Safety and efficacy of a feed additive consisting of Bacillus velezensis NRRL B‐67647, Bacillus pumilus NRRL B‐67648 and Bacillus licheniformis NRRL B‐67649 (Microsaf®) for chickens for fattening, other poultry for fattening and ornamental birds (S.I. Lesaffre)

TL;DR

This study evaluates the safety and effectiveness of a feed additive containing three Bacillus species for poultry and ornamental birds.

Contribution

The study confirms the safety and efficacy of Microsaf® for poultry and ornamental birds using a qualified presumption of safety approach.

Findings

Microsaf® is safe for target species, consumers, and the environment.

The additive improves zootechnical performance in chickens for fattening.

It is compatible with several commonly used feed additives.

Abstract

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of Microsaf® when used as a feed additive for chickens for fattening, other poultry for fattening and ornamental birds. The product under assessment is based on viable cells/spores of Bacillus velezensis NRRL B‐67647, Bacillus pumilus NRRL B‐67648 and Bacillus licheniformis NRRL B‐67649. The three bacterial species are considered by EFSA to be suitable for the qualified presumption of safety approach to safety assessment. As the identity of the strains was clearly established, no acquired antimicrobial resistance determinants of concern were detected and the lack of toxigenic potential was corroborated, the FEEDAP Panel considered the use of the active agents safe for the target species, consumers and the environment. Since no concerns are expected from the other components of the additive, the Panel concluded that Microsaf® is also considered safe for the target species, consumers and the environment. Regarding user safety, the additive is not irritant to the skin or eyes provided it is formulated with only calcium carbonate. The additive should be considered skin and respiratory sensitisers, and any exposure via skin or respiratory tract is considered a risk. The FEEDAP Panel concludes that the additive has the potential to improve the zootechnical performance of chickens for fattening at a proposed minimum inclusion level of 3 × 107 total CFU/kg of complete feedingstuffs. These results are extrapolable to other poultry for fattening and ornamental birds. Microsaf® is compatible with diclazuril, narasin, maduramicin ammonium, salinomycin sodium, monensin sodium, robenidine hydrochloride and the combination narasin/nicarbazin, lasalocid and halofuginone.

Tables

TABLE 1. Data on the batch‐to‐batch variation, impurities and physical properties of Microsaf GT 3B and Microsaf GT 3B‐C. The data presented are average values and (range) for batch‐to‐batch variation, and ranges for all other parameters. The number of batches analysed or groups of parameters is indicated in [].

	Microsaf GT 3B	Microsaf GT 3B‐C
Specifications
Total CFU/g	2 × 108	5 × 109
Batch to batch variation	[5]	[5]
Total (CFU/g)	1.9 × 108 (1.4–2.2 × 108)	1.2 × 1010 (4.0 × 109–3.4 × 1010)
B. licheniformis (CFU/g)	7.6 × 107 (5.7 × 107–1.1 × 108)	5.0 × 109 (8.5 × 108–1.7 × 1010)
B. velezensis (CFU/g)	7.7 × 107 (2.9 × 107–1.2 × 108)	4.3 × 109 (2.2 × 109–1.1 × 1010)
B. pumilus (CFU/g)	4.8 × 107 (1.1 × 107–4.8 × 107)	2.1 × 109 (0.6 × 108–5.9 × 109)
Impurities	[3]	[3]
Lead (mg/kg)	4.4–5.2	3.7–5.5
Mercury (mg/kg)	< 0.02	< 0.02
Cadmium (mg/kg)	< 0.4	< 0.4
Arsenic (mg/kg)	< 0.2	< 0.2
Nickel (mg/kg)	0.2–0.3	0.3
Dioxins and furans (upper bound) 1	[3]	[3]
PCDD/Fs (ng WHO2005‐TEQ/kg)	0.023–0.030	0.046–0.067
PCDD/Fs + PCBs (ng WHO2005‐TEQ/kg)	0.075–0.105	0.108–0.247
nDL‐PCBs (μg/kg)	0.076–0.108	0.088–0.174
Mycotoxins (μg/kg)	[3]	[3]
Aflatoxins B1, B2, G1 and B2	< 0.15	< 0.15
Ochratoxine	<1	<1
Zearalenone	< 5	< 5
Fumonisine B1 and B2	< 10	< 10
Deoxynivalenol	< 5	< 5
HT2 and T2 toxins	< 5	< 5
Microbial contamination	[3]	[3]
Salmonella spp. (per 25 g)	Not detected	Not detected
Enterobacteriaceae (CFU/g)	< 10	< 10
Total coliforms (CFU/g)	< 10	< 10
Escherichia coli (CFU/g)	< 10	< 10
Anaerobic sulfate reducing bacteria (CFU/g)	< 10	< 10
Clostridium perfringens (CFU/g)	< 10	< 10
Coagulase‐positive staphylococci (CFU/g)	< 100	< 100
Yeast and moulds (CFU/g)	< 10	< 10
B. cereus (CFU/g)	< 10	< 10
Physical properties	[3]	[3]
Bulk density (kg/m3)	1426–1433	1427–1468
Dusting potential (Stauber Heubach) (mg/m3)	3002–4080	4940–6070
Particle size distribution (laser diffraction) (% of particles below, v/v)
100 μm	11	9
50 μm	5	3.5
10 μm	2	2
Stability (losses)
Shelf‐life
4–25°C/60%RH ‐ 24 months	< 0.5 log [3]	< 0.5 log [3]
40°C/75%RH ‐ 12 months	< 0.5 log [1]
40°C/75%RH ‐ 18 months	< 0.5 log [2]	< 0.5 log [3]
Stability in premixtures
Vitamin‐mineral premix, 25°/60%RH – 6 months		< 0.5 log [1]
40°/75%RH – 2 months/6 months		< 0.5 log/>1 log [1]
Stability in feed
Complete feeds (mash and pellets)
20°C – 3 months		< 0.5 log [1]
Pelleting 95°C, broiler feed		< 0.5 log [3]
Heat treatments (3 min at 85, 90 or 95°C) – layer feed		< 0.5 log [3]
Homogeneity (coefficient of variation, %)
Broiler mash feed		2 [2]
Broiler pelleted feed		2 [2]

TABLE 2. Trial design and use level of the efficacy trials performed in chickens for fattening.

Trial	Total no of animals (animals × replicate) replicates × treatment	Breed sex	Duration (starter/grower/finisher)	Composition feed (form)	Groups (CFU total bacilli/kg feed)*
					Intended	Analysed (starter/grower/finisher)
1	■■■■■ ■■■■■ ■■■■■	■■■■■ ■■■■■	42 days (1–14/15–28/29–42)	■■■■■	0 3 × 107 ■■■■■	8 × 106/9 × 106/4 × 106 5.7 × 107/3.6 × 107/3.5 × 107 ■■■■■
2	■■■■■ ■■■■■ ■■■■■	■■■■■ ■■■■■	42 days (1–14/15–28/29–42)	■■■■■	0 3.2 × 107	< 5 × 106/< 5 × 106/1.1 × 107 2.8 × 107/2.4 × 107/1.9 × 107
3	■■■■■ ■■■■■ ■■■■■	■■■■■ ■■■■■	42 days (1–14/15–28/29–42)	■■■■■	0 3.2 × 107	2.5 × 106/9.8 × 107/7.5 × 106 1.6 × 108/1.5 × 108/9.9 × 107

TABLE 3. Effects of Microsaf® on the performance of chickens for fattening.

Trial	Groups (CFU/kg feed)	Average daily feed intake (g)	Final body weight (g)*	Average daily weight gain (g/bird)	Feed to gain ratio	Mortality and culling (%)
1	0 3 × 107 ■■■■■	97.9b 107a ■■■■■	2346b 2618a ■■■■■	55.3b 61.3a ■■■■■	1.77 1.75 ■■■■■	3.6 4.3 ■■■■■
2	0 3 × 107	135 134	2950b 2988a	69.2b 70.1a	1.95a 1.91b	3.7 4.0
3	0 3 × 107	134 133	2755b 2860a	65.1b 67.1a	2.07a 1.99b	2.1 2.6

TABLE 4. Effect of coccidiostats on the bacilli counts of ileal contents of chickens fed with B. velezensis NRRL B‐67647, B. pumilus NRRL B‐67648, and B. licheniformis NRRL B‐67649.

Treatment	Ileum counts (log CFU/g)
	B. Velezensis		B. Pumilus		B. Licheniformis
	+ heat treatment	– heat treatment	+ heat treatment	– heat treatment	+ heat treatment	– heat treatment
Microsaf GT 3B	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
Microsaf GT 3B + narasin/nicarbazin1	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
Microsaf GT 3B + maduramicin2	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
Microsaf GT 3B + lasalocid3	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
Microsaf GT 3B + salinomycin4	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
Microsaf GT 3B + monensin5	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
Microsaf GT 3B + robenidine6	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
Microsaf GT 3B + narasin7	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
Microsaf GT 3B + halofuginone8	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■

TABLE 5. Effect of coccidiostats on the bacilli counts of caecum contents of chickens fed with of B. velezensis NRRL B‐67647, B. pumilus NRRL B‐67648 and B. licheniformis NRRL B‐67649.

Treatment	Caecum counts (log CFU/g)
	B. Velezensis		B. Pumilus		B. Licheniformis
	+ heat treatment	– heat treatment	+ heat treatment	– heat treatment	+ heat treatment	– heat treatment
Microsaf GT 3B	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
Microsaf GT 3B + narasin/nicarbazin1	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
Microsaf GT 3B + maduramicin2	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
Microsaf GT 3B + lasalocid3	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
Microsaf GT 3B + salinomycin4	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
Microsaf GT 3B + monensin5	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
Microsaf GT 3B + robenidine6	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
Microsaf GT 3B + narasin7	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■
Microsaf GT 3B + halofuginone8	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■	■■■■■

Full text

INTRODUCTION

1

Background and Terms of Reference

1.1

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 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 S.I. Lesaffre2 for the authorisation of the additive consisting of Bacillus velezensis NRRL B‐67647, Bacillus pumilus NRRL B‐67648, and Bacillus licheniformis NRRL B‐67649 (Microsaf®), when used as a feed additive for chickens for fattening, other poultry for fattening, and ornamental birds (category: zootechnical additives; functional group: gut flora stabilisers and other zootechnical additives).

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). The dossier was received on 09 August 2021 and the general information and supporting documentation are available at https://open.efsa.europa.eu/questions/EFSA‐Q‐2021‐00449. The particulars and documents in support of the application were considered valid by EFSA as of 09 June 2022.

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 feed additive consisting of Bacillus velezensis NRRL B‐67647, Bacillus pumilus NRRL B‐67648, and Bacillus licheniformis NRRL B‐67649 (Microsaf®), when used under the proposed conditions of use (see Section 3.1.3).

Additional information

1.2

The additive is a preparation containing Bacillus velezensis NRRL B‐67647, Bacillus pumilus NRRL B‐67648, and Bacillus licheniformis NRRL B‐67649 (Microsaf®). It is not authorised as a feed additive in the European Union.

DATA AND METHODOLOGIES

2

Data

2.1

The present assessment is based on data submitted by the applicant in the form of a technical dossier3 in support of the authorisation request for the use of Bacillus velezensis NRRL B‐67647, Bacillus pumilus NRRL B‐67648, and Bacillus licheniformis NRRL B‐67649 (Microsaf®) as a feed additive.

In accordance with Article 38 of the Regulation (EC) No 178/20024 and taking into account the protection of confidential information and of personal data in accordance with Articles 39 to 39e of the same Regulation, and of the Decision of EFSA's Executive Director laying down practical arrangements concerning transparency and confidentiality,5 a non‐confidential version of the dossier has been published on Open.EFSA.

According to Article 32c(2) of Regulation (EC) No 178/2002 and to the Decision of EFSA's Executive Director laying down the practical arrangements on pre‐submission phase and public consultations, EFSA carried out a public consultation on the non‐confidential version of the application from 16 November to 07 December 2023, for which no comments were received.

The confidential version of the technical dossier was subject to a target consultation of the interested Member States from 09 June to 09 September 2022; the comments received were considered for the assessment.

EFSA has verified the European Union Reference Laboratory (EURL) report as it relates to the methods used for the control of the active agents in animal feed.6

Methodologies

2.2

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of the product containing B. velezensis NRRL B‐67647, B. pumilus NRRL B‐67648 and B. licheniformis NRRL B‐67649 (Microsaf®) is in line with the principles laid down in Regulation (EC) No 429/20087 and the relevant guidance documents: Guidance on the identity, characterisation and conditions of use of feed additives (EFSA FEEDAP Panel, 2017), Guidance on the assessment of the efficacy of feed additives (EFSA FEEDAP Panel, 2024), Guidance on the characterisation of microorganisms used as feed additives or as production organisms (EFSA FEEDAP Panel, 2018), EFSA statement on the requirements for whole genome sequence analysis of microorganisms intentionally used in the food chain (EFSA, 2021), Guidance on the assessment of the safety of feed additives for the users (EFSA FEEDAP Panel, 2023).

ASSESSMENT

3

The additive under assessment is a preparation containing Bacillus velezensis NRRL B‐67647, Bacillus pumilus NRRL B‐67648, and Bacillus licheniformis NRRL B‐67649 intended for use as a zootechnical additive (functional group: gut flora stabilisers and other zootechnical additives) in feed for chickens for fattening, other poultry for fattening, and ornamental birds. It has the trade name Microsaf®.

Characterisation

3.1

Characterisation of the active agents

3.1.1

The three strains were originally isolated from soil/root.8 All strains are deposited at the Agricultural Research Culture Collection (NRRL), Peoria, Illinois (USA), with the accession numbers NRRL B‐67647, NRRL B‐67648 and NRRL B‐67649 for Bacillus velezensis, Bacillus pumilus and Bacillus licheniformis, respectively.9 According to the applicant, none of the strains has been genetically modified.10

The taxonomic identification of each strain was done by Average Nucleotide Identity (ANI) based on whole genome sequence (WGS) data:

• The highest similarity B. velezensis gave the value of 99.99% compared with Bacillus amyloliquefaciens EBL11, now classified as B. velezensis (Dunlap et al., 2016), and a value of 98.39% compared with the type strain B. velezensis NRRL B‐41580^T^. Additionally, OrthoANI and the alignment‐free genome distance estimation analysis with Mash using MinHash indicated B. amyloliquefaciens EBL11, now classified as B. velezensis, as the closest genome.11
• The highest similarity of B. pumilus NRRL B‐67648 gave the value of 98.60% with B. pumilus 7P and a value of 95.49% compared with the type strain B. pumilus ATCC 7061^T^. Additionally, OrthoANI and the alignment‐free genome distance estimation analysis with Mash using MinHash indicated B. pumilus 7P as the closest genome.12 It harbours one plasmid.
• The highest similarity of B. licheniformis NRRL B‐67649 gave the value of 99.68% compared to the type strain B. licheniformis DSM 13^T^. Additionally, the alignment‐free genome distance estimation analysis with Mash using MinHash indicated Bacillus sp. BT1b_CT2 and the type strain B. licheniformis DSM 13^T^ as the closest genomes.13

The susceptibility of each of the three strains to the antibiotics recommended by the FEEDAP Guidance (EFSA FEEDAP Panel, 2018) was tested ■■■■■.14 All the minimum inhibitory concentration (MIC) values were equal to or fell below the corresponding cut‐off values. Therefore, the three active agents are considered to be susceptible to all relevant antibiotics.

The WGS of the three strains, including plasmids, was interrogated for the presence of antimicrobial resistance genes (AMR) against the ■■■■■ and ■■■■■ databases. A total of one hit (clbA) for B. velezensis NRRL B‐67647,15 two hits (cat86 and blaBPU) for B. pumilus NRRL B‐67648,16 and two hits (rphC and BlaP) for B. licheniformis NRRL B‐6764917 were found exceeding the thresholds recommended by EFSA (EFSA, 2021). Further analysis of these hits following EFSA's criteria (EFSA BIOHAZ Panel, 2023) did not reveal any acquired AMR gene.18 Therefore, the FEEDAP Panel concludes that none of the strains harbour acquired AMR genes, and therefore, they raise no safety concerns.

The WGS of the three strains, including plasmids, was additionally interrogated for the presence of virulence factors against the ■■■■■ database ■■■■■. ■■■■■.19 No hits of concern were identified.

The toxigenic potential of each strain was assessed according to the Guidance on the characterisation of microorganisms used as feed additives or as production organisms (EFSA FEEDAP Panel, 2018).20 No lysis of Vero cells was detected in any of the three strains. Therefore, B. velezensis NRRL B‐67647, B. pumilus NRRL B‐67648, and B. licheniformis NRRL B‐67649 are considered to be non‐toxigenic.

The antimicrobial production for each active agent was determined from pure cell cultures and supernatants using seven microorganisms as indicators.21 ^,^ 22 All three strains tested individually showed antibacterial activity against Bacillus cereus ATCC 2 and Pseudomonas aeruginosa DSM1117. The inhibitory substances were not analysed, but the WGS of each active agent was further analysed for the presence of genes involved in antimicrobial production using ■■■■■ and ■■■■■ databases.23 Some genes coding for bacteriocins were identified. None of these are recognised as critically, highly, or important antimicrobials (WHO, 2024) and therefore are not considered a hazard.

Characterisation of the additive

3.1.2

The three active agents B. velezensis NRRL B‐67647, B. pumilus NRRL B‐67648, and B. licheniformis NRRL B‐67649 are grown by fermentation, harvested by centrifugation, dried, and ground.24 The final additive is then produced and standardised by blending in equal proportion the 3 strains (ratio 1:1:1). Two formulations of the additive are produced, i.e. Microsaf GT 3B (diluted form) and Microsaf GT 3B‐C (concentrated form), with a minimum guaranteed concentration of total bacilli counts of 2 × 10^8^ and 5 × 10^9^ colony‐forming units (CFU) per gram of additive, respectively. Microsaf GT 3B is formulated with ■■■■■ anticaking agent and an average of ■■■■■ carriers, while Microsaf GT 3B‐C is formulated with ■■■■■ anticaking agent and an average of ■■■■■ carriers. The applicant listed among the possible anticaking agents sodium aluminosilicate or colloidal silica and among the possible carriers calcium carbonate. However, no detailed qualitative and quantitative composition of the two forms was provided. The applicant stated that no antimicrobial substances are used during the manufacturing process.25

The data provided by the applicant26 on the batch‐to‐batch variation,27 impurities,28 and physical properties29 of the additive are reported in Table 1.

The data provided by the applicant and described in the table above were derived from additives formulated with only calcium carbonate, except for two batches of Microsaf GT 3B, analysed for the batch‐toto‐batch, which were formulated with calcium carbonate and colloidal silica.

The Panel notes that for three batches of Microsaf GT 3B and four of Microsaf GT 3B‐C, the total counts of bacilli were below the specifications set by the applicant (1.4–1.9 × 10^8^ CFU/g, and 3.2–4.7 × 10^9^ CFU/g, respectively).

The Panel considers that the levels of microbial contamination and impurities analysed in the additive are of no concern.

The Panel also notes that the batches of the additive used in the stability studies had lower total bacilli counts than those set in the specifications by the applicant.

Conditions of use

3.1.3

Microsaf® is intended for use in feed for chickens for fattening, other poultry for fattening, and ornamental birds at a proposed minimum inclusion level of 3 × 10^7^ total CFU/kg of complete feedingstuffs.30

Safety

3.2

Safety for the target species, consumers and the environment

3.2.1

The bacterial species Bacillus velezensis, Bacillus pumilus, and Bacillus licheniformis are considered by EFSA to be suitable for the qualified presumption of safety approach to safety assessment (EFSA BIOHAZ Panel, 2023). This approach requires the identity of the strains to be conclusively established, evidence provided that they do not harbour acquired antimicrobial resistance genes, and lack toxigenic potential. In the view of the FEEDAP Panel, the identity of the active agents as B. velezensis, B. pumilus, and B. licheniformis was established and the compliance with the other qualifications confirmed. Therefore, the FEEDAP Panel concludes that B. velezensis NRRL B‐67647, B. pumilus NRRL B‐67648, and B. licheniformis NRRL B‐67649 do not raise safety concerns for the target species, consumers of products derived from animals fed the additive, and the environment. Since no concerns are expected from the other components of the additive, Microsaf® is also considered safe for the target species, consumers of products derived from animals fed the additive, and the environment.

Safety for the user

3.2.2

Based on the highest dusting potential reported (Section 3.1.2), the FEEDAP Panel considers that the exposure of users through inhalation is likely.

The applicant provided studies on skin and eye irritation with Microsaf GT 3B‐C formulated with only calcium carbonate.

The skin irritation potential of the additive was tested in a study performed according to OECD TG 439.31 The results showed that the additive is not a skin irritant (UN GHS “No Category”).

The eye irritation potential of the additive was tested in a study performed according to OECD TG 438.32 The results showed that the additive is not an eye irritant (UN GHS “No Category”).

The additive contains microorganisms and therefore should be considered as a skin and respiratory sensitiser.

The FEEDAP Panel notes that the additive contains nickel (see Section 3.1.2). The Directive (EU) 2022/43133 set an occupational exposure limit (OEL) of 0.01 and 0.05 mg/m^3^ for both respirable and inhalable fraction, respectively, as nickel meets the criteria for classification as carcinogenic (category 1A). Therefore, to reduce the risk, the FEEDAP Panel considers that the exposure of the users should be minimised. Due to the presence of nickel, the additive should be considered a skin and respiratory sensitiser.

Microsaf GT 3B‐C is not irritant to the skin or eyes. Considering the similarity in composition, the Panel considers that this conclusion can be extended to Microsaf GT 3B provided it is formulated with only calcium carbonate. Both forms of the additive are considered skin and respiratory sensitisers, and any exposure via skin or respiratory tract is considered a risk. The Panel cannot conclude on any other formulations with other unspecified carriers/anticaking agents.

Conclusions on safety

3.2.3

Microsaf® is considered safe for the target species, consumers of products derived from animals fed the additive, and the environment. Regarding user safety, Microsaf GT 3B‐C is not irritant to the skin or eyes. Considering the similarity in composition, the Panel considers that this conclusion can be extended to Microsaf GT 3B, provided it is formulated with only calcium carbonate. Both forms of the additive are considered skin and respiratory sensitisers, and any exposure via skin or respiratory tract is considered a risk. The Panel cannot conclude on any other formulations with other unspecified carriers/anticaking agents.

Efficacy

3.3

Efficacy for chickens for fattening

3.3.1

A total of three long‐term trials were submitted to support the efficacy of the additive on improving the zootechnical performance of chickens for fattening.34 The details on the study design are provided in Table 2 and the main results in Table 3.

In all trials, ■■■■■ The basal diets were either not supplemented (control) or supplemented with Microsaf GT 3B to provide 3 × 10^7^ CFU total Bacillus spp./kg of complete feed.

The health and mortality of the animals were monitored throughout the study. The birds were weighed at the start of the trial. Thereafter, the body weight and feed intake were recorded on a pen basis on days 14, 28, and 42. The average daily feed intake, average daily weight gain, and the feed‐to‐gain ratio were calculated and corrected for mortality for the whole period (1–42 days).

The data were analysed with an analysis of variance (ANOVA) ■■■■■ The significance level was set at 0.05.

In all trials, the health of the animals was good. Mortality and culling were within standard commercial rates, and no differences were observed between groups.

In trial 1, the chickens receiving the additive at the recommended use level of 3 × 10^7^ CFU/kg feed for 42 days showed higher average daily feed intake, average daily gain, and final body weight compared to the control group. ■■■■■

In trials 2 and 3, the chickens in the supplemented group showed improved performance (higher final bodyweight and average daily gain, and better feed‐to‐gain ratio) compared to the control group.

Compatibility with coccidiostats

3.3.2

The applicant provided in vitro studies to support the compatibility of the three strains that compose Microsaf® (B. licheniformis NRRL B‐67649, B. pumilus NRRL B‐67648 and B. velezensis NRRL B‐67647) tested individually against monensin, narasin, salinomycin, maduramicin, lasalocid, decoquinate, diclazuril, robenidine, and the combination of narasin/nicarbazin.35

MICs values against the individual active agents were assessed using a broth microdilution method in aerobic conditions in vitro. Three replicates per treatment were assayed.

The MICs values for diclazuril were greater than four times their maximum authorised level. These results indicate that Microsaf® is compatible with diclazuril. For decoquinate, higher concentrations than 20 mg/kg were not tested due to the product's solubility.

The MIC values for monensin, narasin, salinomycin, maduramicin, lasalocid, robenidine and the combination of narasin/nicarbazin were below four times their maximum authorised dose. Therefore, the incompatibility cannot be excluded and should be assessed in vivo.

The applicant provided in vivo studies to support the compatibility of Microsaf® with monensin, narasin, salinomycin, maduramicin, lasalocid, robenidine, the combination of narasin/nicarbazin, and halofuginone.

The in vivo studies were performed with the additive Microsaf GT 3B as described below.36

■■■■■ The birds were fed ■■■■■ which was supplemented with the additive Microsaf GT 3B at a level of 3.16 × 10^7^ CFU/kg of complete feed and was either not supplemented (control) or supplemented with the corresponding coccidiostat product at the respective maximum authorised content in feed.37 The birds were monitored for general health and mortality. Over the complete trial, 1 mortality was observed in the group treated with Microsaf GT 3B and halofuginone. ■■■■■38 The analysis of the samples was performed with and without heat treatment in order to differentiate between vegetative cells and spores.

The results are given in Tables 4 and 5. The differences between the groups were analysed with an ■■■■■ test, comparing each group against the control diet (supplemented with Microsaf GT 3B but without coccidiostat), and the results are presented as means. Compatibility of Microsaf GT 3B with each of the coccidiostats is assumed if mean differences do not differ more than 1 log_10_ value.

For ileum samples, only the cell counts without heat treatment of the B. velezensis strain in the lasalocid supplemented group showed a reduction larger than 1‐log compared to the values in the Microsaf GT 3B positive control group. And for caecum samples, only the cell counts without heat treatment of the B. pumilus strain in the halofuginone supplemented group showed a reduction larger than 1‐log compared to the values in the Microsaf positive control group.

Although lasalocid and halofuginone may have certain antimicrobial activity against vegetative cells of B. velezensis NRRL B‐67647 and B. pumilus NRRL B‐67648, respectively, the in vivo study reported supports the compatibility of Microsaf GT 3B with all tested coccidiostats.

The FEEDAP Panel notes that decoquinate does not have any antibacterial action (EMEA CVMP, 2000) and, therefore, Microsaf® is considered compatible with this coccidiostat.

Conclusions on efficacy

3.3.3

Based on the efficacy trials submitted, the FEEDAP Panel concludes that the additive has the potential to improve the zootechnical performance of chickens for fattening at a proposed minimum inclusion level of 3 × 10^7^ total CFU/kg of complete feedingstuffs. The conclusion can be extrapolated to other poultry for fattening and ornamental birds.

The FEEDAP Panel concludes that Microsaf® is compatible with diclazuril, narasin, maduramicin ammonium, salinomycin sodium, monensin sodium, robenidine hydrochloride, and the combination narasin/nicarbazin, lasalocid, and halofuginone.

Post‐market monitoring

3.4

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 Regulation39 and Good Manufacturing Practice.

CONCLUSIONS

4

Microsaf® is considered safe for the target species, consumers of products derived from animals fed the additive, and the environment.

Regarding user safety, Microsaf GT 3B‐C is not irritant to the skin or eyes. Considering the similarity in composition, the Panel considers that this conclusion can be extended to Microsaf GT 3B provided it is formulated with only calcium carbonate. Both forms of the additive are considered skin and respiratory sensitisers, and any exposure via skin or respiratory tract is considered a risk. The Panel cannot conclude on any other formulations with other unspecified carriers/anticaking agents.

The FEEDAP Panel concludes that the additive has the potential to be efficacious as a zootechnical additive when added to the feed of chickens for fattening, other poultry for fattening, and ornamental birds at a proposed minimum inclusion level of 3 × 10^7^ total CFU/kg of complete feedingstuffs.

Microsaf® is compatible with diclazuril, narasin, maduramicin ammonium, salinomycin sodium, monensin sodium, robenidine hydrochloride, and the combination narasin/nicarbazin, lasalocid, and halofuginone.

ABBREVIATIONSAMRantimicrobial resistanceCFUcolony‐forming unitCVMPCommittee for Medicinal Products for Veterinary UseDMdry matterECEuropean CommissionEURLEuropean Union Reference LaboratoryFEEDAPEFSA Scientific Panel on Additives and Products or Substances used in Animal FeedLODlimit of detectionLOQlimit of quantificationMICminimum inhibitory concentrationQPSqualified presumption of safetyWHOWorld Health OrganisationWGSwhole genome sequence

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2021‐00449

COPYRIGHT FOR NON‐EFSA CONTENT

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.

PANEL MEMBERS

Roberto Edoardo Villa, Giovanna Azimonti, Eleftherios Bonos, Henrik Christensen, Mojca Durjava, Birgit Dusemund, Ronette Gehring, Boet Glandorf, Maryline Kouba, Marta López‐Alonso, Francesca Marcon, Carlo Nebbia, Alena Pechová, Miguel Prieto‐Maradona, Ilen Röhe, and Katerina Theodoridou.

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 EFSA. The full output has been shared with the European Commission, EU Member States (if applicable) and the applicant. The blackening may be subject to review once the decision on the confidentiality requests is adopted by EFSA and in case it rejects some of the confidentiality requests.