This is a joint FSA and FSS publication.
1. Introduction
The FSA and FSS have undertaken an assessment for a feed additive containing Bacillus licheniformis (also known as Bacillus paralicheniformis) DSM5749 and Bacillus subtilis DSM5750 (BioPlus® 2B, Chr. Hansen A/S, 10-12 Boege Allé, 2970, Hoersholm, Denmark) under assimilated Regulation (EC) No 1831/2003 (EC, 2003) in the category of ‘zootechnical additives’ and functional group ‘gut flora stabilisers’, for its use in calves for fattening, other growing ruminants at the corresponding developmental stage, and suckling piglets during the period in which solid feed is given.
To support the safety assessment by FSA and FSS, the ACAF provided advice to the FSA and FSS outlined in this document.
In line with Article 8 of 1831/2003, the assessment has considered and concluded that the feed additive complies with the conditions laid down in Article 5, including: safety considerations for human, animal and environmental health; efficacy of the additive for its intended effect; potential impairment of the distinctive features of animal products. This, and the guidance put in place by EFSA for the evaluation of feed additive applications, has formed the basis and structure for the assessment.
The dossier was evaluated by ACAF at their July 2023 meeting, after which a request for information was communicated to the applicant. The response to this request and subsequent requests was reviewed by the ACAF at their July 2024 meeting.
The views of ACAF have been taken into account in this safety assessment which represents the opinion of the FSA/FSS.
2. Assessment
2.1. Section II: Identity, characterisation and conditions of use
The additive is a preparation of non-GMO Bacillus licheniformis DSM5749 and Bacillus subtilis DSM5750, at a ratio of 1:1, and a minimum concentration of 3.2 x 1010 CFU/g of both microorganisms combined. The applicant provided data from several batches supporting the values outlined below (Table 2):
Requests were made to the applicant to provide updated data on composition and impurities, as well as whole genome sequence (WGS) data analysis for the identification and characterisation of the bacterial strains that comprise the active substances of the additive. The Committee concluded that the WGS presented in the request for information confirmed the identity of B. licheniformis DSM5749 and B. subtilis DSM5750 as being QPS microorganisms. The additive showed stability in water, premixtures and feed. Stability was shown up to 95oC after 10 minutes of exposure through pelleting. Homogeneity was also demonstrated. A shelf-life of 2 years was proposed and demonstrated for the additive. The proposed conditions of use of the additive are described in Table 3:
2.1.1. Conclusions on Section II: Identity, characterisation and conditions of use
The ACAF concluded the additive was correctly characterised. No concerns were raised for Section II of the dossier.
2.2. Section III: Safety
Bacillus licheniformis DSM5749 and Bacillus subtilis DSM5750 were included in the Qualified Presumption of Safety (QPS) list in 2023. The species B. licheniformis and B. subtilis were included in the original QPS list in 2007, at a time when the UK was part of the EU. The ACAF raised no concern over the use of the QPS approach for the presumption of safety of these two strains.
2.2.1. Safety for the target species and consumers
No evaluation of the safety for the target species and consumers was required due to the QPS status confirmation of both Bacillus licheniformis DSM5749 and Bacillus subtilis DSM5750. The additive is presumed safe for the target species and consumers.
2.2.2. Safety for the user/worker
No new studies to evaluate the safety for the user were presented in this application. As the active substances are microorganisms, these are presumed to be respiratory sensitisers. In the absence of data, the additive was concluded to be a potential skin and eye irritant, as well as a skin sensitiser. Given the high dusting potential of the final formulation, the use of protective equipment in well-ventilated areas is recommended to minimise exposure.
2.2.3. Safety for the environment
As both microorganisms are QPS, the additive is presumed to be safe for the environment. The other components of the additive do not raise cause for concern from an environmental safety standpoint. No further testing was carried out.
2.2.4. Conclusions on safety
The ACAF concluded that the additive is safe for the target species, consumers and the environment, given the QPS status of the microorganisms that act as active substances of the additive. The ACAF raised no concern over the use of the QPS approach for the presumption of safety of these two bacterial strains.
The active substances are presumed to be respiratory sensitisers. In the absence of data, the additive should be considered a potential skin and eye irritant, and a skin sensitiser. It is recommended that methods are adopted to minimise worker exposure to the additive.
2.3. Section IV: Efficacy
2.3.1. Efficacy in calves for fattening and other growing ruminants
The applicant presented data from three studies previously evaluated by EFSA in their 2016 opinion (FEEDAP, 2016), where it was concluded that the additive has the potential to improve performance of calves for rearing. The applicant proposed to extrapolate the data to calves for fattening and other growing ruminants at the same developmental stage. The committee noted the disparity in stages of development as defined in legislation and guidance texts, and considered the validity of the shorter trials presented (56 days). It was concluded that the physiological status of calves would be very similar regardless of their purpose being for fattening or rearing, therefore the extrapolation of conclusions was accepted. It was concluded that the additive has the potential to be efficacious in calves for fattening and other growing ruminants at the same developmental stage.
2.3.2. Efficacy in piglets
The applicant presented data from three studies previously evaluated by EFSA in their 2016 opinion (FEEDAP, 2016), where it was concluded that the additive has the potential to improve performance of weaned piglets. After a request for further information, the applicant clarified that they proposed to extrapolate these conclusions to suckling piglets to the period in which solid feed is given, without the requirement to give the additive to lactating sows simultaneously. The Committee evaluated the proposal and concluded that the studies presented showed the additive has the potential to be efficacious for weaned piglets, and that these conclusions can be extrapolated to suckling piglets for the period in which solid feed is given. Members agreed with the current EU authorisation suggestion that the simultaneous feeding of the additive to sows should not be a requirement, but still remain as a recommendation.
2.3.3. Conclusions on efficacy
The ACAF concluded that BioPlus 2B has the potential to be efficacious for calves for fattening and other growing ruminants at the same developmental stage, as well as suckling piglets during the period in which solid feed is given. For suckling piglets, it is recommended to feed the additive to lactating sows simultaneously.
3. Analytical methods evaluation
Conclusions on the analytical methods are presented here as an extract from the Evaluation Report of the European Union Reference Laboratory (EURL) for Feed Additives on the Method(s) of the Analysis for BioPlus® 2B (EURL, 2010). Reference is made to the Community Reference Laboratory (CRL), which is the predecessor body to the EURL:
"Furthermore, the applicant suggested a minimum content of BioPlus® 2B ranging from 11 to 270 g (depending on the species) per 1000 animals in drinking water. For the enumeration of the sum of the two strains of the active agents in feed additive, premixtures, feedingstuffs and water the applicant proposes the ring trial validated spread plate method (EN 15784) using tryptone soya agar. The performance characteristics of the CEN method reported after logarithmic transformation of measured CFU values are:
- a standard deviation for repeatability (sr) ranging from 0.07 to 0.09 log10 CFU/g
- a standard deviation for reproducibility (sR) ranging from of 0.32 to 0.35 log10 CFU/g, and
- a limit of detection (LOD) of 105 CFU/kg of feedingstuffs.
Based on these performance characteristics, the CRL recommends for official control the ring trial validated method EN 15784 for the determination of Bacillus subtilis DSM 5750 and Bacillus licheniformis DSM 5749 in feed additive, premixtures, feedingstuffs and water.
Molecular methods were used by the Applicant for identification of the active agent. The CRL recommends for official control, Pulsed Field Gel Electrophoresis (PFGE), a generally recognised standard methodology for microbial identification."
FSA/FSS accepts the EURL analytical method evaluation reports. FSA/FSS determined the analytical methods proposed as appropriate for official controls for this feed additive. The analytical methods were approved in 2010, at the time in which the UK was still an EU member.
4. Conclusions
The ACAF concluded the additive was correctly characterised and both microorganisms were confirmed to be QPS. No further concerns were raised for Section II of the dossier.
The ACAF concluded that the proposed level of inclusion in feed is safe for the target species, consumers and the environment.
The active substances are presumed to be respiratory sensitisers. In the absence of data, the additive should be considered a potential skin and eye irritant, and a skin sensitiser. It is recommended that methods are adopted to minimise worker exposure to the additive.
The ACAF concluded that BioPlus® 2B, when used at the proposed doses of 1.3 x 109 CFU/kg of complete feed (12% moisture) and 6.5 x 108 CFU/L of drinking water, has the potential to be efficacious for calves for fattening and other growing ruminants at the same developmental stage, as well as suckling piglets during the period in which solid feed is given. For suckling piglets, it is recommended to feed the additive to lactating sows simultaneously.
Abbreviations
Acknowledgements
With thanks to the members of the ACAF during the course of the assessment, who were: Professor Nicholas Jonsson, Martin Briggs, Professor Emily Burton, Professor Katrina Campbell, Professor Matthew Fisher, Hannah Kane, Susan MacDonald, Dr. Oonagh Markey, Christine McAlinden, Dr. Donald Morrison, Derek Renshaw, Dr. Michael Salter, Dr. Adam Smith, Christel Wake, Dr. Helen Warren and Dr. Nick Wheelhouse. Martin Briggs declared an indirect conflict of interest and remained in the meetings for discussion.