The EFSA Journal (2006) 406, 1-11

Transcription

1 The EFSA Journal (26) 46, 1-11 Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on the safety and efficacy of the microbiological product 35, a preparation of Bacillus subtilis, as a feed additive for chickens for fattening in accordance with Regulation (EC) No 1831/23 (Question N o EFSA-Q ) Adopted on 17 October 26 SUMMARY The product 35 is a microbial feed additive based on a single strain of Bacillus subtilis. It is proposed as a zootechnical feed additive for chickens for fattening up to slaughter age at a dose range of 8 x x 1 9 CFU kg -1 complete feedingstuff. The European Food Safety Authority has been requested to deliver 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 at the minimum recommended dose has been demonstrated in four of seven studies, in which a statistically significant improvement in the final weight was seen. The in vitro data provided by the applicant on compatibility with coccidiostats can be considered indicative but would need to be substantiated in vivo before reaching a conclusion. The FEEDAP Panel concludes that 35 is safe at the highest recommended level for chickens for fattening, with at least 1-fold margin of safety. The lack of toxigenic properties of the B. subtilis strain has been experimentally demonstrated. The consumer safety aspects were considered during the assessment for authorisation of BioPlus 2B, a product which contains the production strain and another Bacillus strain. The FEEDAP Panel is not aware of any new information that would require it to reconsider this assessment. As a proteinacious substance 35 may have a sensitizing potential, and adequate protection is recommended when handling the product. Since bacilli are common soil organisms and abundantly present in the environment, the product 35 is not of environmental concern. Key words: 35, Bacillus subtilis, chickens for fattening, efficacy, safety

2 Opinion on 35 for chickens for fattening 2/11 BACKGROUND Regulation (EC) No 1831/23 1 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 Chr Hansen A/S 2 for authorisation of the microbial product 35, which is a preparation of Bacillus subtilis (DSM 17299) 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. This product has not been previously authorised. According to Article 7(1) of Regulation (EC) No 1831/23, 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 9 of January of 26. The product 35 is a preparation of Bacillus subtilis (DSM 17299). This Bacillus subtilis strain (deposited as DSM 575) is authorised, together with Bacillus licheniformis (DSM 5749) as a feed additive (BioPlus 2B) for use in piglets, sows, pigs for fattening, turkeys for fattening and calves up to three months (E 17). The safety of Bacillus subtilis (DSM 575) for the target animals pigs, chickens for fattening, turkeys and calves, as well as for the consumer and user was assessed when used in the product BioPlus 2B by the Scientific Committee on Animal Nutrition (SCAN) on 22 June 2. 3 TERMS OF REFERENCE According to Article 8 of Regulation (EC) No 1831/23 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 microbiological product 35, which is a preparation of Bacillus subtilis (DSM 17299) when used under the conditions described in Table 1. 1 OJ L 268, , p Chr. Hansen A/S Boege Allé, DK-297 Hoersholm, Denmark 3

3 Opinion on 35 for chickens for fattening 3/11 Table 1. Register entry as proposed by the applicant. Additive 35 Registration number/ec No/No Category of additive Zootechnical Functional group of additive Gut flora stabiliser Description Composition, description Chemical formula Purity criteria Method of analysis Min. 1.6 x 1 9 CFU g -1 of Bacillus subtilis DSM Whey permeate as carrier. Not applicable Complies with EU legislation on microbial quality, heavy metals, toxins and undesirable substance. Microbial counting technique based on plating on Tryptose Blood Agar, TBA (later on Tryptic Soya Agar, TSA). Trade name Name of the holder of authorisation Chr. Hansen A/S, Denmark Conditions of use Species or category of animal Chickens for fattening Maximum Age Minimum content Maximum content CFU kg -1 of complete feedingstuffs Withdrawal period Slaughter age 8 x x 1 9 Not applicable 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 None Face mask, gloves and goggles should be worn when handling the product to reduce contact with dust. Post-market monitoring is included in the general handling system of incoming complaints. Qualified employees will be handling each reported case, and evaluate if any adverse effects are seen by the use of the product, and take the needed actions. Data and samples of each produced batch are kept for at least five years if further tests should be needed. The production sites producing 35 are GMP, Fami-qs and/or ISO certified which includes the principles of HACCP. Dosage in final complete feedingstuff should be x 1 9 CFU per kg. Marker residue Maximum Residue Limit (MRL) Species or category of animal Target tissue(s) or food products Maximum content in tissues

4 Opinion on 35 for chickens for fattening 4/11 Not applicable Not applicable Not applicable Not applicable

5 Opinion on 35 for chickens for fattening 5/11 ASSESSMENT 1. Introduction The product 35, a microbial feed additive based on a single strain of Bacillus subtilis (DSM 17299) is proposed for use in feed for chickens for fattening up to slaughter age (functional group: gut flora stabilisers). The Bacillus subtilis (DSM 17299) strain is used together with a Bacillus licheniformis strain in a product already authorised (BioPlus 2B). 2. Characterisation of the product 2.1. Product description The product is composed of Bacillus subtilis DSM spore concentrate (.5%), sodium aluminosilicate (1.%) and whey permeate (98.5%). The product is a freeflowing powder, with less than 1.3% of particles with a particle size below 1 µm (determined by Laser scattering/diffraction technique). The dusting potential has been characterised by Stauber-Heubach method as 6 g per m 3 of air. The product has been shown to mix homogenously in the feed. The production includes a two stage fermentation process, harvesting of the spores by centrifugation and spray drying of the spore concentrate. The spray dried spore concentrate is subsequently blended with the carrier materials. The product is monitored for the presence of Salmonella, coliforms, B. cereus and yeasts and moulds, and the counts were shown to be below the detection levels of the standard microbiological analytical methods. The content of heavy metals (As, Cd, Hg and Pb) are <.5 mg kg -1, and that of aflatoxin B1 <.2 mg kg -1. Dioxins were not detected in the product Identity of the active agent The Bacillus subtilis strain from the product 35 was originally isolated from soy bean mash. It has subsequently been deposited in Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM) with deposition number DSM 17299, and the species identification has been performed by the DSM Identification Service. The genetic stability of the strain has been assessed by restriction enzyme digestion patterns of chromosomal DNA after up to 35 generations of growth. The strain does not contain plasmids and is not genetically modified Incompatibilities with other additives The spores and vegetative cells of DSM have been tested against a number of coccidiostats using a plate assay. The concentrations in the medium corresponded with the maximum amounts allowed in feeds. The strain tolerated diclazuril, halofuginone and robenidine, but was sensitive to maduramicin, monensin, lasalocid sodium and salinomycin. These in vitro data can be considered indicative but would need to be substantiated in vivo before reaching a conclusion on the compatibility of this product with the coccidiostats tested Intended use of the product The product is intended for use in feed for chickens for fattening at the inclusion levels of 8 x x 1 9 spores per kg of feedstuff Stability The stability of aluminium foil packed product has been studied at three different storage temperatures (5, 3 and 37ºC) for 24 months. Although there was a slight

6 Opinion on 35 for chickens for fattening 6/11 reduction in spore viability at higher temperatures, 82% of the spores survived at 37ºC at the end of the trial. The product has been shown to be stable for as much as 35 months (survival 81% at the end of the trial) in commercial packages and stored at ambient temperatures. The product tolerates pelletisation at 84ºC, without compromising the viability (survival 86.7%). These results are partly based on previous trials with BioPlus 2B, which has a similar composition, but contains also B. licheniformis, as stated above. In a study on a mineral premixture (1 g of mixture in foil bags at 25ºC) the spore counts remained unaffected for three months. The stability in final feeds was demonstrated in pelletised chicken feed stored at 25ºC for up to 28 months of storage. At the end of the trial the viable spore count was 71% of the original (no reduction was seen during the first 18 months of storage). In a similar study the spore counts were not affected by the inclusion of 6 mg kg -1 salinomycin sodium in the feed Evaluation of the analytical 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 report can be found in the Appendix. 3. Efficacy The company reports seven trials with chickens for fattening, in which different doses of 35 have been used. The doses were verified by microbiological analysis in trials 1, 2, 3, 4 and 5, while with trials 6 and 7 this information is lacking. Two of the trials (trials 6 and 7) have been performed outside Europe (in the USA and Brazil, respectively). All the trials were well conducted with proper experimental design and statistical analysis. The results are summarized in Table 2. Statistically significant improvements in the final weight of birds were seen at the minimum recommended dose (8. x 1 8 CFU kg -1 ) in trials 2, 4, 6 and 7, and at the maximum recommended dose (1.6 x 1 9 CFU kg -1 ) in trials 1 and 2. In trials 3 and 5 the weight gain improvements in 35 treated groups (either at the minimum or maximum recommended dose) were not statistically significant. Regarding the feed conversion rate (FCR), the effects were variable. In trials 1, 3, 4, 5 and 7, FCR was not affected by 35 supplementation. In trial 2, FCR was statistically significantly higher and in trial 6 significantly lower in 35-treated groups in comparison to controls. Conclusions on efficacy The applicant has provided four studies showing a statistically significantly higher final weight in birds fed with 35 at the minimum recommended dose level. Thus the product can be considered efficacious at the lowest recommended dose (8. x 1 8 CFU kg -1 ).

7 Opinion on 35 for chickens for fattening 7/11 Table 2. Trial No Efficacy studies performed with 35 on chickens for fattening 35 (CFU kg -1 feed) Animals (replicates) per treatment x 1 8 (8) 1.6 x x x x (8) x 1 8 (8) 1.6 x 1 9 Sex Trial period (days) Mixed - 42 Males - 35 Males - 35 Average body weight at slaughter (g) 2272 a 247 b 2438 b 2159 a 2275 b 2261 b 2275 b Feed conversion (kg kg -1 ) a 1.57 b 1.59 b 1.58 b Males - 35 a x 1 8 (6) 1936 b Females x 1 8 (6) Mixed - 42 a 2.23 a 8. x 1 8 (1) 1999 b 1.99 b Males - 42 a x 1 8 (1) 2614 b 1.74 a, b: Means within a column in a trial with different superscripts are significantly different at P.5 4. Safety for the target species 4.1. Tolerance for the target species A tolerance trial was done in connection with the efficacy trial 1, in which two of the four treatment groups (eight replicates of 35 birds per treatment) were supplemented with high concentrations of 35 (2.4 x 1 1 and 2.1 x 1 11 CFU kg -1, corresponding to 15X and 131X maximum recommended dose). The trial lasted 42 days, starting with oneday-old chickens. Supplementation with 15X or 131X overdose did not result in any adverse effects on performance (Table 3) or health of the animals. Table 3. Performance parameters of chickens fed an overdose of (CFU kg -1 feed) Actual dose as multiple of recommended dose Average body weight at slaughter (g) 2272 a 2438 b 2399 b 2419 b Feed conversion (kg kg -1 ) x x x a, b: Means within a column in a trial with different superscripts are significantly different at P.1 A second tolerance trial was performed, in which the experimental design and doses were the same as in the tolerance trial reported above, but with only one replicate of 55 4 Technical dossier/section III/Annex 3 5 Technical dossier/section III/Annex 31 6 Technical dossier/section III/Annex 33 7 Technical dossier/section III/Annex 32 8 Technical dossier/section III/Annex 34 9 Supplementary information May 26/Annex 1 1 Supplementary information May 26/Annex 2

8 Opinion on 35 for chickens for fattening 8/11 birds (mixed sex) per treatment. Individual body weight changes, morbidity and mortality were recorded. At the age of three weeks, tissue samples from Bursa Fabricii and small intestine were collected from five birds per treatment for histological examination. Weights of liver, spleen and heart were recorded at the end of the trial from 3 birds of the control and the highest dose group, respectively. The absence of replication does not allow the evaluation of the performance data. Mortality was reported not to be affected by treatment. There were no relevant differences in the weights of liver, heart and spleen between the control group and the group receiving the highest dose. No differences were observed between the groups in the pathological examination of Bursa Fabricii and small intestine Effects on the intestinal microbiota Microbiological investigations were carried out in connection with the tolerance study at the age of three weeks from the caeca and ilea of seven birds from the control group and the group receiving the highest dose, respectively. The spore counts were analysed from the caeca of all the animals at day 42. The microbial groups analysed from both the caeca and small intestine were total viable anaerobes, streptococci, lactobacilli, sulphite reducers, clostridia, aerobic spore formers (both vegetative cells and spores), Escherichia coli and Salmonella. The aerobic spore formers were, as expected, four logs higher in the treated group than in the controls indicating the survival of the strain. Otherwise the only difference between the groups was the very low number of sulphite reducers in the caeca of the treatment group compared to controls. This difference was not, however, seen in the ileal samples. At day 42 the counts of aerobic spore formers in the caeca of the treated groups ranged from 1.36 x 1 4 to 2.7 x 1 7 CFU g -1, depending of the dose level, compared to 3.8 x 1 3 CFU g -1 in the controls Conclusions for the safety for the target species The performance data from the first tolerance study showed that the animals tolerated the dose of 2.1 x 1 11 CFU kg -1 representing 131 times the maximum recommended dose (1.6 x 1 9 CFU kg -1 ). This observation was supported by the lack of differences in gross pathology in the second study. The microbiological studies confirm the survival and passage of the product strain, and do not indicate any imbalance of the analysed microbial groups as a result of 35 supplementation. The FEEDAP Panel concludes that 35 is safe at the highest recommended level for chickens for fattening. 5. Safety for the consumer The safety of the Bacillus subtilis strain used in 35 has been assessed by SCAN as component of the product BioPlus 2B (see background). The FEEDAP Panel is not aware of any new information that will lead to revise this opinion. The MIC values of Bacillus subtilis DSM against the antibiotics listed in the Opinion of the FEEDAP Panel (EFSA, 25) have been assessed using the NCCLS guidelines, and found to be below the defined breakpoints. The absence of toxigenic potential of the strain has been confirmed during the assessment for authorisation of BioPlus 2B using the boar spermatozoa motility inhibition assay, ileal loop test on pigs, cytotoxicity assays and PCR screen for B. cereus enterotoxin genes hbl, nhe, and EntT.

9 Opinion on 35 for chickens for fattening 9/11 6. Safety for the user Although the respirable fraction of 35 is small (approximately 1.3%), 35, as a proteinacious substance, may induce respiratory sensitisation. 7. Safety for the environment Since bacilli are common soil organisms and abundantly present in the environment, the FEEDAP Panel considers that the product 35 does not pose a risk to wider environment, and does not require environmental safety assessment. 8. Post-market monitoring The applicant describes a 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 The applicant has provided four studies showing an improved weight gain in chickens fed with 35 at the minimum recommended dose (8 x 1 8 CFU kg -1 ). Thus the product can be considered efficacious for chickens for fattening. The in vitro data provided by the applicant on compatibility with coccidiostats can be considered indicative but would need to be substantiated in vivo before reaching a conclusion. The FEEDAP Panel concludes that 35 is safe at the highest recommended level for chickens for fattening. The consumer safety has been previously assessed by SCAN, and particularly its nontoxigenicity has been demonstrated already during the assessment for authorisation of the nearly identical product BioPlus 2B. In absence of specific data on user safety, and taking into account its proteinaceous nature, the product should be treated as a potential respiratory sensitizer. Since bacilli are common soil organisms and abundantly present in the environment, the product 35 is not of environmental concern. REFERENCES EFSA (European Food Safety Authority). 25. Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on the updating of the criteria used in the assessment of bacteria for resistance to antibiotics of human or veterinary importance. DOCUMENTATION PROVIDED TO EFSA 1. Dossier on 35 for use in feedingstuff for chickens for fattening. September 25. Submitted by Chr. Hansen A/S Supplementary information FAD Answers to questions put buy EFSA ( ). December 25. Submitted by Chr. Hansen A/S Supplementary information FAD Answers to questions put buy EFSA ( ). May 26. Submitted by Chr. Hansen A/S. 4. Evaluation report of the Community Reference Laboratory feed additives authorisation on the methods(s) of analysis for 35 for chickens for fattening.

10 Opinion on 35 for chickens for fattening 1/11 5. Comments from the Member States received via the EFSAnet. SCIENTIFIC PANEL MEMBERS Georges Bories, Paul Brantom, Joaquim Brufau de Barberà, Andrew Chesson, Pier Sandro Cocconcelli, Bogdan Debski, Noël Dierick, Anders Franklin, Jürgen Gropp, Ingrid Halle, Christer Hogstrand, Joop de Knecht, Lubomir Leng, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Miklós Mézes, Carlo Nebbia, Walter Rambeck, Guido Rychen, Atte von Wright and Pieter Wester

11 Opinion on 35 for chickens for fattening 11/11 APPENDIX Executive Summary of the Evaluation Report of the Community Reference Laboratory Feed Additives Authorisation on the Method(s) of Analysis for 35 for chickens for fattening In the current application authorisation is sought for the feed additive 35 under the category zootechnical additives 4(b) gut flora stabilisers, according to Annex I of Regulation (EC) No 1831/23. Specifically, authorisation is sought to use 35 for chickens for fattening. The feed additive 35 is a yellowish free-flowing powdery product containing at least 1.6 x 1 9 colony forming units (c.f.u.) per gram of the additive and it is proposed for use in premixtures and feedingstuffs obtaining a concentration ranging from x 1 9 c.f.u. of active agent per kg complete feedingstuffs. For the determination of the active agent (Bacillus subtilis DSM 17299) in the feed additive, premixtures and feedingstuffs, a surface plate count method is proposed. The method uses Tryptose Blood Agar (TBA) base with inclusion of 5% defibrinated calf or sheep blood. The limit of quantification (LOQ) of this method is 1. x 1 3 c.f.u./g (1. x 1 6 c.f.u./kg) which is well below the anticipated minimum target level of application in the animal feedingstuffs. This method is very similar to another method that was ringtrial validated [J.AOAC Int. 23, 86, ] and which uses Tryptose Soya Agar (TSA) as medium. The applicant provides data which confirm that the use of TBA as medium results in comparable performance as when using TSA, and that there are no statistically significant differences between the two methods. The method performance characteristics for the ring trial validated method include relative standard deviations for repeatability (RSDr) and between-laboratory reproducibility (RSDR) of around 1% and 6%, respectively. These performance characteristics are considered acceptable. Therefore, in the opinion of the CRL, the ring trial validated method is suitable for official control purposes. The purity and correct identity of the Bacillus subtilis strain (DSM 17299) is examined by molecular DNA fingerprinting methodology, which is considered to be appropriate for the intended purpose. Pulsed field gel electrophoresis would be considered a suitable technique for official control purposes. Standard and/or official methods are proposed by the applicant for the determination of impurities (heavy metals, microbiological agents) in the feed additive. The methods are considered suitable for the intended purposes. On the basis of supplied documentation, no supplementary experimental work (testing or method validation) is required.