opinion of the scientific panel on additives and products

The EFSA Journal (2005) 272, 1-8

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 product Bacillus licheniformis (DSM 5749) and Bacillus subtilis (DSM 5750) (BioPlus 2B)

authorised as a feed additive in accordance with Council Directive 70/524/EEC

(Question No EFSA-Q-2004-174)

Adopted on 22 September 2005

SUMMARY The European Food Safety Authority (EFSA) received a request to assess the impact of the mixing of the micro-organism product BioPlus 2B with the growth promoter potassium diformate (Formi LHS) in pig feed or the coccidiostat lasalocid A sodium (Avatec 15%) in turkey feed.

An in vitro study aimed at determining the susceptibility to potassium diformate of the bacilli vegetative cells was performed using a viable plate count technique. The results showed similar bacilli counts in media with or without potassium diformate 0.5%. However the buffering capacity of the medium used to perform the study may have masked the expected acidic environment.

An in vivo growth study involving four experimental groups of 250 one-day old turkeys (control, BioPlus 2B, coccidiostat, BioPlus 2B + coccidiostat) was performed in order to determine the compatibility between the bacterial strains (Bacillus licheniformis, Bacillus subtilis) and the coccidiostat. Despite the numerous zootechnical, biochemical or microbiological parameters studied, the experiment did not make it possible to demonstrate the survival of the bacilli in presence of lasalocid A sodium.

In conclusion, the compatibility of BioPlus 2B with either potassium diformate or lasalocid A sodium was not established.

Keywords: Potassium diformate, coccidiostat, compatibility, lasalocid, Bacillus, pig, turkey

Opinion on the additive BioPlus 2B 2/7

TERMS OF REFERENCE

BACKGROUND

The European Commission received a request from Chr Hansen A/S3 to modify the Annex entry of Directive 70/524/EEC to allow the use of the product in pig feed containing the growth promoter potassium diformate (Formi LHS) and in turkey feed containing the coccidiostat lasalocid sodium (Avatec 15%) 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 EFSA. 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 03 of March 2005.

Regulation (EC) No 1831/20031 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 additive Bioplus 2B is a mixture of Bacillus licheniformis (DSM 5749) and Bacillus subtilis (DSM 5750) in a 1:1 ratio. This product is already permanently authorised for use in piglets, sows and pigs for fattening (E 1700), and is currently undergoing assessment for permanent authorisation for turkeys for fattening (former authorisation expired on 23 November 2004).

The Scientific Committee on Animal Nutrition (SCAN) issued an opinion on the use of BioPlus 2B as feed additive on 22 June 20002. On 12 November 2003, the European Food Safety Authority (EFSA) issued an opinion on the use of this product in feeds for chickens for fattening and turkeys for fattening containing the coccidiostat “maduramicin ammonium”.

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, human user and consumer and the environment of the micro-organism product Bacillus licheniformis (DSM 5749) and Bacillus subtilis (DSM 5750) (BioPlus 2B) when used under the conditions described in Table 1, taking into account the background and the information submitted by the applicant. Taking into consideration the previous assessments of BioPlus 2B (safety of this product for the target species evaluated with positive outcome by the SCAN, product permanently approved for all pig categories, product currently undergoing evaluation for permanent approval for turkeys for fattening), the current assessment shall focus only on the impact of the mixing of the micro-organism product “BioPlus 2B” with the growth promoter potassium diformate (Formi LHS) in pig feed and with the coccidiostat lasalocid sodium (Avatec 15%) in turkey feed, when used under the conditions set out in Table 1.

1 OJ L 268, 18.10.2003, p.29. 2 http://europa.eu.int/comm/food/fs/sc/scan/out49_en.pdf 3 Chr. Hansen A/S, 10-12 Boge Allé. P.O. Boks 407 DK – 2970 Horsholm Denmark.

http://europa.eu.int/comm/food/fs/sc/scan/out49_en.pdf


Table 1. Proposal for inclusion of BioPlus 2B in the annex of Directive 70/524/EEC (proposed modifications in bold).

Minimum content

Maximum content EC No

Additive Chemical formula, Description

Species or category of animal

Maximum age CFU kg-1 of complete

feedingstuff

Other provisions Period of authorisation

Piglets __ 1.28x109 3.2x109

In the directions for use of the additive and premixture, indicate the storage temperature, storage life and stability to pelleting. For piglets until approximately 35 kg May be used in compound feed containing the permitted growth promoter: potassium diformate

Sows __ 1.28 x109 1.28 x109

In the directions for use of the additive and premixture, indicate the storage temperature, storage life and stability to pelleting. For sows two weeks before farrowing and during the lactation. May be used in compound feed containing the permitted growth promoter: potassium diformate

E 1700

Bacillus licheniformis (DSM 5749) and Bacillus subtilis

(DSM 5750) [in a 1/1 ratio]

Mixture of Bacillus licheniformis and Bacillus subtilis

containing a minimum of 3.2 x 109 CFU g-1 of

the additive (1.6x109 CFU g-1 of each

bacterium)

Pigs for fattening __ 1.28 x109 1.28 x109

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 growth promoter: potassium diformate

Without time limit

4/7

20 Turkeys

for fattening

__ 1.28x109 3.2x109

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: diclazuril, halofuginone, lasalocid sodium, monensin sodium and robenidine.

23.11.2004

Opinion on the additive BioPlus 2B


ASSESSMENT

1. Compatibility with potassium diformate

To determine the compatibility between the bacterial strains of BioPlus 2B (Bacillus licheniformis and Bacillus subtilis) and the approved acidic growth promoter potassium diformate (Formi LHS), an in vitro study aimed at determining the susceptibility to potassium diformate of the Bacillus vegetative cells was performed using plate count technique. The media used for this study was tryptose blood agar with or without potassium diformate. To simulate the conditions of the gastrointestinal tract, the viability of the bacilli was determined in response to 0.5 % potassium diformate, corresponding to the feed level when corrected by the water to feed ratio (2-2.5). The results of this study showed no significant differences between the bacilli counts of the media with or without potassium diformate. Thus, the bacilli vegetative cells did survive in the potassium diformate acidic environment of this study.

However the buffering capacity of the tryptose blood agar may have masked the acidic effect of the additive. Thus, this in vitro study does not allow to mimic the survival of the bacilli cells in presence of potassium formate in in vivo conditions.

2. Compatibility with lasalocid sodium

To determine the compatibility between the bacterial strains of BioPlus 2B (Bacillus licheniformis and Bacillus subtilis) and lasalocid A sodium (Avatec 15%), an in vivo trial was conducted with 1006 one-day old turkeys until slaughter at 16 weeks of age. They were allocated to four groups (10 replicates per group) : an untreated control group (T1), a group treated with Bioplus 2B (1.28x109 CFU kg-1 feed, T2), a group treated with 125 mg kg-1 lasalocid A sodium (T3), and a group treated with both BioPlus 2B and the coccidiostat (T4). The parameters studied were:

- Zootechnical parameters (feed intake, body weight) - Mortality and morbidity - Dry matter in excrement - Carcass evaluation - Caecal biochemical and microbiological parameters (pH, ammonia, glycolytic

activity, volatile fatty acids (VFA), counts of bacilli)

The results were analysed using a one-way ANOVA and differences between groups were determined by the Duncan's multiple range test.

Zootechnical parameters indicated a significant improvement in feed conversion ratio in the treatment group T4 receiving both additives compared to turkeys receiving only BioPlus 2B. However, performance of T4 animals were not different from control animals.

Table 2. Effect of BioPlus 2B with or without Lasalocid A sodium on performance parameters of turkeys (0-16 weeks)

Treatment Weight gain (kg)

Feed intake (kg bird-1) Feed gain ratio

Control (T1) 13.82 35.84 2.60ab

BioPlus 2B (T2) 13.84 36.78 2.65a

Lasalocid (T3) 13.87 35.50 2.58ab

BioPlus 2B + Lasalocid (T4) 14.03 35.43 2.53 b

a,b: Mean values within a column with no common superscripts differ significantly (P<0.05)

When considering caecal biochemical parameters, significant differences were found between the groups. However these differences did not appear to be related to the additives used.


The recovery of bacilli spores from T2 animals was 3.97 x 104 CFU g-1 and 1.24 x 103

CFU g-1 in T1 animals. No bacilli enumeration was provided for caecal contents of T3 and T4 animals. Thus, no comparison could be done between caecal bacilli counts from T2 (BioPlus 2B) and T4 (BioPlus 2B + lasalocid A sodium). Despite of the numerous parameters studied, this study did not make it possible to demonstrate the survival of the bacilli from BioPlus 2B and lasalocid A sodium.

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. An extract of the Executive Summary of the CRL can be found in Annex A.

CONCLUSIONS Compatibility with potassium diformate

The buffering capacity of the tryptose blood agar may have masked the acidic effect of the additive. Thus, the in vitro study does not allow to mimic the survival of the bacilli cells in presence of potassium formate in in vivo conditions. It does not allow FEEDAP to conclude on the potential effects of potassium diformate on BioPlus 2B.

Compatibility with lasalocid A sodium in turkey feed

The in vivo study involved different dietary treatments and in particular animals treated with BioPlus 2B and animals treated with BioPlus 2B and lasalocid A sodium. Despite the numerous zootechnical, biochemical and microbiological parameters studied, the experiment did not make it possible to demonstrate the survival of the bacilli in the presence of lasalocid A sodium. In the absence of such data, it is not possible to conclude on the potential effects of lasalocid A sodium on BioPlus 2B.

DOCUMENTATION PROVIDED TO EFSA 1. Supplementary dossier vol. 31. Additional information on BioPlus 2B. Compatibility

with the permitted growth promoter potassium diformate (Formi LHS) in feedingstuffs for pigs and the permitted coccidiostat lasalocid A sodium (Avatec 15% cc) in feedingstuffs for turkeys. November 2004. Submitted by Chr. Hansen A/S.

2. Supplementary information FAD-04-001. Additional information on BioPlus 2B. February 2005. Submitted by Chr. Hansen A/S.

3. Letter of reply of Chr Hansen (14/06/05).

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.


ANNEX A.

Extract of the Executive Summary of the Evaluation Report of the Community Reference Laboratory Feed Additives Authorisation on the Method(s) of Analysis for BioPlus 2B

BioPlus 2B is a feed additive consisting of B. subtilis and B. licheniformis belonging to zootechnical additives, category 4.

The current application for BioPlus 2B seeks the following two extensions of its use in animal feeds:

- when used for piglets, pigs for fattening and sows, to use BioPlus 2B together with feed containing the acidic growth promoter potassium diformate (FormiTMLHS); and

- when used for turkeys for fattening, to use BioPlus 2B together with feed containing the coccidiostat lasalocid A sodium 15g/100g (Avatec 15%).

Concerning the determination of the active substances of BioPlus 2B (Bacillus subtilis and Bacillus licheniformis) per se (individually and together), in premixtures and feedingstuffs, a surface plate count method was proposed by the applicant to determine viable counts of the preparation. The method is quantitative and uses tryptone blood agar base (TBA) with inclusion of 5% defibrinated blood. This method is very similar to a previously validated method (by full ring trial, according to international guidelines). The previously validated method uses tryptone soya agar (TSA) as medium and is used for quantification of the additive per se, in premixtures and animal feed. The applicant compared the use of TBA as medium with the use of TSA. Method performance characteristics for the method using TSA as medium included relative standard deviations for repeatability (RSDr) and reproducibility (RSDR) of around 1% and 6%, respectively, according to a peer reviewed scientific journal. Upon request, the applicant provided additional documentation concerning the method validation study of TBA. According to this, the method has a limit of quantification of 1000 colony forming units (c.f.u) per gram (g) sample.

Taking into account (1) the target level of application which ranges between 105 – 109

c.f.u./g of feed sample, (2) the systematic and well performed validation studies for 2 the quantitative microbiological enumeration methodology used in the BioPlus 2B dossier showing equivalence of TBA and TSA, (3) the latter of which has acceptable performance characteristics of RSD values, in the opinion of the CRL this method is fit for purpose and suitable for official control purposes.

In summary, and taking into account the compatibility studies where the number of colonies was similar with and without the presence of Formi LHS and Avatec 15%, indicating that these substances do not affect the method performance, the CRL finds that the proposed methods fulfill the requirements to quantitatively determine the colony forming units present in BioPlus 2B in the proposed concentration range.

On the basis of the supplied documentation, no supplementary experimental work (testing or method validation) is required by the CRL.

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