Safety and efficacy of ProEquo^® (Lactobacillus plantarum DSM 11520) as a feed additive for horses

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 ProEquo^® as a feed additive for horses. This additive is a preparation single strain of Lactobacillus plantarum intended for use as a zootechnical additive in feed for horses. The identity of the bacterial strain was not established. Consequently, the qualified presumption of safety approach to safety assessment cannot be applied, and separate studies on target animals, consumer and environmental safety are required. The tolerance trial provided could not be considered due to non‐compliance with the minimum requirements. No additional studies or data were provided for the other safety aspects. Therefore, in the absence of data, the FEEDAP Panel was not in the position to conclude on the safety of ProEquo^® for the target animals, consumers of products derived from animals fed the additive and the environment. ProEquo^® should be considered 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 on its dermal sensitisation potential. The FEEDAP Panel is not in the position to conclude on the efficacy of ProEquo^® for horses.

1 Introduction

1.1 Background and Terms of Reference as provided by the requestor

Regulation (EC) No 1831/200311 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.
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 ProEquo AB22 ProEquo AB, Research Park Ideon, House Delta 6, Scheelevägen 19F, SE‐223 70, Sweden.
for the authorisation of ProEquo^® (Lactobacillus plantarum DSM 11520) when used as a feed additive for horses (category: zootechnical additives; functional group: gut flora stabilisers).33 The applicant originally submitted an application including the re‐evaluation of the authorisation as a silage additive (Art 10). However, in the course of the assessment the applicant withdrew this request.

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 23 October 2018.

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 ProEquo^® (L. plantarum DSM 11520), when used under the proposed conditions of use (see Section 3.1.4).

1.2 Additional information

The additive ProEquo^® is a preparation of L. plantarum DSM 11520. It is not currently authorised in the European Union.

2 Data and methodologies

2.1 Data

The present assessment is based on data submitted by the applicant in the form of a technical dossier44 FEED dossier reference: FAD‐2017‐0004.
in support of the authorisation request for the use of ProEquo^® as a feed additive.

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.55 The full report is available on the EURL website: https://ec.europa.eu/jrc/sites/jrcsh/files/finrep_fad-2017-0004_proequo.docx_.pdf
Due to lack of information the EURL was not able to fully assess the suitability of the method of analysis proposed by the applicant for the enumeration of the active agent in the additive or in feedingstuffs.

2.2 Methodologies

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of ProEquo^® is in line with the principles laid down in Regulation (EC) No 429/200866 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.
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) and Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance (EFSA FEEDAP Panel, 2012c).

3 Assessment

ProEquo^® is a preparation of a single strain of L. plantarum intended for use as a zootechnical additive (gut flora stabiliser) in feed for horses.

3.1 Characterisation

3.1.1 Characterisation of the active agent

The strain was isolated from a healthy horse. It is deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) under the accession number DSM 11520.77 Technical dossier/Section II/Annex II‐1 and Supplementary information November 2018/Annex 2.1.
^,88 Lactobacillus plantarum JI: 1 is an in‐house identifier.
It has not been genetically modified.

Species identity is described ■■■■■99 Technical dossier/Section II/Annex II‐1 and Supplementary information November 2018/Annex 2.2b.
■■■■■1010 Technical dossier/Section II/Annex II‐1 and Supplementary information November 2018/Annex 2.2a.
However REA and RAPD are fingerprinting techniques not suitable for species identification. Furthermore, the original reports were not made available. Therefore, no conclusions can be drawn on the identification of the active agent.

The susceptibility of the active agent to the antibiotics recommended by FEEDAP for the L. plantarum/pentosus group was tested by broth microdilution following the method of the Clinical and Laboratory Standards Institute (CLSI).1111 Technical dossier/Section II/Annex II.2.2.2.4.Conf and Supplementary information November 2018/Annex 2.3.
All of the minimum inhibitory concentration (MIC) values found fell below the corresponding cut‐off values (EFSA FEEDAP Panel, 2012c), except for kanamycin which was one dilution higher than the cut‐off value ■■■■■ Exceedance of the cut‐off value by one dilution is considered to be within the normal range of variation and thus, not a matter of concern. However, in the absence of a proper identification of the active agent at species level, no conclusion can be drawn on its susceptibility to the relevant antibiotics.

3.1.2 Manufacturing process and characterisation of the additive

The additive is produced in two forms, a liquid and a solid form. ■■■■■1212 Technical dossier/Section II/Annex II‐2 and 12.
■■■■■1313 Technical dossier/Section II/Annex II‐6.
^,1414 Technical dossier/Supplementary information November 2018.

The two formulations are:

• ProEquo^® Sticks: a powder product sold in aluminium foil sticks (as a feed supplement) composed of the culture ■■■■■ plus maltodextrin ■■■■■ and beta‐glucan ■■■■■ and with a minimum declared content of 8 × 10¹⁰ colony forming units (CFU)/stick (dose serving of 6 g), equivalent to 1.3 × 10¹⁰ CFU/g,1515 Technical dossier/Section II/Annex II‐8.
  and
• ProEquo^® Liquid: composed of the inoculum and its fermentation broth ■■■■■1616 Technical dossier/Section II/Annex II‐5.
  No minimum declared content of L. plantarum DSM 11520 was described. This form is to be stored refrigerated (< 8°C).

At the time of submission of the application, the applicant declared that the ProEquo^® Sticks was the only formulation on the market and provided analysis of five batches showing counts ranging 1.0–1.3 × 10¹¹ CFU/stick (equivalent to 1.7–2.7 × 10¹⁰ CFU/g).1717 Technical dossier/Section II/Annex II‐9.
Analysis of eight batches of the cells concentrate (an intermediate preparation with specifications described as > 6 × 10¹¹ CFU/g) showed counts in the range 8.3–14 × 10¹¹ CFU/g.1818 Technical dossier/Section II/Annex II‐7.
No data were provided for the ProEquo^® Liquid, for which as stated above, no specifications were described in the dossier.

Microbial contamination is monitored at various points in the manufacturing process and in the final product. ■■■■■1414 Technical dossier/Supplementary information November 2018.
■■■■■1919 ■■■■■

No data on the dusting potential or particle size distribution of the solid form of the additive were provided.1414 Technical dossier/Supplementary information November 2018.

3.1.3 Stability

The viability of the active agent in the solid form ■■■■■ was measured ■■■■■1414 Technical dossier/Supplementary information November 2018.
■■■■■

No data on stability in feed were provided. The applicant claimed that ProEquo^® is intended to be poured on the top of the feed and immediately consumed by the animal.

3.1.4 Conditions of use

The additive is intended to be added as a top dressing to the feed for horses in a daily dose, from 1 to 3 sticks per day. The stick is described as containing 8 × 10¹⁰ CFU/stick, equivalent to 1.3 × 10¹⁰ CFU/g. Therefore, the proposed use level ranges from 8 to 24 × 10¹⁰ CFU/head per day. No recommended dose of the liquid form has been described and in the absence of specifications, no estimation can be made.

3.2 Safety

The species L. plantarum is considered by EFSA to be suitable for the Qualified Presumption of Safety (QPS) approach to safety assessment (EFSA, 2007; EFSA BIOHAZ Panel, 2020). This approach requires the identity of the strain to be conclusively established and evidence that it does not carry acquired antimicrobial genes. In the view of the FEEDAP Panel, the identity of the strain was not unambiguously established. Consequently, the QPS approach to safety assessment cannot be applied and separate studies on target animals, consumers and environmental safety are required.

The applicant submitted a tolerance trial with horses. However, it was not further considered as it did not comply with the minimum requirements for a tolerance trial in terms of design and reporting ■■■■■2020 Technical dossier/Section III/Annex III‐2.

No additional studies or data were provided to support the safety of ProEquo^® for the consumers or the environment.

The FEEDAP Panel is not in the position to conclude on the safety of ProEquo^® for the target species, consumers and the environment.

No studies were provided on the irritancy or sensitisation potential of the additive. In the absence of data, no conclusions on skin or eye irritancy or on dermal sensitisation can be drawn. In the absence of data on dusting potential and owing to the proteinaceous nature of the active agent, the additive should be considered a respiratory sensitiser.

3.3 Efficacy

Four efficacy trials were provided to support the efficacy of the additive. However, none could be further considered due to weaknesses in the experimental design and/or reporting ■■■■■2020 Technical dossier/Section III/Annex III‐2.
^,2121 ■■■■■
■■■■■■■■■■2323 Technical dossier/Section IV/Annexes IV‐2 and IV‐3.
■■■■■ Therefore, the FEEDAP Panel is not in the position to conclude on the efficacy of ProEquo^® for horses.

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 Regulation2424 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.
and Good Manufacturing Practice.

4 Conclusions

The identity of the active agent as L. plantarum was not established. Consequently, the QPS approach to safety assessment cannot be applied and separate studies on target animals, consumer and environmental safety are required. In the absence of data, the FEEDAP Panel is not in the position to conclude on the safety of ProEquo^® for the target animals, consumers of products derived from animals fed the additive and the environment.

ProEquo^® should be considered 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 on its dermal sensitisation potential.

The FEEDAP Panel is not in the position to conclude on the efficacy of ProEquo^® for horses.

Documentation as provided to EFSA/Chronology

Date	Event
03/01/2017	Dossier received by EFSA. Lactobacillus plantarum JI:1 (DSM 115 20). Submitted by ProEquo AB
03/01/2017	Reception mandate from the European Commission
23/10/2017	Application validated by EFSA – Start of the scientific assessment
30/11/2017	Request of supplementary information to the applicant in line with Article 8(1)(2) of Regulation (EC) No 1831/2003 – Scientific assessment suspended. Issues: methods of analysis
06/12/2017	Request of supplementary information to the applicant in line with Article 8(1)(2) of Regulation (EC) No 1831/2003 – Scientific assessment suspended. Issues: characterisation, conditions of use, safety and efficacy
23/01/2018	Comments received from Member States
15/11/2018	Reception of supplementary information from the applicant ‐ Scientific assessment still on hold
21/01/2020	Reception of supplementary information from the applicant ‐ Scientific assessment re‐started
29/01/2020	Reception of the Evaluation report of the European Union Reference Laboratory for Feed Additives
07/05/2020	Opinion adopted by the FEEDAP Panel. End of the Scientific assessment

Abbreviations

CFU

colony forming unit

CLSI

Clinical and Laboratory Standards Institute

DSMZ

Deutsche Sammlung von Mikroorganismen und Zellkulturen

EURL

European Union Reference Laboratory

FEEDAP

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

MIC

minimum inhibitory concentration

PFGE

pulsed field gel electrophoresis

QPS

Qualified Presumption of Safety

Annex A – Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for ProEquo^®

In the current application authorisation is sought under Article 4(1) (authorisation of a new feed additive) for a preparation of Lactobacillus plantarum JI:1 (DSM 115 20) 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 horses.

According to the Applicant, the feed additive contains as active substance viable cells of the non‐genetically modified strain Lactobacillus plantarum JI:1 (DSM 115 20) . The feed additive is to be marketed as liquid preparation (ProEquo ^® liquid ) or as a 6 g dry feed‐stick (ProEquo ^® Sticks ). The dry form of the feed additive has a minimum Lactobacillus plantarum JI:1 (DSM 115 20) content of 13 × 109 Colony Forming Unit (CFU)/g. The feed additive is intended to be added poured on top of other feed.

For the enumeration of Lactobacillus plantarum JI:1 (DSM 115 20) in the feed additive the Applicant submitted the NMKL method No. 140 (1991) published by the Nordic Committee on Food Analysis. Nevertheless, the Applicant did not provide any experimental data demonstrating the fitness for purpose of the proposed method for the enumeration of Lactobacillus plantarum JI:1 (DSM 115 20) in the feed additive .

The EURL requested the Applicant to apply and verify the ring‐trial validated spread plate CEN method EN 15787 for the enumeration of lactobacilli in the two commercial products (ProEquo ^® liquid and ProEquo ^® Sticks ). However the Applicant did not follow the EURL request and confirmed the submission of the incomplete information as provided in the original dossier. Therefore the EURL cannot conclude on the suitability of the proposed methods of analysis (i.e. EN 15787 and/or NMKL method No. 140/1991) for the official control of Lactobacillus plantarum JI:1 (DSM 115 20) in the feed additive .

For the enumeration of Lactobacillus plantarum JI:1 (DSM 115 20) in the feedingstuff the Applicant submitted a procedure based on the abovementioned NMKL method No. 140 (1991). However the Applicant did not provide any supporting experimental data. Therefore, the EURL cannot evaluate and is unable to recommend any method for official control to quantify Lactobacillus plantarum JI:1 (DSM 115 20) in feed .

For the identification of Lactobacillus plantarum JI:1 (DSM 115 20) , the EURL recommends for official control Pulsed Field Gel Electrophoresis (PFGE), a generally recognised methodology for the genetic identification of bacterial strains.

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, as last amended by Regulation (EU) 2015/1761) is not considered necessary.