The EFSA Journal (2006) 336, 1-15

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1 The EFSA Journal (2006) 336, 1-15 Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on the safety and efficacy of the product Calsporin, a preparation of Bacillus subtilis, as a feed additive for chickens for fattening in accordance with Regulation (EC) No 1831/2003 SUMMARY (Question N o EFSA-Q ) Adopted on 8 of March 2006 Calsporin is a microbial feed additive based on a single strain of Bacillus subtilis. The Applicant is seeking authorization for the use of the product as a zootechnical feed additive for chickens for fattening at a recommended dose of 1 x 10 9 cfu kg -1 complete feedingstuff. The European Food Safety Authority has been requested by the European Commission to give an opinion on the efficacy of the additive and its safety for the target animals the consumer, the user of the product and the environment. Efficacy was demonstrated in three of four trials with a statistically significant benefit shown over the whole trial period manifested as an improvement in final weight, daily weight gain and/or feed to gain ratio. The statistical analysis of the pooled data from these four trials gives an indication of the magnitude of the improvement in zootechnical parameters which might be expected from the inclusion of Calsporin in broiler feed (2-3%). A 36-day tolerance study included a Calsporin treated group given the product at a minimum of ten-times the recommended dose. No adverse effects of treatment were seen either in performance characteristics or after necropsy. Microbiological analysis of the gut flora of birds given Calsporin at the recommended dose did not show any numerical differences amongst the organisms measured which might indicate a source of concern. Published evidence pointed to the ability of Calsporin to reduce numbers of Salmonella and Campylobacter in artificially and naturally affected birds and consequently to reduce carcass contamination. The production strain was shown to be non-toxigenic and the complete product (Calsporin) to be negative for genotoxicity in two in vitro assays and not to cause adverse effects in a repeat dose chronic toxicity study in rats. The FEEDAP Panel concludes that the use of this product in chickens for fattening will not be harmful for consumers of chicken meat or meat products. Specific tests for irritancy (dermal and ocular) and for skin sensitization were negative indicating that skin contact is unlikely to cause problems for those handling the product. However, in the absence of specific data, and given the small particle size and propensity for dust formation, sensitization via a respiratory route cannot be excluded. Bacillus subtilis is ubiquitous in the environment and use of the product as a feed additive would not measurably increase the concentration of the organism or otherwise disturb the local environment. Consequently no environmental risk assessment is considered necessary. The FEEDAP Panel concludes that Calsporin has the potential to be efficacious, that the additive is well tolerated by chickens for fattening with at least a ten-fold margin of safety and that use of the product will not introduce any hazards for consumers of

2 Opinion on Calsporin for chickens for fattening 2/15 chicken meat or meat products or for the wider environment. There is, however, a potential risk of sensitisation by a respiratory route for those handling the product. Key words: Calsporin, Bacillus subtilis, probiotic, chickens for fattening, efficacy, safety assessment

3 Opinion on Calsporin for chickens for fattening 3/15 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 the company Calpis Co Ltd 2 for authorisation of the product Calsporin to be used as a feed additive for chickens for fattening (category: zootechnical additives; functional group: gut flora stabilisers) 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. 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 12 of September of The additive Calsporin is a preparation of Bacillus subtilis (C-3102). This product has not been previously authorised in the Community. 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 efficacy and the safety for the target animals, user and consumer and the environment of the product Calsporin, which is a preparation of Bacillus subtilis (C-3102) when used under the conditions described in Table 1. 1 OJ L 268, , p Calpis Co Ltd. (Feed Division) 4-1, 2-chome, Ebisu-Minami, Tokyo , Japan; represented in the EU by Orffa International Holding B.V., Industriepark Ambachtsstraat 6, 1840 Londerzeel, Belgium

4 Opinion on Calsporin for chickens for fattening 4/15 Table 1. Register entry as proposed by the applicant. Additive Registration number/ec No/No (if appropriate) Category of additive Calsporin Pending Zootechnical additives Functional group of additive Gut flora stabiliser Composition, description 1 x viable spores (CFU) of Bacillus subtilis C-3102 per gram, in a calcium carbonate carrier Chemical formula - Description Purity criteria (if appropriate) Complies with EU law in relation to microbial quality, heavy metals, toxins and undesirable substances Method of analysis (if appropriate) Standard microbial counting technique adapted from ISO 4833 Trade name (if appropriate) Name of the holder of authorisation (if appropriate) Calsporin Calpis Co. Ltd. (Feed Division), Japan, represented in the EU by Orffa International Holding BV Conditions of use Species or category of animal Maximum Age Minimum content Maximum content CFU kg -1 of complete feedingstuffs Withdrawal period (if appropriate) Chickens for fattening Slaughter age 1 x x 10 9 Not appropriate Other provisions and additional requirements for the labelling 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) None R42, potential respiratory sensitiser Post-marketing monitoring will be carried out jointly by Calpis Co. Ltd. and their EU distributor (Orffa International Holding B.V.) in compliance with EU Regulations concerning Feed Hygiene and Feed and Food Controls, namely HACCP and Traceability, routine post-marketing sampling and analysis, and formal monitoring of customer feedback through product or service complaints. Dosage used should supply 1 x 10 9 CFU kg -1 final complete feedingstuff Marker residue Maximum Residue Limit (MRL) (if appropriate) Species or category of animal Target tissue(s) or food products Maximum content in tissues

5 Opinion on Calsporin for chickens for fattening 5/15 ASSESSMENT 1. Introduction Calsporin is the trade name for a feed additive based on viable spores of a strain of Bacillus subtilis and is intended for use with chickens for fattening to improve growth and the efficiency of feed conversion. The Company has applied for an authorisation for a period of ten years under the category of a zootechnical additive (functional group: gut flora stabiliser). The product and its active component have not previously been assessed for use as feed additive in the European Community, although it is presently authorised for use in a number of Asian countries including Japan, the USA and Brazil. 2. Identity, characterisation and conditions of use 2.1 Production and composition of the product The product is the result of a simple fermentation of B. subtilis in an aqueous suspension of defatted soybean (non-gm). The medium is not sterilised prior to use but after fermentation is completed the medium and cells are dried and pasteurised to kill vegetative cells. The pasteurised ferment is then ground to pass a 0.33 mm mesh and mixed with calcium carbonate. At this stage samples are taken for microbiological examination and enumeration of viable B. subtilis spores. On the basis of this enumeration sufficient additional amounts of calcium carbonate are added to produce a final product containing 1.15 x cfu g -1 of B. subtilis. Typically the final product contains 25-30% of the dried ferment and calcium carbonate to 100%. It is dry freeflowing powder with an average particle size of about 23 µm. HACCP procedures are specified and all raw materials are sourced from GMP suppliers and comply with EU legislation in respect to contamination by heavy metals, dioxins and PCBs (calcium carbonate) and mycotoxins (soybean). Results for the analysis of 59 production batches of Calsporin for heavy metals all fell below 2 ppm arsenic and 20 ppm total heavy metals. Data on the analysis of a further three batches showed detectable but negligible amounts of mycotoxins (aflatoxins, zearalone, ochratoxin and DON) and fungal contaminants (10-20 cfu g -1 product). Numbers of vegetative bacterial contaminants (enterobacteriaceae, Salmonella and staphylococci) were below detection levels. 2.2 Characterisation of the active agent The single strain of B. subtilis was originally isolated from soil in Japan and is deposited in Europe in the German Collection of Micro-organisms and Cell Cultures (DSMZ) with the accession number DSM It has not been genetically engineered and does not harbour plasmids. Identity was established using biochemical, 16S rdna sequence and restriction pattern analysis. Both biochemical and 16S rdna analysis gave a 99+% probability of similarity with B. subtilis. However comparison of the restriction pattern with those obtained from other relevant Bacillus spp. lodged with the German Collection of Micro-organisms and Cell Cultures, showed the highest similarity with a strain of Bacillus amyloliquefaciens. This apparently anomalous result was investigated by examination of the genes encoding amylase which are known to be highly conserved and specific for each species. Probes specific for the B. amyloliquefaciens-type amylase genes and B. subtilis type amylase gene were developed. The B. amyloliquefaciens probe gave positive results in all strains of B. amyloliquefaciens tested but not in any other strain, while the B. subtilis probe was positive for all B. subtilis strains tested and for the Calsporin production strain. The FEEDAP Panel agrees with the conclusion of the applicant that the weight of evidence strongly favours a B. subtilis identity.

6 Opinion on Calsporin for chickens for fattening 6/ Genetic stability The potential for genetic drift was studied by comparison of isolates from the current production strain, the deposit made in the EU in 2003 and the same strain laid down in 1992 (originating from the original isolation made in 1985). The PGFE profiles of all three isolated were identical and it was concluded that DSM is genetically stable Toxigenic potential The production strain was tested for its cytotoxic potential following the recommendations made by SCAN (2000) using, where appropriate, a positive control. Commercial ELISA-based tests designed to detect B. cereus enterotoxins (NHE and Hbl) proved negative when applied to a supernatant of the Calsporin strain and PCR failed to detect the presence of any component of the genes encoding the tripartite toxins (nhe, hbl) or the killer toxin (cytk). In case any toxins produced by the Calsporin strain lacked homology with the known B. cereus toxins and so escaped detection, a ten-fold concentrated supernatant from the Calsporin strain was applied to Vero cells in a cell culture cytotoxicity test. Reduction in labelled leucine uptake, used as the measure of cell toxicity, did not differ markedly from the appropriate control. Finally a sperm motility test was included to test for the presence of an emetic toxin (cereulide-like toxin). No change in sperm motility was seen in the presence of a suspension of agargrown B. subtilis, in contrast to the complete inhibition of mobility seen in the presence of B. cereus cells (the positive control) Antibiotic resistance and antibiotic production The susceptibility of the production strain to the 13 antibiotics recommended by SCAN in its revised Opinion of 2003 (SCAN, 2003) was tested using MIC plates containing serial dilutions of the antibiotics. NCCLS-recommended reference strains were included as positive controls (NCCLS, 2003). All of the MIC values fell below the SCAN defined breakpoints. The production strain of B. subtilis produced a substance inhibitory to the growth of B. cereus when directly co-cultured on agar or when a filter paper impregnated with supernatant was as applied to a lawn culture. A more limited and marginal response was also seen towards Staphylococcus aureus but not against the other four indicator strains tested. The observations are consistent with the production of a bacteriocin-like substance and do not indicate the production of an antimicrobial agents relevant to antibiotics in present clinical use. 2.3 Stability of the additive Three batches of the manufactured product were stored for 36 months at 25ºC and 60% relative humidity and for six months at 40ºC and 75% relative humidity. However, the data presented covers only the first 12 months although there is a stated intention to provide data of survival after 18, 24 and 36 months. In addition, no zero time counts were available for bacterial counts made by one external organisation. Parallel measurements made in-house included zero time values but only covered a nine month period. Nonetheless, despite the experimental inadequacies, as would be expected given the nature of the product, Calsporin is evidently stable at 25ºC for at least one year and at 40ºC for at least 6 months. Good photostability was also demonstrated for one batch. The same three batches of the additive used above were also incorporated into typical broiler premixes and pelleted feed and stored for six and three months respectively under the two sets of conditions described above. Recoveries from premixes after six months were between 91 and 123% of the time zero counts when stored at the lower temperature but only 79-88% of the zero time counts when stored at the higher temperature. Consequently the applicant advises that these higher temperatures should

7 Opinion on Calsporin for chickens for fattening 7/15 be avoided for periods longer than one month. No losses were seen in the pelleted feed samples after one month storage at 25ºC, but counts were reduced by approximately one-third after three months. The product was not stable in pelleted feed held at 40ºC. The feed used in the stability experiment was conditioned/pelleted at a relatively low temperature (70ºC). In a separate series of experiments investigating stability during feed preparation, recovery was also found to be greatest from feed conditioned and pelleted at 70ºC than at higher temperatures. However even at 90ºC the average recovery was 75% of that added (compared to an average of 83% at 70ºC). Appropriate multiple samples were taken from premixes and complete feed and used to measure homogeneity. The data presented demonstrates that Calsporin is capable of being evenly distributed throughout premixes, mash and pelleted feed. 2.4 Incompatibilities An in vivo compatibility study with 204 broiler chicks was made for three weeks and involved 15 separate treatments (12 chicks per treatment). One group was fed the basal diet without Calsporin, a second group Calsporin only and a further 13 groups Calsporin plus one of a representative range of approved additives or medicinal substances (Table 2). An ash marker was included in the basal diet and used to derive expected bacterial counts assuming that the Bacillus spores acted as inert particles. This value was compared with the observed faecal counts on day 10 and day 21. Table 2. Effect of representative feed additives or medicinal substances on the survival of Calsporin Treatment Calsporin count (log10 cfu g -1 ) (Calsporin +) Expected Observed 10 days Observed 21 days No Calsporin Calsporin only Formic acid Narasin + nicarbazin Maduramycin Lasolacid Semduramycin Oxytetracycline Enrofloxacin Colistin Trimethoprim Amoxycillin * 5.63 Monensin Salinomycin Diclazuril * Significantly different from treatment with Calsporin only (P<0.05) Overall, only two observations were significantly different (P<0.05) but only in one case (Calsporin plus amoxicillin at day 10) was the faecal count significantly lower than corresponding count for Calsporin alone. In all other cases no significant reduction in numbers of bacilli were observed. 2.5 Proposed conditions of use Calsporin is intended for use as a zootechnical feed additive at a recommended dose of 1 x 10 9 cfu kg -1 complete feedingstuffs. No withdrawal period is proposed. 2.6 Control methods The plate count method for the enumeration of viable spores of B. subtilis in the complete additive, premixes and feedingstuffs has been accepted by the CRL as

8 Opinion on Calsporin for chickens for fattening 8/15 suitable for purpose. EFSA has verified the report submitted by CRL, the Executive Summary of which can be found attached as Annex A. 3. Efficacy Evidence for the efficacy of the additive Calsporin is presented in the form of four efficacy studies made with broilers, all completed in the same year (2004) in four different Member States of the EU. The study design was broadly similar in each case allowing the results from the individual trials to be pooled and subject to further statistical analysis (meta-analysis). In each case the trial consisted of two treatments, a control group fed a normal broiler diet and a test group given the same diet supplemented with Calsporin at the recommended dose (1 x 10 9 cfu kg -1 complete feedingstuffs). The intended dose was confirmed by direct measurement. The duration of the trials was 42 days. The trial involving the smallest number of birds (Trial 4, 1248 birds) had 24 replicates of 26 broilers per treatment and the trial with the greatest number of birds (Trial 2, 2600 birds) had 26 replicates of 50 birds for the control group and 22 replicates for the test group (Table 3). Grower (0-21 days) and finisher diets (22-42 days) were formulated based on wheat and soybean as the major ingredients but with variations in composition reflecting local practice. In three trials feed was supplied as pellets and in one (Trial 1) as a mash. Feed was free of antibiotic growth promoters and in three cases coccidiostat. Only in Trial 1 was diclazuril included. Ross broilers were used in three trials and Cobb (Trial 2) in the remaining study. Mortality rates were consistently low ranging from % with deaths randomly distributed between the two treatments. Table 3. Summary of the zootechnical parameters for the four trials (Day 1-42, males and females combined). Trial Final weight Feed intake Weight gain Treatment (No of birds) (g) (g day -1 ) (g day -1 ) Feed/gain Trial 1 Control (1800) Calsporin * Trial 2 Control (2600) Calsporin 2073 ** ** 1.91 ** Trial 3 Control (2560) Calsporin * 1.77 Trial 4 Control (1248) Calsporin *P<0.05; ** P<0.001 compared to Control values In Trial 1 there was an overall small (~1%) but significant improvement in the efficiency of feed conversion in the treated group compared to the control group. In the second trial highly significant improvements in the efficiency of feed conversion (~3.3%), mean daily weight gain (+3.5%) and final weight (+3.6%) were seen in the treated group. Results from the third trial also showed a significant increase in mean daily weight gain in the Calsporin treated group (~+6.0% in females and +5.4% for males), but this was not accompanied by an overall significant improvement in feed conversion. Intake increased significantly in female birds only. No overall significant treatment-related improvements were seen in Trial 4. The data from the four trials were tested for homogeneity, pooled and analysed with supplementation, sex and trial considered as the main effects. The results are summarised for the whole growing period in Table 4. Table 4. Summary of the meta-analysis applied to data from the four efficacy trials. Treatment Final body Average daily Average daily Feed/gain

9 Opinion on Calsporin for chickens for fattening 9/15 weight (g) gain (g) feed intake (g) Control Calsporin SEM (n=104) Probability Conclusions In three of the four trials there was a statistically significant benefit over the whole trial period manifested as an improvement in final weight, daily weight gain and/or feed to gain ratio. The meta-analysis gives a strong indication of the magnitude of the improvement in zootechnical parameters which might be expected from the inclusion of Calsporin in broiler feed. Total body weight, daily gain and the efficacy of feed conversion were all improved by 2.7%. These changes were not a result of increased feed intake implying that the product has a beneficial effect on feed utilization or retention. 4. Studies concerning the safety of the product 4.1. Safety of the product for the target species Tolerance of target species A tolerance study was made with 192 Ross 308 broilers per treatment group (equal numbers of males and females) with the birds randomly allocated to single-sex floor pens of 12 birds per pen. Birds were fed either a standard broiler mash or the same feed supplemented with 1.5 x cfu kg -1 complete feed Calsporin, representing 15X the recommended dose. Analysis confirmed that in all cases the Calsporin was supplied at least 10X the recommended dose. Observations were made from day 5 to day 40 (duration 36 days). In addition to observations of general health and the measured zootechnical parameters, blood was taken from four birds per sex and treatment group for haematology and blood chemistry. Necropsies were performed on four birds per sex per treatment group at the end of the study Seven birds died or were culled on trial, five from the control group and two from the test group (1.8% mortality) for reasons unrelated to the product under test. Otherwise, no adverse health problems were encountered. There were no statistically significant differences between control and test groups with respect to weight gain or feed conversion ratio. Necropsies did not reveal any pathological abnormalities. The blood chemistry values were within the expected range and, with the exception of the haematocrit values, did not differ significantly between treatments. Haematocrit values for control and test birds (27.3 vs. 26.0%, respectively) were significantly different (P<0.05), although all values fell within the normal physiological range Effects on the gut microflora Advantage was taken of a large efficacy study made with 8160 broilers (equal numbers of each sex) to examine the effects of the additive on the gut flora and to determine persistence of the production strain after removal from feed. Faecal samples were taken from 60 birds per treatment (control and supplemented) on days 20 and 37 and analysed for numbers of the B. subtilis production strain, enterococci, lactobacilli, Clostridium perfringens, Salmonella and Campylobacter. To measure persistence an additional 96 day-old birds were placed on the control diet and housed separately from the efficacy study. On day 20, 96 birds receiving the Calsporin diet were withdrawn from the efficacy study, transferred to a separate facility and placed on the control diet. Faecal samples were collected on days 20, 23, 27 and 30 from 60 birds per treatment group and numbers of B. subtilis measured. All diets contained coccidiostats (robenidine in the starter phase and halofuginol in the grower phase).

10 Opinion on Calsporin for chickens for fattening 10/15 As would be expected, numbers of B. subtilis at day 20 and the end of the experiment were significantly higher in birds receiving the additive, otherwise no significant differences in numbers of enterococci, lactobacilli and C. perfringens were seen. Salmonella and Campylobacter could not be detected in any faecal sample. After withdrawal of Calsporin from feed, numbers of B. subtilis in faeces returned to the level seen in control birds within three days. The higher numbers of Bacillus spores recovered from the treated group was taken as evidence of compatibility with robenidine and halofuginol. Reference is also made to two publications, the first of which (Maruta et al., 1996) describes two laboratory studies with the Calsporin strain of B. subtilis made with broiler chicks deliberately infected with either Salmonella or Campylobacter and the microbiology of three field studies in Japan. However, in both this paper and the subsequent, the dose used was 3 x 10 8 cfu kg -1 feed, approximately one fifth of the presently recommended dose. In the laboratory challenge studies numbers of infected birds were reported to be significantly lower in the supplemented group compared to the control group. In each of the three field trials (two experimental designs) there was a significant decrease in the numbers of Campylobacter and in the detection rate for both Campylobacter and Salmonella in birds receiving the additive. The second published study (Fritts et al., 2000) describes two American trials with the same experimental design in which the effects of Calsporin on performance and the microbiological quality of the carcass was compared with untreated controls. Analysed over both studies, a significant reduction in numbers of Campylobacter were seen in the carcasses of treated birds. Half of the carcasses of treated birds scored positive for Salmonella compared to all of the control carcasses Conclusions on safety for the target species Calsporin was well tolerated by chickens for fattening at a ten-fold overdose. The difference in haematocrit values is not considered an issue of concern since all values remained within the normal physiological range. Numbers of the main groups of bacteria measured contributing to the flora of the digestive tract in chickens were not significantly affected by the inclusion of Calsporin at the recommended dose. However, there is published evidence of the ability of Calsporin to reduce numbers of Salmonella and Campylobacter in artificially and naturally affected birds and consequently to reduce contamination of carcasses. The production strain did not colonise the digestive tract; numbers falling to the background level within three days of withdrawal from feed Safety for the consumer Genotoxicity studies including mutagenicity Two in vitro genotoxicity tests were completed using a production batch of Calsporin. Both studies followed internationally recognised protocols and were GLP compliant. The test material was sterilised on the day of use (115ºC for 10 or 15 min). A bacterial reverse mutation (Ames) test was made with five strains of Salmonella Typhimurium (TA1535, TA1537, TA102, TA98 and TA100) with and without metabolic activation. The test material caused no visible reduction in bacterial growth and so was tested to the maximum recommended dose of 5000 µg plate -1. No significant increases in the frequency of revertant colonies were observed with any of the tested doses. Consequently Calsporin was considered non-mutagenic under the conditions of the test. The second genotoxicity test reported was test for chromosome aberrations in human lymphocytes. A preliminary test showed that the maximum recommended dose level for the test (5000 µg ml -1 ) was non-toxic to the cells. Three dose levels including the

11 Opinion on Calsporin for chickens for fattening 11/15 maximum recommended were used with and without (S9) metabolic activation. Vehicle controls had frequencies of cell with aberrations within the range expected for normal lymphocytes and the positive control (Mitomycin C) induced the expected significant increase in numbers of cells with aberrations. Calsporin behaved as the vehicle control and thus was considered non-clastogenic to human lymphocytes Oral toxicity A 90-day repeat dose oral toxicity test was made with 96 rats, four weeks of age at the start of the study. The study followed an internationally recognised protocol and was GLP compliant. Twelve rats of each sex were assigned to each of four treatment groups. The first group was a control fed a basal diet without inclusion of Calsporin. In the remaining three groups the basal diet was supplemented with 1 x 10 8 cfu kg -1, 1 x cfu kg -1 or 5 x cfu kg -1 feed B. subtilis supplied in the form of Calsporin (equivalent to 1/10 th, 10X and 500X of the dose recommended for chickens for fattening). Three deaths were recorded (one male in the low dose group and two males in the high dose group), but these were considered unrelated to the administration of the test substance. A significant increase in urinary sodium and chloride was seen in all animals receiving Calsporin but not in the control animals. As there were no corresponding abnormalities in kidney function or morphology, it was considered that these changes were diet related but were not adverse effects. There were also significant increases in serum albumin/globulin ratio and serum albumin in the medium and high dose females. However, there was no change in total serum protein and no tendency towards haemoconcentration (i.e., normal haematocrit and haemoglobin concentrations). No adverse effects were seen on gross pathology, histopathology or ophthalmology. On the basis of this chronic study a NOAEL of 5.0% Calsporin (5 x viable spores kg -1 complete feed) is proposed Safety for the user The product is marketed in the form of a free-flowing powder with moderate dusting potential ( % in a Heubach dustometer) and with a particle size distribution suggesting that about 90% of the product is inhalable (<100 µm diameter) and approximately 10% respirable (<10 µm diameter). Consequently, the most likely route of exposure for those handling the product is via an airborne route Irritancy Although, given the nature of the additive, irritancy is not regarded as a major hazard, two GLP-compliant irritancy tests (skin and eye) were done with albino New Zealand rabbits using samples from a commercial batch of Calsporin following internationally recognised protocols. Calsporin was not found to be corrosive or an irritant for the skin of a rabbit or, after instillation, for the eye Skin sensitisation A skin sensitisation test with 15 guinea pigs was completed, again using a sample from a commercial batch of Calsporin and following an internationally recognised protocol. One guinea pig died at the start of the study before exposure and a second of unknown cause during the second phase of the study. No erythema or oedema was seen in any of the surviving animals after the challenge phase and so it was concluded that the product did not produce a delayed dermatitis under the conditions of the test Toxic effects on or via the respiratory system. The possible toxic effects of the product via a respiratory route was no directly determined. Consequently it has to be assumed that Calsporin, in common with many

12 Opinion on Calsporin for chickens for fattening 12/15 other proteinaceous products, would act as a respiratory sensitizer and the product should be labelled and handled accordingly. This is made more likely by the relative small particle size and dusting potential of Calsporin. The 90-day study indicates that the product does not have a systemic toxicity. No toxic events have been reported from the plants manufacturing Calsporin Conclusions on safety for consumers and workers The production strain was shown to be non-toxigenic and Calsporin to be negative for genotoxicty in two in vitro assays and not to cause adverse effects in a repeat dose chronic toxicity study in rats. The FEEDAP Panel concludes that the use of product in chickens for fattening will not be harmful for consumers of chicken meat or meat products. Exposure is most like via a dermal or respiratory route for those handling the product. Specific tests for irritancy (dermal and ocular) and for skin sensitization were negative indicating that skin contact is unlikely to cause problems. However, in the absence of specific data, and given the small particle size and propensity for dust formation, sensitization via a respiratory route cannot be excluded Safety for the Environment Bacillus subtilis is ubiquitous in the environment, particularly soils from which this particular strain originated. Use of the product as a feed additive would not measurably increase the concentration of the organism or otherwise disturb the local environment. Consequently no environmental risk assessment is considered necessary. 5. Post-market monitoring The applicant describes the routine quality control procedures in place to ensure the continuing consistency and safety of the product, the complaint-handling procedure and mechanism for product recall. The FEEDAP Panel considers these procedures a necessary part of good manufacturing practice and to be adequate to deal with any problems that might be reasonably anticipated. CONCLUSIONS AND RECOMMENDATIONS CONCLUSIONS Efficacy was demonstrated in three of four trials with a statistically significant benefit shown over the whole trial period manifested as an improvement in final weight, daily weight gain and/or feed to gain ratio. The statistical analysis of the pooled data from all four trials gives an indication of the magnitude of the improvement in zootechnical parameters which might be expected from the inclusion of Calsporin in broiler feed (2-3%). Calsporin is well tolerated by chickens for fattening at a ten-fold overdose. Numbers of the main groups of bacteria measured contributing to the flora of the digestive tract in chickens were not significantly affected by the inclusion of Calsporin at the recommended dose. However, there is published evidence of the ability of Calsporin to reduce numbers of Salmonella and Campylobacter in artificially and naturally affected birds and consequently to reduce carcass contamination. The production strain did not colonise the digestive tract; numbers falling to the background level within three days of withdrawal from feed. The production strain was shown to be non-toxigenic and Calsporin to be negative for genotoxicty in two in vitro assays and not to cause adverse effects in a repeat dose chronic toxicity study in rats. The FEEDAP Panel concludes that the use of product in

13 Opinion on Calsporin for chickens for fattening 13/15 chicken for fattening will not be harmful for consumers of chicken meat or meat products. Exposure is most likely via a dermal or respiratory route for those handling the product. Specific tests for irritancy (dermal and ocular) and for skin sensitization were negative indicating that skin contact is unlikely to cause problems. However, in the absence of specific data, and given the small particle size and propensity for dust formation, sensitization via a respiratory route cannot be excluded. Bacillus subtilis is ubiquitous in the environment and use of the product as a feed additive would not measurably increase the concentration of the organism or otherwise disturb the local environment. Consequently no environmental risk assessment is necessary. RECOMMENDATION The fermentation process used for the production of Calsporin is non-sterile and depends initially on the size of the inoculum to out-complete with unwanted bacteria and then on a pasteurization step to kill any non-spore formers that manage to develop. However, there remains a potential for other aerobic spore-formers to survive and multiply alongside the production strain and to contaminate the final product. The FEEDAP Panel recognizes that the majority of other spore-formers are innocuous and likely to be present only in low number. A possible exception is Bacillus cereus. Consequently, the FEEDAP Panel recommends that the final product should be routinely monitored for the specific presence of B. cereus as part of the quality control process. REFERENCES Fritts, C.A., Kersey, J. H., Motl, M.A., Kroger, E.C., Yan, F., Si, J., Jiang, Q., Campos, M.M., Waldroup, A.L. and Waldroup, R.W Bacillus subtilis C-3102 (Calsporin) improves live performance and microbiological status of broiler chickens. J. Appl. Poult. Res. 9, Maruta, K., Miyazaki, H., Masuda, S., Takahashi, M., Marubashi, T., Tadano, Y., and Takahashi, H Exclusion of intestinal pathogens by continuous feeding with Bacillus subtilis C-3102 and its influence on the intestinal microflora in broilers. Anim. Sci. Technol. (Jpn.) 67 (3), NCCLS (National committee for clinical laboratory standards) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard sixth edition M7-A6, vol 23 N 2, Wayne, Pa: National committee for clinical laboratory standards. Villanova, PA. SCAN (Scientific Committee on Animal Nutrition), Opinion of the Scientific Committee on Animal Nutrition on the safety of use of bacillus species in animal nutrition. SCAN (Scientific Committee on Animal Nutrition), Opinion of the Scientific Committee on Animal Nutrition on the criteria for assessing the safety of microorganisms resistant to antibiotics of human clinical and veterinary importance. DOCUMENTATION PROVIDED TO EFSA 1- Dossier on CALSPORIN (Bacillus subtilis C-3102) in chickens for fattening. June Submitted by Calpis Co Ltd. 2- Evaluation report of the Community Reference Laboratory feed additives authorisation on the methods(s) of analysis for Calsporin for chickens for fattening.

14 Opinion on Calsporin for chickens for fattening 14/15 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 ACKNOWLEDGEMENT The Scientific Panel on Additives and Products or Substances used in Animal Feed wishes to thank Professor Atte von Wright for the contribution to this opinion.

15 Opinion on Calsporin for chickens for fattening 15/15 ANNEX A Executive Summary of the Evaluation Report of the Community Reference Laboratory Feed Additives Authorisation on the Method(s) of Analysis for Calsporin for chickens for fattening In the current application authorisation is sought for the probiotic, Calsporin, under the category zootechnical additives, group 4, according to the classification system of Annex I of Regulation (EC) No 1831/2003. Specifically, the use of Calsporin as a gut flora stabiliser for chickens for fattening (broilers), to improve growth and feed efficiency, is requested. Calsporin consists of viable spores of Bacillus subtilis C-3102 (as active agent) and calcium carbonate (as carrier). The appearance of the additive is a dry and granular powder. The feed additive is intended to be mixed into compound feedingstuffs at a final concentration of 1 x 10 9 c.f.u./kg (c.f.u. = colony-forming units). For the determination of the active agent in the feed additive, premixtures and feedingstuffs, a surface plate count method is proposed by the applicant to enumerate viable spores of Bacillus subtilis C Vegetative cells are inactivated by a heat treatment and not taken into account. The method is quantitative and uses trypticase soy agar (TSA) as medium. The plate count method s performance characteristics for enumerating the active agent in the feed additive include a relative standard deviation of repeatability (RSDr) and within-laboratory relative standard deviation reproducibility (RSDR) values in the 8 10 % range. The limit of quantification (LOQ) for the method is 2.5 x10 3 c.f.u./kg. The performance characteristics are considered acceptable. For the enumeration of the active agent in premixtures and feedingstuffs the method s performance characteristics include RSDr and within-laboratory RSDR values in the % range. The LOQ of the method is 2.5 x 10 4 c.f.u./kg. The performance characteristics obtained for premixtures and feedingstuffs are considered acceptable. However, for official control purposes a fully ring-trial validated, peer reviewed and published method for enumeration of bacilli spores including those of B. subtilis, is recommended [J.AOAC Int , ]. Methods performance characteristics for the AOAC method using samples of premixtures and feedingstuffs include a RSDr and a between-laboratory RSDR of around 1% and 6%, respectively. For identification of the active agent a standardised, automated molecular method based on restriction analysis of DNA is proposed and appears suitable for the purpose of analysis. Further testing or validation is not considered necessary, as appropriate methods for official control are available.