Abstract
Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of a feed additive consisting of two bacilli strains (tradename: BioPlus® 2B) when used in suckling piglets, calves for fattening and other growing ruminants. BioPlus® 2B is composed of viable cells of Bacillus subtilis DSM 5750 and Bacillus licheniformis DSM 5749. In the course of the current assessment, the latest strain was reclassified as Bacillus paralicheniformis. BioPlus® 2B is intended for use in feedingstuffs and water for drinking for the target species at the minimum inclusion level of 1.3 × 109 CFU/kg feed and 6.4 × 108 CFU/l water, respectively. B. paralicheniformis and B. subtilis are considered eligible for the qualified presumption of safety (QPS) approach. The identity of the active agents was established, and the qualifications regarding the lack of acquired antimicrobial resistance genes, toxigenic potential and bacitracin production ability were complied with. Following the QPS approach, B. paralicheniformis DSM 5749 and B. subtilis DSM 5750 are presumed safe for the target species, consumers and the environment. Since no concerns are expected from the other components of the additive, BioPlus® 2B was also considered safe for the target species, consumers and the environment. BioPlus® 2B is not irritant to the eyes or skin but should be considered a respiratory sensitiser. The Panel could not conclude on the skin sensitisation potential of the additive. BioPlus® 2B when supplemented at 1.3 × 109 CFU/kg complete feed and 6.4 × 108 CFU/l water for drinking has the potential to be efficacious in suckling piglets, calves for fattening and other growing ruminants (e.g. sheep, goat, buffalo) at the same developmental stage.
Keywords: zootechnical additive, BioPlus® 2B, Bacillus paralicheniformis DSM 5749, Bacillus subtilis DSM 5750, safety, QPS, efficacy
1. Introduction
1.1. Background and Terms of Reference as provided by the requestor
Regulation (EC) No 1831/2003 1 establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lays down that any person seeking authorisation for a feed additive or for a new use of a feed additive shall submit an application in accordance with Article 7.
The European Commission received a request from Chr. Hansen A/S 2 for authorisation of the additive consisting on Bacillus paralicheniformis 3 DSM 5749 and Bacillus subtilis DSM 5750 (tradename: BioPlus® 2B), when used as a feed additive for calves for fattening, other growing ruminants, piglets (suckling and weaned), other minor growing and reproductive porcine species, turkeys for breeding and other minor poultry species (category: zootechnical additive; functional group: gut flora stabilizer). In the course of the assessment, the applicant withdrew the request for authorisation for other minor growing and reproductive porcine species, turkeys for breeding and other minor poultry species. 4
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. The particulars and documents in support of the application were considered valid by EFSA as of 19 March 2020.
According to Article 8 of Regulation (EC) No 1831/2003, 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. EFSA shall deliver an opinion on the safety for the target animals, consumer, user and the environment and on the efficacy of the additive consisting on Bacillus paralicheniformis DSM 5749 and Bacillus subtilis DSM 5750 (BioPlus® 2B), when used under the proposed conditions of use (see Section 3.1.3).
1.2. Additional information
The subject of the assessment is the product consisting of viable spores of B. paralicheniformis DSM 5749 and B. subtilis DSM 5750. EFSA issued several opinions on the compatibility of this product with coccidiostats (EFSA, 2004, 2005, 2007a,b, 2008; EFSA FEEDAP Panel, 2005, 2010, 2011a, 2019a), one opinion on the use of the additive for sows (EFSA FEEDAP Panel, 2011b) and one opinion on the use of the additive in water for drinking for piglets, pigs and turkeys for fattening, sows and calves (EFSA FEEDAP Panel, 2016).
The additive is currently authorised as a zootechnical feed additive (gut flora stabiliser) for sows, suckling piglets, 5 weaned piglets, pigs for fattening, calves for rearing and turkeys for fattening (4b1700i). 6 The current authorisation for use in suckling piglets prescribes the simultaneous use with lactating sows. With the current application the applicant seeks the extrapolation of the data from weaned piglets to suckling piglets to allow an independent use of the additive in sows and suckling piglets, and from calves for rearing to calves for fattening and other growing ruminants (e.g. sheep, goat, buffalo) at the same developmental stage.
2. Data and methodologies
2.1. Data
The present assessment is based on data submitted by the applicant in the form of a technical dossier 7 in support of the authorisation request of product consisting of viable cells of B. paralicheniformis DSM 5749 and B. subtilis DSM 5750 as a feed additive. The dossier was received on 12/11/2019 and the general information and supporting documentation is available at https://open.efsa.europa.eu/questions/EFSA-Q-2019-00736.
The confidential version of the technical dossier was subject to a target consultation of the interested Member States from 22 March to 22 June 2020 for which received comments that were considered for the assessment.
The European Union Reference Laboratory (EURL) considered that the conclusions and recommendations reached in the previous assessment regarding the methods used for the control of the active substance/agent in animal feed/marker residue in tissues are valid and applicable for the current application. 8
2.2. Methodologies
The approach followed by the FEEDAP Panel to assess the safety and the efficacy of the product consisting of viable spores of B. paralicheniformis DSM 5749 and B. subtilis DSM 5750 is in line with the principles laid down in Regulation (EC) No 429/2008 9 and the relevant guidance documents: Guidance on studies concerning the safety of use of the additive for users/workers (EFSA FEEDAP Panel, 2012), Guidance on the identity, characterisation and conditions of use of feed additives (EFSA FEEDAP Panel, 2017a), Guidance on the characterisation of microorganisms used as feed additives or as production organisms (EFSA FEEDAP Panel, 2018a), Guidance on the assessment of the safety of feed additives for the target species (EFSA FEEDAP Panel, 2017b), Guidance on the assessment of the safety of feed additives for the consumer (EFSA FEEDAP Panel, 2017c), Guidance on the assessment of the efficacy of feed additives (EFSA FEEDAP Panel, 2018b), Guidance on the assessment of the safety of feed additives for the environment (EFSA FEEDAP Panel, 2019b).
3. Assessment
The subject of the assessment is a product containing viable spores of Bacillus paralicheniformis DSM 5749 and Bacillus subtilis DSM 5750, with the tradename BioPlus® 2B. It is intended for use as a zootechnical additive (functional group: gut flora stabiliser) in feed and water for drinking for calves for fattening, other growing ruminants and piglets (suckling and weaned). It will be hereafter referred to as BioPlus® 2B.
3.1. Characterisation
3.1.1. Characterisation of the active agents
The strains were originally isolated from soil 10 and soybean mash. 11 , 12 Both strains are deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen under the accession numbers DSM 5749 (B. paralicheniformis) and DSM 5750 (B. subtilis). 13 The strains have not been genetically modified.
The full genome of both active agents was sequenced and used for identification purposes. The taxonomic identification of DSM 5749 as B. paralicheniformis was established by digital DNA–DNA hybridisation (dDDH) which showed 92.3% similarity compared with the genome sequence of B. paralicheniformis KJ‐16T. 14 These results were confirmed by multilocus sequence analysis comparing the partial gyrA and rpoB sequences. 15 Similarly, the DSM 5750 strain was identified at species level as B. subtilis by dDDH with 87.8% similarity compared with the genome sequence of B. subtilis ATCC 6051T. 16 These results were also confirmed by multilocus sequence analysis comparing the partial sequences of groEL, gyrA, polC, purH and rpoB. 15
The susceptibility of the two bacterial strains was tested against the list of antibiotics recommended by EFSA (EFSA FEEDAP Panel, 2018). All the minimum inhibitory concentration (MIC) values for DSM 5749 were equal to or fell below the corresponding cut‐off values, except for chloramphenicol, erythromycin and clindamycin. 17 The MIC value for chloramphenicol is one dilution above the cut‐off value. Exceeding the cut‐off value by one dilution is considered to be within the normal range of variation, and thus, not a matter of concern. The MIC values for erythromycin and clindamycin are at least two dilutions above the corresponding cut‐off values (MIC >8 mg/l and 16 mg/l, respectively, cut‐off: 4 mg/l). 18 Therefore, the strain is susceptible to the relevant antibiotics, except to erythromycin and clindamycin. All the MIC values for DSM 5750 were equal to or fell below the corresponding cut‐off values. Therefore, the strain is susceptible to the relevant antibiotics.
The whole genome sequence (WGS) of both strains was interrogated for the presence of antimicrobial resistance (AMR) genes against the ResFinder ■■■■■ and NCBI ■■■■■ databases. A threshold of ■■■■■ was set at protein level. The search for DSM 5749 (B. paralicheniformis) did not identify any hit of concern. 19 Although the strain was resistant to erythromycin and clindamycin, since no acquired AMR genes were found in the WGS, these resistances do not raise safety concerns. No hits of concern for DSM 5750 (B. subtilis) were identified. 20
A cytotoxicity test with Vero cells for each strain was conducted in accordance with the provisions of the FEEDAP guidance on the characterisation of microorganisms used as feed additives or as production organisms (EFSA FEEDAP Panel, 2018). 21 No cytotoxicity was observed.
To exclude the capacity of B. paralicheniformis DSM 5749 to produce bacitracin, the supernatants from the culture were tested against reference strains. 22 , 23 No inhibition by DSM 5749 was observed in any of the reference strains, denoting the lack of antimicrobial production, including bacitracin.
Additionally, the applicant provided a study ■■■■■ 24 ■■■■■. The results would support the conclusion of the phenotypic assay.
3.1.2. Characterisation of the additive
BioPlus® 2B is a powder containing a 1:1 mixture of the two active agents, B. paralicheniformis DSM 5749 and B. subtilis DSM 5750, each present at a minimum concentration of 1.6 × 1010 colony forming units (CFU) per gram of additive, and with a minimum content of total spores in the additive of 3.2 × 1010 CFU/g.
BioPlus® 2B has the same formulation (spores concentrate ~6%, limestone/calcium carbonate as carrier ~92.9% and kieselgur 25 ~1.1% as anti‐caking agent) and method of manufacture 26 as that considered in the previous opinion (EFSA FEEDAP Panel, 2016). Thus, the data pertaining to composition, physical properties and shelf‐life still apply.
Data on five batches of BioPlus® 2B showed that the minimum specification was exceeded in all samples (mean value 5.2 × 1010 CFU/g, range 4.9–6.1 × 1010 CFU/g) and that the 1:1 ratio between the two active agents was complied with in all the analysed batches. 27
The additive is routinely monitored for microbial and chemical contamination at various points in the manufacturing process and in the final product. Limits are set for total coliforms (< 100 CFU/g), Escherichia coli (< 10 CFU/g), yeasts and filamentous fungi (< 100 CFU/g), and Salmonella spp. (no detection in 25 g). Analysis of five recent batches of the additive demonstrated compliance with these limits. 27 The Panel notes that no information on the potential contamination with Bacillus cereus and Enterobacteriaceae was provided. Apparently, no specifications are set for undesirable substances, but the applicant declares that levels are in accordance with legal limits, when these exist. The analysed values on three batches of aflatoxin B1 (< 0.46 μg/kg), mercury (0.0067–0.0075 mg/kg), lead (1.03–1.10 mg/kg), cadmium (0.144–0.174‐mg/kg) and arsenic (1.19–1.26 mg/kg) do not raise concerns. 28
The stability and homogeneous mixing of the additive with feed and premixtures for several animal species were established in the previous opinion (EFSA FEEDAP Panel, 2016). No new data have been provided in the current application, except for an experiment where bacilli counts of three batches of the additive stored at ambient temperature (~25°C) were monitored over 24 months. Results showed no losses (< 0.5 log) over the experimental period. 29
3.1.3. Conditions of use
The additive is intended for use in complete feed and water for drinking for calves for fattening, other growing ruminants and piglets (suckling and weaned) at the minimum recommended inclusion level of 1.3 × 109 CFU/kg feed and 6.4 × 108 CFU/l water. 30
3.2. Safety
3.2.1. Safety for the target species, consumers and environment
Both bacterial species are considered by EFSA to be eligible for the Qualified Presumption of Safety (QPS) approach (EFSA, 2007c; EFSA BIOHAZ Panel, 2020, 2023). This approach requires the identity of the strains to be conclusively established and evidence that they do not show acquired resistance to antibiotics of human and veterinary importance or toxigenic potential. Moreover, the B. paralicheniformis strain should also lack the ability to produce bacitracin. In the view of the FEEDAP Panel, the identity of the strains as B. paralicheniformis and B. subtilis was established, and the qualifications as regards the lack of acquired antimicrobial resistance genes and of toxigenic potential have been met. Moreover, the QPS qualification related with bacitracin has been achieved for DSM 5749, which showed a negative phenotype related to bacitracin production, supported by a number of non‐conservative amino acid substitutions in the active domains of the NRPS enzymes. Consequently, B. paralicheniformis DSM 5749 and B. subtilis DSM 5750 are presumed safe for the target species, consumers and the environment. Since no concerns arise from other components of the additive, BioPlus® 2B is also presumed safe for the target animals, consumers and the environment.
3.2.2. Safety for the user
In a former opinion (EFSA FEEDAP Panel 2016), the dusting potential of the additive (6.3 g/m3) indicated that exposure via inhalation is possible. Given the proteinaceous nature of the active agents, BioPlus® 2B should be considered a respiratory sensitiser.
The skin irritation potential of the additive was studied in vitro according to OECD TG 439 ‘the Reconstructed Human Epidermis Test Method’. 31 The results obtained indicated that the additive is non‐irritant to the skin and according to the UN GHS is classified as ‘No Category’.
The eye irritation potential of the additive was investigated in vitro according to OECD TG 492 the ‘Reconstructed Human Cornea‐like Epithelium (RhCE) test method’. 32 The results of the study showed that the additive is non‐irritant to eyes and according to UN GHS is classified as ‘No Category’.
The FEEDAP Panel notes that the OECD test guidelines available at present are designed to assess the skin sensitisation potential of chemical substances only and that currently no validated assays for assessing the sensitisation potential of microorganisms are available. 33 Therefore, no conclusion can be drawn on the skin sensitisation potential of the additive.
3.2.3. Conclusions on safety for the user
BioPlus® 2B is not irritant to the eyes or skin but should be considered a respiratory sensitiser. The Panel is not in the position to conclude on the skin sensitisation potential of the additive.
3.3. Efficacy
The efficacy BioPlus® 2B for weaned piglets, for piglets during the suckling and post‐weaning phase and for calves for rearing was established in a previous opinion (EFSA FEEDAP Panel, 2016).
The current authorisation for use in suckling piglets 5 prescribes that the additive shall be fed to lactating sows and suckling piglets simultaneously. With the current application the applicant seeks the extrapolation of the data from weaned piglets to suckling piglets to allow for an independent use of the additive in sows and suckling piglets.
Considering the above and since the applicant proposes the use of the same inclusion level (1.3 × 109 CFU/kg complete feed and 6.4 × 108 CFU/l water for drinking) for piglets during the suckling phase only, for calves for fattening and for other growing ruminants (e.g. sheep, goat, buffalo) at the same developmental stage, the conclusions reached in weaned piglets and calves for rearing can be extrapolated/extended to these species/categories at the same use level.
3.4. Post‐market monitoring
The FEEDAP Panel considers that there is no need for specific requirements for a post‐market monitoring plan other than those established in the Feed Hygiene Regulation 34 and Good Manufacturing Practice.
4. Conclusions
BioPlus® 2B is safe for the target animals, consumers and the environment.
BioPlus® 2B is not irritant to the eyes or skin but should be considered a respiratory sensitiser. The Panel is not in the position to conclude on the skin sensitisation potential of the additive.
BioPlus® 2B when supplemented at 1.3 × 109 CFU/kg complete feed and 6.4 × 108 CFU/l water for drinking has the potential to be efficacious in suckling piglets, calves for fattening and other growing ruminants (e.g. sheep, goat, buffalo).
Abbreviations
- CFU
colony forming unit
- EURL
European Union Reference Laboratory
- LOD
limit of detection
- LOQ
limit of quantification
- MIC
minimum inhibitory concentration
Suggested citation: EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed) , Bampidis V, Azimonti G, Bastos ML, Christensen H, Dusemund B, Durjava M, Kouba M, López‐Alonso M, López‐Puente S, Marcon F, Mayo B, Pechová A, Petkova M, Ramos F, Sanz Y, Villa RE, Woutersen R, Prieto M, Anguita M, García‐Cazorla Y, Innocenti ML, López‐Gálvez G, Pettenati E, Revez J, Vettori MV and Brozzi R, 2023. Scientific Opinion on the safety and efficacy of a feed additive consisting of Bacillus paralicheniformis DSM 5749 and Bacillus subtilis DSM 5750 (BioPlus® 2B) for piglets, calves for fattening and other growing ruminants (Chr. Hansen A/S). EFSA Journal 2023;21(3):7859, 10 pp. 10.2903/j.efsa.2023.7859
Requestor European Commission
Question number EFSA‐Q‐2019‐00736
Panel members Vasileios Bampidis, Giovanna Azimonti, Maria de Lourdes Bastos, Henrik Christensen, Birgit Dusemund, Mojca Durjava, Maryline Kouba, Marta López‐Alonso, Secundino López Puente, Francesca Marcon, Baltasar Mayo, Alena Pechová, Mariana Petkova, Fernando Ramos, Yolanda Sanz, Roberto Edoardo Villa and Ruud Woutersen.
Legal notice Relevant information or parts of this scientific output have been blackened in accordance with the confidentiality requests formulated by the applicant pending a decision thereon by the European Commission. The full output has been shared with the European Commission, EU Member States and the applicant. The blackening will be subject to review once the decision on the confidentiality requests is adopted by the European Commission.
Declarations of interest If you wish to access the declaration of interests of any expert contributing to an EFSA scientific assessment, please contact interestmanagement@efsa.europa.eu.
Acknowledgements The Panel wishes to thank the following for the support provided to this scientific output: Natalia Alija Novo and the Microbiology Working Group.
EFSA may include images or other content for which it does not hold copyright. In such cases, EFSA indicates the copyright holder and users should seek permission to reproduce the content from the original source.
Adopted: 31 January 2023
Notes
Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition. OJ L 268, 18.10.2003, p. 29.
Chr. Hansen A/S, 10–12 Boege Allé, DK‐2970 Hoersholm, Denmark.
Originally designated as Bacillus licheniformis DSM 5749.
Technical dossier/Supplementary information January 2021/Annex 1.Answers_EFSA_Qs_CH_CONF.
Commission Implementing Regulation (EU) 2017/2308 of 13 December 2017 concerning the authorisation of the preparation of Bacillus subtilis (DSM 5750) and Bacillus licheniformis (DSM 5749) as a feed additive. OJ L 3319, 14.12.2017, p. 19.
Commission Implementing Regulation (EU) 2017/447 of 14 March 2017 concerning the authorisation of the preparation of Bacillus subtilis (DSM 5750) and Bacillus licheniformis (DSM 5749) as a feed additive. OJ L 69, 15.3.2017, p. 18.
FEED dossier reference: FAD‐2019‐0069.
Evaluation report received on 29/10/2010 and available on the EU Science Hub https://joint-research-centre.ec.europa.eu/eurl-fa-eurl-feed-additives/eurl-fa-authorisation/eurl-fa-evaluation-reports_en
Commission Regulation (EC) No 429/2008 of 25 April 2008 on detailed rules for the implementation of Regulation (EC) No 1831/2003 of the European Parliament and of the Council as regards the preparation and the presentation of applications and the assessment and the authorisation of feed additives. OJ L 133, 22.5.2008, p. 1.
DSM 5749.
DSM 5750.
Technical dossier/Section II.
Technical dossier/Section II/Annex II 2.1.2a and Supplementary information January 2021/Annex B Q1.
Technical dossier/Supplementary information January 2021/Annex_B_Q2_1.
Technical dossier/Section II/Annex II2.1.2.b.
Technical dossier/Supplementary information January 2021/Annex_B_Q2_2.
Technical dossier/Supplementary information January 2021/Annex_B_Q4 1 and Supplementary information May 2021/1.Answers_EFSA_Qs_BP_05.2021_CONF.pdf and Annex_MICs_DSM5750_2021.
Technical dossier/Supplementary information January 2021/Annex_B_Q42.
Technical dossier/Supplementary information January 2021/Annex_B_Q4_3_DSM5749.
Technical dossier/Supplementary information January 2021/Annex_B_Q4 4 DSM5750 and Supplementary information May 2021/1.Answers_EFSA_Qs_BP_05.2021_CONF.pdf and Annex_MICs_DSM5750_2021.
Technical dossier/Supplementary information January 2021/Annex_B_Q3_1_DSM5749 and Annex_ B_Q3_2_DSM5750.
Staphylococcus aureus ATCC 25923, S. aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Enterococcus faecium ATCC 19434, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853.
Technical dossier/Supplementary information March 2022/Report_inhibitory_activity_DSM5749.
Technical dossier/Supplementary information August 2022/3.Characterization_active_agent_07.2022.
Currently under re‐evaluation (FAD‐2010‐0284).
Technical dossier/Section II/Annex_II_3_2a_Manufact_process_CONF and Annex_II_3_2b_Flowchart_CONF.
Technical dossier/Supplementary information January 2021/Annex_C_Q1.
Technical dossier/Supplementary information January 2021/Annex_C_Q2.
Technical dossier/Section II/Annex_II_4_1_Stab_BP_Conc.
Technical Dossier/Section II.
Technical dossier/Supplementary information August 2022/4.895–439‐6012_Final Report_ec_BP_Skin.
Technical dossier/Supplementary information August 2022/5.895–492‐6164_ Final Report_ec_BP_Eye.
Regulation (EC) No 183/2005 of the European Parliament and of the Council of 12 January 2005 laying down requirements for feed hygiene. OJ L 35, 8.2.2005, p. 1.
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