This is a joint FSA and FSS publication.
1. Introduction
The FSA and FSS have undertaken an assessment for a feed additive (PB6) containing Bacillus velezensis ATCC PTA-6737 (Kemin Europa N.V. in Toekomstlaan 42, Herentals, 2200, Belgium) under Assimilated Regulation (EC) No 1831/2003. The application sought a renewal authorisation for use in sows, weaned piglets and weaned minor porcine species, as well as an extension of use to all pig species. The additive falls under the category ‘zootechnical additive’, functional group ‘gut flora stabiliser’.
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 the Advisory Committee on Animal Feedingstuffs (ACAF) at its January 2024 meeting, after which a request for further information was communicated to the applicant. The applicant’s response to this request and subsequent requests were evaluated by ACAF at its April and June 2024 meetings.
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 active ingredient of PB6 are spores of Bacillus velezensis ATCC PTA-6737 at a minimum concentration of 8.0 x 1010 CFU spores per gram. PB6 is a dry powder consisting of 75 – 95% sodium bicarbonate, used as a carrier, and 5 – 25% dried ferment. The applicant provided data from five batches supporting the identification values outlined below (Table 2):
The applicant provided clarification regarding the presence of AMR genes, as well as the absence of plasmids, allowing the Committee to agree that the strain had been fully characterised using whole genome sequencing. Members were also able to conclude on the identity and characterisation of the additive after the applicant provided more recent data demonstrating batch-to-batch variation and impurity testing. Supporting documentation and certification relating to quality assurance and further information on Hazard Analysis and Critical Control Point (HACCP) principles, including critical control points, were also provided upon request.
Members noted a discrepancy in the dusting potential values given. Clarification provided by the applicant confirmed that the dusting potential values for this additive are quite high, as these values range from 1.045 to 1.540 g/m3. The additive consists of particles of inhalable size (< 100 µm) and has a high proportion of particles of respirable size (< 10 µm).
The additive was shown to be stable for 12 months at 40°C and 6 months at 55°C. There were no statistically significant differences between the counts at the start of the study and after 18 months of storage at room temperature. PB6 was also demonstrated to be stable for 6 months when added to both a mineral premix and a finished mash feed, and remained stable for 3 months after pelleting at a maximum of 90°C for 45 seconds. Homogeneity in premixture and in feed samples was also demonstrated.
The Committee noted that the additive is authorised for use in weaned piglets at a minimum content of 1 x 107 CFU/kg complete feed and is intended for use in growing pigs at a minimum content of 1 x 107 CFU/kg complete feed, and in sows and minor reproductive species at 1 x 108 CFU/kg complete feed. The applicant was asked to clarify how they concluded on the dose for growing pigs, as efficacy trials were performed using different doses for weaned piglets and sows. In their response, the applicant explained that the efficacy data from piglets was extrapolated to growing pigs, in line with guidance, hence growing pigs would receive the same dose as piglets. Members could not accept the applicant’s specific request for extrapolation from piglets to all growing pigs due to the lack of evidence for efficacy when fed at the lower dose (1 x 107 CFU/kg) proposed in piglets and considering the difference in physiological age between piglets and pigs for fattening. However, given the presence of three significant piglet trials and three significant sow trials, the Committee acknowledged that the studies provided could provide supporting evidence of efficacy in all pigs when a minimum content of 1 x 108 CFU/kg was utilised for pigs in the fattening phase. The Committee decided on this higher dose of 1 x 108 CFU/kg for pigs for fattening due to physiological state (i.e. they are more mature animals), and an uncertainty in the additive’s efficacy in older animals; therefore the higher dose was more appropriate to ensure efficacy in pigs for fattening. Consequently, the Committee concluded that the additive has the potential to be efficacious in all growing pigs, but use in pigs for fattening would be at the higher dose of 1 x 108 CFU/kg complete feed.
The proposed conditions of use of the additive are described in Table 3.
The ACAF concluded on a higher dose of 1 x 108 CFU/kg of complete feedingstuff as being potentially efficacious for use in pigs for fattening. This is discussed further in Section IV.
2.1.1. Conclusions on Section II
The ACAF concluded that the additive was fully identified and characterised. Members were able to conclude positively on the stability and homogeneity of the additive.
Regarding conditions of use, the ACAF were able to conclude positively on those outlined by the applicant, however use in pigs for fattening would be at the higher dose of 1 x 108 CFU/kg of complete feedingstuff. No further concerns were raised for Section II of the dossier.
2.2. Section III: Safety
2.2.1. Safety for the target species
No evaluation of the safety for the target species was required given the Qualified Presumption of Safety (QPS) status confirmation of Bacillus velezensis ATCC PTA-6737. The additive is therefore presumed safe for the target species.
2.2.2. Safety for the consumer
No evaluation of the safety for the consumer was required given the QPS status confirmation of Bacillus velezensis ATCC PTA-6737; however, the applicant provided additional data relating to safety for the consumer to support its QPS status. The applicant provided the following tests: an acute oral toxicity test, an erythrocyte micronucleus assay in mice and a 28-day repeat dose oral toxicity study using doses of up to 1,000 mg PB6/kg bw/day. The Committee concluded that the toxicological studies provided demonstrated no adverse effects and that the additive could be considered safe for the consumer.
2.2.3. Safety for the user
The applicant presented the following tests to evaluate the safety of the additive for the user/worker:
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Acute inhalation test, following OECD protocol 403;
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Acute dermal irritation/corrosion study, following OECD protocol 404;
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Acute eye irritation/corrosion study, following OECD protocol 405.
The Committee concluded that PB6 is not irritant to skin or eyes, but that it should be treated as a potential skin sensitiser as the applicant has provided insufficient evidence proving otherwise. The ACAF also concluded that as a microbial product PB6 is regarded as a potential respiratory sensitiser and it could deposit in the respiratory tract of exposed workers; therefore inhalation should be avoided.
2.2.4. Safety for the environment
No evaluation of the safety for the environment was required given the QPS status confirmation of Bacillus velezensis ATCC PTA-6737. The additive is therefore presumed safe for the environment.
2.2.5. Conclusions on safety
The ACAF concluded that the additive is presumed safe for the target animals, the consumers, and the environment.
The additive is not irritant to skin or eyes but should be treated as a potential skin and respiratory sensitiser.
2.3. Section IV: Efficacy
The applicant presented four efficacy studies in weaned piglets and four studies in sows to allow for extrapolation to all pig species at all growing phases. The applicant concluded that supplementation with Bacillus velezensis ATCC PTA-6737 (PB6) in the diet of growing piglets at dosages of 1 x 107 CFU/kg and 5 x 107 CFU/kg results in improvements in zootechnical parameters. The applicant also concluded that supplementation with the additive in sow diets at a dosage of 1 x 108 CFU/kg significantly improves the performance of sows and their piglets.
The Committee assessed the efficacy trials provided for weaned piglets and concluded that the additive is efficacious in weaned piglets at a dose of 1 x 107 CFU/kg complete feed. The Committee noted several inconsistencies within the sow trials and only two of the trials appeared to demonstrate efficacy; however, three trials are needed to provide a conclusion. Further clarification was therefore requested regarding efficacy in sows and reproductive stages of minor porcine species and on the proposed doses for use in pigs for fattening. An additional trial could not be provided by the applicant for use in sows, and therefore the ACAF concluded that the additive has the potential to be efficacious in sows at a dose of 1 x 108 CFU/kg complete feed.
After assessing the information provided the Committee also concluded that the data provided was suitable for extrapolation to all pigs in line with the guidance, as the applicant had provided sufficient studies for weaned piglets and sows. The Committee therefore concluded that the additive is efficacious in weaned piglets at a dose of 1 x 107 CFU/kg complete feed and has the potential to be efficacious in sows and pigs for fattening at a dose of 1 x 108 CFU/kg complete feed.
2.3.1. Conclusions on efficacy
The ACAF concluded that Bacillus velezensis ATCC PTA-6737 is efficacious in weaned piglets at a dose of 1 x 107 CFU/kg complete feed and has the potential to be efficacious in sows and pigs for fattening at a dose of 1 x 108 CFU/kg complete feed.
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 for Feed Additives on the Method(s) of the Analysis for Bacillus subtilis ATCC PTA-6737 (EURL 2009):
"For the enumeration of Bacillus subtilis ATCC PTA-6737 in the feed additive, premixtures and feedingstuffs, the applicant proposes the draft CEN method – prEN 15784:2008 E – an internationally recognised spread plate method. This method was ring-trial validated using the premixtures and feedingstuffs samples containing Bacillus subtilis spores. The performance characteristics of the draft CEN method reported after logarithmic transformation of measured values (CFU) are:
For the premixtures: (1) a standard deviation for repeatability (sr) of 0.09 log10 and (2) a standard deviation for between-laboratory reproducibility (sR) of 0.32 log10.
For the feedingstuffs: (1) a sr of 0.07 log10 and (2) a sR of 0.35 log10.
The applicant used the above mentioned spread plate method to analyse the various matrices containing Bacillus subtilis ATCC PTA-6767 spores and reported the following results: (a) 1 x 109 to 1.5 x 1011 CFU/g of feed additive; (b) 1 x 107 to 1.5 x 109 CFU/kg for premixtures and (c) 1 x 107 to 1.5 x 108 for feedingstuffs. The results obtained for feed additives and premixtures are considered acceptable; this method is therefore recommended for official controls for the feed additives and premixtures in the frame of the authorisation.
As regards feedingstuffs, the CRL notes that the limit of quantification reported by the applicant upon request (LOQ = 1 x 107 CFU/kg feedingstuffs) is identical to the minimum dose proposed and is below the LOQ reported in the draft CEN method (2 x 107 CFU/kg). On the basis of the available information, the draft CEN method is recommended for official control of the feedingstuffs containing Bacillus subtilis PB6 at the dosages above the LOQ reported by CEN. Below 2 x 107 CFU/kg the CRL is not able to conclude on the suitability of this method for official control purposes.
Molecular methods were used by the applicant for identification of the active agent. For official controls pulsed field gel electrophoresis (PFGE), a generally recognised standard methodology for microbial identification, is recommended.
Further testing or validation is not considered necessary."
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.
4. Conclusions
The ACAF concluded that the additive was fully identified and characterised. Members concluded positively on the stability and homogeneity of the additive.
The additive is presumed safe for the target animals, the consumer and the environment. It is not irritant to skin or eyes but should be treated as a potential skin and respiratory sensitiser.
The ACAF concluded that Bacillus velezensis ATCC PTA-6737 is efficacious in weaned piglets at a dose of 1 x 107 CFU/kg complete feed. The Committee also concluded that the additive has the potential to be efficacious in sows at a dose of 1 x 108 CFU/kg complete feed. As suitable efficacy trials were submitted for both sows and piglets, the data provided could be extrapolated to all pigs in line with the guidance, however the Committee noted that when fed to pigs during the fattening phase the higher dose of 1 x 108 CFU/kg should be utilised.
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 Professor Nick Wheelhouse.