, it is recommended to use a larger quantity of feed samples than the proposed 2 g, such as for example 50 g of feed samples to take account of potential sample heterogeneity. For official control purposes, the CRL recommends a fully ring-trial validated method for enumeration of bacilli spores in premixtures and feedingstuffs which was published in a peer reviewed journal (Journal of AOAC International (2003) 86, ). The study demonstrated acceptable methods performance characteristics, since the RSDr was about 1 % and the RSDR was about 6%. On the basis of the supplied documentation, no supplementary experimental work (testing or method validation) is required.

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