Safety and efficacy of ZM16 10 (Bacillus amyloliquefaciens DSM 25840) as a feed additive for weaned piglets and minor porcine species

Abstract

Following a request from the European Commission, the EFSA 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 ZM16 10 for weaned piglets and minor porcine species. The additive is a preparation containing viable spores of a strain of Bacillus amyloliquefaciens intended for use in feed at the proposed dose of 5 × 10⁸ CFU/kg complete feedingstuffs and in water for drinking at 1.7 × 10⁸ CFU/L. The additive exists in two forms, ZM16 and ZM16 10, which contain the bacterium in concentrations of 1.25 × 10⁹ CFU/g additive and 1.25 × 10¹⁰ CFU/g additive, respectively. The two formulations are considered equivalent when used to deliver the same dose. B. amyloliquefaciens is considered by EFSA to be suitable for the qualified presumption of safety (QPS) approach to establishing safety. The active agent fulfils the requirements of the QPS approach to the assessment of safety. Consequently, the additive can be presumed safe for the target animals, consumers of products from treated animals and the environment. Given the proteinaceous nature of the active agent, the additive should be considered a potential respiratory sensitiser. In the absence of data, the FEEDAP Panel cannot conclude on the irritancy of the additive to skin and eyes or its dermal sensitisation. Insufficient evidence was provided to conclude on the efficacy of the additive in weaned piglets or minor weaned porcine species.

1. Introduction

1.1. Background and Terms of Reference

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 Chr. Hansen A/S2 for authorisation of the product ZM16 10 (Bacillus amyloliquefaciens DSM 25840), when used as a feed additive for weaned piglets and weaned minor porcine species (category: Zootechnical additives; functional group: gut flora stabilisers).

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 particulars and documents in support of the application were considered valid by EFSA as of 13 January 2017.

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 product ZM16 10 (Bacillus amyloliquefaciens DSM 25840), when used under the proposed conditions of use (see Section 3.1.4).

1.2. Additional information

The additive ZM16 10 is a preparation containing viable spores of B. amyloliquefaciens DSM 25840. It has not been previously authorised as a feed additive in the EU.

2. Data and methodologies

2.1. Data

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 ZM16 10 as a feed additive. The technical dossier was prepared following the provisions of Article 7 of Regulation (EC) No 1831/2003, Regulation (EC) No 429/20084 and the applicable EFSA guidance documents.

EFSA has verified the European Union Reference Laboratory (EURL) report as it relates to the methods used for the control of the active agent in animal feed. The Executive Summary of the EURL report can be found in Annex A.5

2.2. Methodologies

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of ZM16 10 is in line with the principles laid down in Regulation (EC) No 429/2008 and the relevant guidance documents: Guidance on zootechnical additives (EFSA FEEDAP Panel, 2012a), Technical guidance: Tolerance and efficacy studies in target animals (EFSA FEEDAP Panel, 2011), Guidance on studies concerning the safety of use of the additive for users/workers (EFSA FEEDAP Panel, 2012b), Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition (EFSA FEEDAP Panel, 2014) and Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance (EFSA FEEDAP Panel, 2012c).

3. Assessment

The additive is a preparation containing viable spores of B. amyloliquefaciens DSM 25840 intended to be used in feed and water for drinking for weaned piglets and minor weaned porcine species to improve growth.

3.1. Characterisation

3.1.1. Characterisation of the active agent

B. amyloliquefaciens DSM 25840 was isolated from the faeces of healthy adult pigs and has not been genetically modified.6 The strain has been deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen with the accession number DSM 25840.7

Taxonomical identification of the product strain as B. amyloliquefaciens was achieved using the nearly complete 16S rRNA gene sequence and multilocus sequence typing of partial sequences of the genes groEL, gyrA, polC, purH and rpoB, and by comparison with reference databases. Strain‐specific identification was based on the use of pulsed‐field gel electrophoresis after cleavage with restriction enzymes NotI and SpeI used individually.8

B. amyloliquefaciens DSM 25840 was tested for antibiotic susceptibility using twofold broth dilutions. The battery of antibiotics tested was that recommended by EFSA (EFSA FEEDAP Panel, 2012c).9 All minimum inhibitory concentration values fell below the corresponding cut‐off values defined by the FEEDAP Panel, therefore the strain is considered to be susceptible to relevant antibiotics.

The toxigenic potential of B. amyloliquefaciens DSM 25840 was assessed according to the Technical Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition (EFSA FEEDAP Panel, 2014).10 No lysis of Vero cells was detected, so B. amyloliquefaciens DSM 25840 is considered to be not toxigenic.

3.1.2. Characterisation of the additive11

The manufacturing process of the additive is detailed in the dossier.12 ZM16 10 is produced with a minimum guaranteed concentration of 1.25 × 10¹⁰ colony forming units (CFU) per gram of additive. Batch‐to‐batch variation was measured in five batches of the additive and found to be consistently compliant with specifications (mean: 1.9 × 10¹⁰ CFU/g, range: 1.8–2.0 × 10¹⁰ CFU/g).13 The applicant mentions in the dossier a second formulation called ZM16, with a minimum concentration of 1.25 × 10⁹ CFU/g additive. This formulation was used in some stability and efficacy studies.

The additive is routinely monitored for microbial and chemical contamination. Limits are set for total coliforms (< 10³ CFU/g), yeasts and filamentous fungi (< 10³ CFU/g), Escherichia coli (< 10 CFU/g), Salmonella (absent in 25 g), aflatoxin B1 (< 0.005 mg/kg), lead (< 5.0 mg/kg), cadmium (< 0.5 mg/kg), mercury (< 0.1 mg/kg) and arsenic (< 2.0 mg/kg). Analyses of five batches showed compliance with the limits for microbial impurities13 and the absence of Bacillus cereus contamination.14 ^, 15 Analyses of three batches of the additive showed values compliant with specifications for chemical contaminants, or levels not giving rise to concerns (lead ≤ 0.192 mg/kg, cadmium < 0.005 mg/kg, mercury ≤ 0.0137 mg/kg, arsenic ≤ 0.337 mg/kg, aflatoxin B1 < 0.64 μg/kg, dioxins and polychlorinated biphenyls (PCBs): octachlorodibenzo‐p‐dioxin (OCDD) < 0.153 ng/kg, octachlorodibenzofuran (OCDF) < 0.161 ng/kg, WHO‐polychlorinated dibenzodioxins/dibenzofurans‐toxic equivalent (WHO‐PCDD/F‐TEQ) < 0.186 ng/kg and WHO‐PCDD/F‐PCB‐TEQ < 0.198 ng/kg.16

The particle size distribution of ZM16 10 was tested in three batches by laser diffraction. Results showed that 8% (v/v) of the additive consists of particles with diameters less than 50 μm and 4% less than 10 μm.17 The dusting potential of the same three batches of the additive, tested with a Heubach Dustmeter, showed an mean value of 13.5 g/m³.18

3.1.3. Stability and homogeneity

The stability of three batches of ZM16 10 packed in impermeable bags was monitored at 25, 30 and 37°C for 12 months.19 Losses of B. amyloliquefaciens counts after this period were negligible (< 0.5 log) at all conditions.

Three batches of ZM16 mixed with two vitamin–mineral premixtures (not containing choline chloride) for piglets according to the conditions of use were stored for 6 months at 20–25°C.20 Microbial counts after this period showed a recovery > 100% in both cases.

To test stability to pelleting conditions, three batches of ZM16 were mixed with a pig mash feed, in accordance with the conditions of use, and subjected to pelleting temperatures of 75, 85 and 95°C.21 Total counts of bacilli showed a recovery of 95% at all temperatures.

To test stability in feed for piglets, three batches of ZM16 were mixed with mash and pelleted feed (at 95°C), in accordance with the conditions of use. Samples were stored for 6 months at 20–25°C.22 No viability losses were detected in the mash feed or in the pelleted feed.

The stability of three batches of ZM16 suspended in water for drinking at 20–25°C was tested after 1 and 2 days, respectively. Total recovery exceeded 90% at both time points.23

The capacity of ZM16 10 to homogeneously distribute in pelleted feed for piglets (based on 10 subsamples) according to the conditions of use was investigated in one study. Analyses of total counts showed a coefficient of variation of 0.4%.24

3.1.4. Conditions of use

The product is intended for use in feed for weaned piglets and weaned minor porcine species at a dose of 5 × 10⁸ CFU/kg complete feedingstuffs and of 1.7 × 10⁸ CFU/L of water for drinking.

3.2. Safety

3.2.1. Safety for the target species, consumers and environment

The bacterial species B. amyloliquefaciens is considered by EFSA to be suitable for the qualified presumption of safety (QPS) approach to safety assessment (EFSA, 2007; EFSA BIOHAZ Panel, 2017). This approach requires the identity of the strains to be conclusively established and evidence that the strains lack toxigenic potential and do not show resistance to antibiotics of human and veterinary importance.

In the view of the FEEDAP Panel, the identity of the active agent is established as B. amyloliquefaciens and the toxigenic potential and the antibiotic resistance qualifications have been met. Therefore, the strain is presumed safe for the target species, consumer and the environment. ZM16 10 is also considered safe for target animals, consumers and the environment.

3.2.2. Safety for the user

No information was provided on the inhalation toxicity of the additive. The dustiness of the preparations tested indicated a potential for users to be exposed via inhalation. A significant fraction of the product consists of fine particles that have the potential to reach the alveoli when inhaled. Given the proteinaceous nature of the active agent, the additive should be considered a potential respiratory sensitiser. No data are available on skin/eye irritation or skin sensitisation for the additive ZM16 10.25 In the absence of data, the FEEDAP Panel cannot conclude on the irritancy potential of the additive to skin and eyes or on its dermal sensitisation.

3.3. Efficacy

3.3.1. Efficacy for weaned piglets26

Six efficacy studies were performed in four Member States and are described in the dossier.27

In the four studies considered, supplementation of ZM16 led to a numerical lower feed to gain ratio, however, reaching significance only in two cases (ZM16 = 1.55 g/g vs control = 1.64 g/g in one study and ZM16 = 1.57 g/g vs control = 1.65 g/g in the other study; p < 0.05).

Significant effects on faecal scores28 were observed only in one of these (ZM16 10 = 7.43 vs control = 7.23, p < 0.05).

Significant effects were found only in two studies. Therefore, there is insufficient evidence to conclude on the efficacy of ZM16 in weaned piglets.

3.3.2. Efficacy for weaned minor porcine species

In the absence of a demonstration of efficacy for weaned piglets, no conclusions can be drawn for minor porcine species.

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 Regulation29 and Good Manufacturing Practice.

4. Conclusions

The active agent fulfils the requirements of the QPS approach to the assessment of safety, consequently, ZM16 10 is presumed safe for the target animals, consumers of products from treated animals and the environment.

The additive should be considered to have the potential to be a respiratory sensitiser. In the absence of data, the FEEDAP Panel cannot conclude on the irritancy potential of the additive to skin and eyes or its dermal sensitisation.

Insufficient evidence was provided to conclude on the efficacy of the additive in weaned piglets or minor weaned porcine species.

Documentation provided to EFSA

1. ZM16 (Bacillus amyloliquefaciens, DSM 25840) Zootechnical feed additive for weaned piglets and other weaned minor porcine species + use in drinking water. November 2016. Submitted by Chr. Hansen A/S

2. ZM16 (Bacillus amyloliquefaciens, DSM 25840) Zootechnical feed additive for weaned piglets and other weaned minor porcine species + use in drinking water. Supplementary information. July 2017. Submitted by Chr. Hansen A/S.

3. Evaluation report of the European Union Reference Laboratory for Feed Additives on the Methods(s) of Analysis for ZM16 10.

4. Comments from Member States.

Abbreviations

CFU

colony forming unit

EURL

European Union Reference Laboratory

FEEDAP

EFSA Panel on Additives and Products or Substances used in Animal Feed

OCDD

octachlorodibenzo‐p‐dioxin

OCDF

octachlorodibenzofuran

PCB

polychlorinated biphenyl

PCDD

polychlorinated dibenzodioxin/dibenzofuran

QPS

qualified presumption of safety

TEQ

toxic equivalent

Annex A – Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Methods of Analysis for ZM16 10

1.

In the current application authorisation is sought under Article 4(1) for Bacillus amyloliquefaciens DSM 25840 under the category/functional group 4(b) ‘zootechnical additives’/‘gut flora stabilisers’, according to Annex I of Regulation (EC) No 1831/2003. Authorisation is sought for the use of the feed additive for weaned piglets and other weaned minor porcine species.

According to the Applicant, the feed additive contains as active substance viable spores of the non‐genetically modified strain Bacillus amyloliquefaciens DSM 25840. The feed additive is to be marketed as a dry powder containing a minimum Bacillus amyloliquefaciens DSM 25840 content of 1.3 × 10¹⁰ Colony Forming Unit (CFU)/g. The feed additive is intended to be used in drinking water at a minimum dose of 1.7 × 10⁸ CFU/L, included through premixtures or added directly into feedingstuffs at a minimum dose of 5 × 10⁸ CFU/kg complete feedingstuffs.

For the identification of Bacillus amyloliquefaciens DSM 25840, the EURL recommends for official control Pulsed Field Gel Electrophoresis (PFGE), a generally recognised methodology for genetic identification of bacterial strain.

For the enumeration of Bacillus amyloliquefaciens DSM 25840 in the feed additive, in premixtures, feedingstuffs and water the Applicant submitted the ring‐trial validated spread plate CEN method 15784. Based on the performance characteristics available, the EURL recommends this method for official control.

Further testing or validation of the methods to be performed through the consortium of National Reference Laboratories as specified by Article 10 (Commission Regulation (EC) No 378/2005) is not considered necessary.

Suggested citation: EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed) , Rychen G, Aquilina G, Azimonti G, Bampidis V, Bastos ML, Bories G, Chesson A, Cocconcelli PS, Flachowsky G, Gropp J, Kolar B, Kouba M, López‐Alonso M, López Puente S, Mantovani A, Mayo B, Ramos F, Villa RE, Wallace RJ, Wester P, Brozzi R and Saarela M, 2018. Scientific Opinion on the safety and efficacy of ZM16 10 (Bacillus amyloliquefaciens DSM 25840) as a feed additive for weaned piglets and minor porcine species. EFSA Journal 2018;16(4):5200, 9 pp. 10.2903/j.efsa.2018.5200