Assessment of the feed additive consisting of Enterococcus lactis DSM 10663 NCIMB 10415 (Oralin®) for calves for rearing, piglets, chickens for fattening, turkeys for fattening, cats and dogs for the renewal of its authorisation (Chevita Tierarzneimittel GmbH)

TL;DR

This paper assesses the safety of a feed additive containing Enterococcus lactis for various animals and concludes it remains safe under current conditions.

Contribution

The study provides updated safety evaluation of Oralin® for continued authorization as a feed additive.

Findings

The additive is safe for target animals, consumers, and the environment under authorized conditions.

The additive is a skin and respiratory sensitiser, posing risks via dermal and respiratory exposure.

The coated form of the additive may have eye irritancy potential, but this could not be conclusively assessed.

Abstract

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the assessment of the application for renewal of authorisation of the additive Oralin®, a preparation containing viable cells of Enterococcus lactis DSM 10663 (formerly identified as Enterococcus faecium), as a zootechnical feed additive for calves for rearing, piglets, chickens for fattening, turkeys for fattening, cats and dogs. The applicant has provided evidence that the additive currently in the market complies with the existing conditions of authorisation. The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the additive remains safe for the target animals, consumers and the environment under the authorised conditions of use. Regarding user safety, the additive in all forms should be considered as a skin and respiratory sensitiser. Exposure by dermal and respiratory routes is considered a risk. The additive is not irritant to eyes in the powder (Oralin® 350 P), granulated (Oralin® 350 G) and liquid (Oralin® 100 S) preparations. The FEEDAP Panel is not in the position to conclude on the irritancy potential for eyes of the coated form (Oralin® 200 C). There is no need for assessing the efficacy of the additive in the context of the renewal of the authorisation.

Tables

TABLE 1. Data on the batch‐to‐batch variation and impurities of the additive Oralin®. The data presented are average values and (range) for batch‐to‐batch variation, and ranges for all other parameters. The number of batches analysed are indicated in [].

	Oralin® 350 G	Oralin® 350 P	Oralin® 200 C	Oralin® 100 S
Specifications 1
Enterococcus lactis DSM 10663 (CFU/g)	≥3.5 × 1010	≥3.5 × 1010	≥2 × 1010	≥1 × 1010
Batch‐to‐batch variation	[6]	[5]	[5]	[5]
Enterococcus lactis (CFU/g)	■■■■■	■■■■■	■■■■■	■■■■■
	■■■■■	■■■■■	■■■■■	■■■■■
Impurities (mg/kg)	[3]	[3]	[3]	[4]
Lead	< 0.50	< 0.50	< 0.50	< 0.50
Mercury	< 0.02	< 0.02	< 0.02	< 0.02
Cadmium	< 0.20	< 0.20	< 0.20	< 0.20
Arsenic	< 0.50	< 0.50	< 0.50	< 0.50
Fluor	< 40	< 40	< 40	< 40
Nickel	–	–	–	<0.50–0.69 [3]
Dioxins and furans (upper bound) 2	[3]	[3]	[3]	[4]
PCDD/Fs (ng WHO2005‐TEQ/kg)	0.30	0.30	0.30	0.30
PCDD/Fs + PCBs (ng WHO2005‐TEQ/kg)	0.16	0.16	0.16	0.16
nDL‐PCBs (μg/kg)	0.14	0.14	0.14	0.14
Mycotoxins (μg/kg)	[3]	[3]	[3]	[4]
Aflatoxins B1, B2, G1, G2 (individual)	< 1	< 1	< 1	< 1
Deoxynivalenol	< 50	< 50	< 50	< 50
Zearalenone	< 5	< 5	< 15	< 15
Diacetoxyscirpenol	< 10	< 10	< 10	< 10
Fumonisins B1, B2, B3 (individual)	< 40	< 100	< 100	< 100
HT‐2 Toxin	< 10	< 10	< 20	< 20
T‐2 Toxin	< 5	< 10	< 10	< 10
Nivalenol	< 50	< 50	< 150	< 100
Fusarenon X	< 50	< 50	< 50	< 50
Microbial contamination	[3]	[3]	[3]	[3]
Salmonella spp. (per 25g)	Not detected	Not detected	Not detected	Not detected
Yeast and moulds (CFU/g)	< 10	< 10–1.14 × 102	< 10	< 10
Total coliforms (CFU/g)	< 10	< 10	< 10	< 10
Anaerobic spore‐formers (CFU/g)	< 10	< 10	< 10	< 10
Escherichia coli (per 1 g)	Not detected	Not detected	Not detected	Not detected
Pseudomonas aeruginosa (per 1 g)	Not detected	Not detected	Not detected	Not detected
Staphylococcus aureus (per 1 g)	Not detected	Not detected	Not detected	Not detected
Listeria monocytogenes (per 25 g)	Not detected	Not detected	Not detected	Not detected
Shigella spp. (per 25 g)	Not detected	Not detected	Not detected	Not detected
Vibrio spp. (per 25 g)	Not detected	Not detected	Not detected	Not detected

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 14(1) of that Regulation lays down that an application for renewal shall be sent to the Commission at the latest one year before the expiry date of the authorisation.

The European Commission received a request from Chevita Tierarzneimittel GmbH2 for the renewal of the authorisation of the additive consisting of a preparation of Enterococcus lactis DSM 106633 (Oralin®), when used as a feed additive for calves for rearing, piglets, chickens for fattening, turkeys for fattening, cats and dogs (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 14(1) (renewal of the authorisation). The dossier was received on 19 July 2024 and the general information and supporting documentation are available at https://open.efsa.europa.eu/questions/EFSA‐Q‐2024‐00476. The particulars and documents in support of the application were considered valid by EFSA as of 22 October 2024.

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 Oralin®, when used under the proposed conditions of use (see Section 3.1.3).

Additional information

1.2

The additive is a preparation containing viable cells of E. lactis DSM 10663 (Oralin®). The additive is currently authorised as a zootechnical additive (functional group: gut flora stabilisers) for use in calves for rearing, piglets (suckling and weaned), chickens for fattening, turkeys for fattening, cats and dogs (4b1707).4 EFSA has issued several opinions on the safety and efficacy of this product (EFSA FEEDAP Panel 2010, 2014a, 2014b).

DATA AND METHODOLOGIES

2

Data

2.1

The present assessment is based on data submitted by the applicant in the form of a technical dossier5 in support of the authorisation request for the use of Oralin® as a feed additive.

The confidential version of the technical dossier was subject to a target consultation of the interested Member States from 25 October 2024 to 25 January 2025; the comments received were considered for the assessment.

In accordance with Article 38 of the Regulation (EC) No 178/20026 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,7 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 technical dossier 9 April 2025 to 30 April 2025 for which no comments were received.

The European Union Reference Laboratory (EURL) considered that the conclusions and recommendations reached in the previous assessment regarding the methods used for the control of Oralin® in animal feed are valid and applicable for the current application.8

Methodologies

2.2

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of Oralin® is in line with the principles laid down in Regulation (EC) No 429/20089 and the relevant guidance documents: Guidance on the renewal of the authorisation of feed additives (EFSA FEEDAP Panel, 2021).

ASSESSMENT

3

The additive Oralin® is a preparation containing viable cells of E. lactis DSM 10663 and is currently authorised as a zootechnical additive (functional group: gut flora stabiliser) for calves for rearing, piglets, chickens for fattening, turkeys for fattening, cats and dogs. The assessment regards the renewal of the authorisation of the feed additive for those target species.

Characterisation

3.1

Characterisation of the additive

3.1.1

The following four preparations of the additive Oralin® are currently authorised:

• Oralin® 350 P: powder formulation with a minimum content of the active agent of 3.5 × 10^10^ CFU/g of additive, ■■■■■ (■■■■■) and lactose and/or saccharose (■■■■■).
• Oralin® 350 G: granulated formulation with a minimum content of the active agent of 3.5 × 10^10^ CFU/g of additive, ■■■■■ (■■■■■) and lactose and/or saccharose (■■■■■).
• Oralin® 200 C: coated formulation with a minimum content of the active agent of 1 × 10^10^ CFU/g of additive, ■■■■■ (■■■■■) and saccharose/maize starch (■■■■■).
• Oralin® 100 S: liquid formulation with a minimum content of the active agent of 1 × 10^10^ CFU/g of additive, ■■■■■ (■■■■■), glycerol polyethylene ricinoleate (■■■■■), silicon dioxide E551b10 (■■■■■), lactose and/or saccharose (■■■■■) and peanut oil (■■■■■).

The applicant declared that the manufacturing process has not been modified since the previous authorisation. ■■■■■.11 No antimicrobials are used during the manufacturing process.12

The data provided by the applicant on the batch‐to‐batch variation13 and impurities14 of the additive are reported in Table 1.

The data provided showed compliance with the specifications set in the authorising regulation.15 The FEEDAP Panel considers that the microbial contamination and the amounts of the detected impurities do not raise safety concerns, except for nickel in the Oralin® 100 S preparation, which is addressed in Section 3.2. The FEEDAP Panel notes that analysis for the detection of Enterobacteriaceae were not provided. No new data were provided regarding the physico‐chemical properties or stability of the additive.16 Since no changes were introduced in the manufacturing process and composition, the data described in the previous opinions (EFSA FEEDAP Panel, 2014a, 2014b) are considered still valid.

Characterisation of the active agent

3.1.2

The active agent was originally isolated from the faeces of a healthy infant and is deposited in the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) with the accession number DSM 10663.17 No proof of deposit in the British National Collection of Industrial and Marine Bacteria (NCIMB) has been provided in the current application. The applicant claims that the active agent has not been genetically modified.18

The active agent, originally assigned to the Enterococcus faecium species (EFSA FEEDAP Panel, 2014b), was identified as Enterococcus lactis based on bioinformatic analysis of the whole genome sequence (WGS) data. The taxonomic assignment was based on digital DNA–DNA hybridisation (dDDH) and average nucleotide identity (ANI) analyses.19 ■■■■■

The susceptibility of the DSM 10663 strain to antimicrobials was tested using a broth microdilution method and including the set of antibiotics recommended by EFSA (EFSA FEEDAP Panel, 2018). The minimum inhibitory concentration (MIC) values of the strain were compared with the defined EFSA cut‐off values for the closest related species E. faecium.20 All the MIC values were below or equal to the corresponding cut‐off values, and therefore, the strain is considered to be susceptible to all the relevant antibiotics.

The WGS data of the DSM 10663 strain, ■■■■■ was interrogated for the presence of antimicrobial resistance (AMR) genes by a search against the NCBI and ResFinder databases.21 The search resulted in ■■■■■ exceeding the EFSA thresholds (EFSA, 2024). Further analysis of these hits following EFSA criteria (EFSA BIOHAZ Panel, 2023) revealed that these are not acquired AMR genes. Therefore, the FEEDAP Panel concludes that the strain harbours no acquired AMR genes.

The antimicrobial production was analysed in culture supernatants of the active agent using five microorganisms as indicators.22 ^,^ 23 None of the reference strains was inhibited, demonstrating the absence of antimicrobial activity.

The safety of E. faecium should be assessed demonstrating the absence of genetic markers typical of the clinical isolates E. faecium clade A (IS16, esp, hylEfm) and the susceptibility to ampicillin. Considering the recent allocation of E. faecium clade B strains to the E. lactis species, the FEEDAP Panel considers these criteria are also applicable to E. lactis strains. The active agent DSM 10663 was susceptible to ampicillin and none of the three genetic determinants were detected by BLASTn analysis of its WGS data.24

Conditions of use

3.1.3

The additive is currently authorised at the minimum level of 1 × 10^9^ CFU/kg complete feed for calves for rearing, piglets (suckling and weaned), chickens for fattening, turkeys for fattening, cats and dogs. Under other provisions of the authorisation, it is specified that:

• In the directions for use of the additive and premixture, indicate the storage conditions and stability to pelleting.
• Recommended dose for suckling piglets: 1 × 10^9^ CFU/piglet/day.
• The use is permitted in feed for chickens for fattening and turkeys for fattening containing the authorised coccidiostats: semduramicin sodium, diclazuril, robenidine hydrochloride, maduramicin ammonium, decoquinate, lasalocid A sodium or halofuginone.
• For use in weaned piglets until approximately 35 kg.

The applicant intends to maintain the same conditions of use in the authorisation.25

Safety

3.2

In the previous assessments (EFSA FEEDAP Panel, 2014a, 2014b), the FEEDAP Panel concluded that Oralin® was considered safe for calves for rearing, piglets, chickens for fattening, turkeys for fattening, cats and dogs, consumers and the environment. Regarding user safety, the Panel concluded that the additive is not an eye/skin irritant or a skin sensitiser but should be considered as a potential respiratory sensitiser.

The applicant declared that no incidents or safety issues for target animals, consumers, users and/or the environment have been documented or reported regarding the additive since its approval.26

In the context of the current application, the identity of the active agent has been reassigned from E. faecium to E. lactis, and evidence was provided that the strain does not harbour acquired AMR genes and was shown not to contain marker genes typical of hospital‐associated isolates responsible for clinical infections. The FEEDAP Panel considers the criteria to assess the safety of E. faecium to be applicable also to E. lactis strains. In addition, the manufacturing process of the additive, its composition and the conditions of use have not been modified. The only modification done is in the composition of Oralin® 200C (i.e. ■■■■■) and it would not raise safety concerns. Consequently, the previous conclusions are still valid, and the Panel considers that the additive remains safe for the target species, consumers and the environment under the authorised conditions of use.

In support of the safety of the additive, the applicant submitted the results of a literature search that covered the period from 2010 to 27 May 2024.27 Four databases were searched (AGRICOLA, EMBASE, MEDLINE, TOXCENTER). The terms included the trade name of the additive, the active agent and covered the safety for the target species and user in two independent searches. Exclusion and inclusion criteria were reported. A total of 108 references were retrieved. None of them were considered relevant.

Regarding the safety for the user, the FEEDAP Panel notes that the OECD test guidelines available at present are designed to assess the skin sensitisation potential of chemical substances only and that currently no validated assays for assessing the sensitisation potential of microorganisms are available. The additive contains a microorganism and therefore should be considered as a skin and respiratory sensitiser (EFSA FEEDAP Panel, 2023). The Panel reiterates its previous conclusions for the powder (Oralin® 350 P), granulated (Oralin® 350 G) and liquid (Oralin® 100 S) preparations of the additive, that they are not irritant to eyes. No specific studies for the coated formulation were provided. Therefore, the FEEDAP Panel is not in the position to conclude on the irritancy potential for eyes for the coated form Oralin® 200 C.

The FEEDAP Panel notes that the preparation Oralin® 100 S of the additive contains nickel (see Section 3.1.1). The Directive (EU) 2022/43128 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). Due to the presence of nickel, the additive should be considered as a skin and respiratory sensitiser.

Conclusions on safety

3.2.1

The FEEDAP Panel concludes that Oralin® remains safe for the target species, consumers and the environment at the current conditions of the authorisation. Regarding user safety, the additive in all forms should be considered as a skin and respiratory sensitiser. Exposure by dermal and respiratory routes is considered a risk and should be minimised. The powder (Oralin® 350 P), granulated (Oralin® 350 G) and liquid (Oralin® 100 S) preparations of the additive are not irritant to eyes. The FEEDAP Panel is not in the position to conclude on the eye irritation potential of the coated form (Oralin® 200 C).

Efficacy

3.3

The present application for renewal of the authorisation does not include a proposal for amending or supplementing the conditions of the original authorisation that would have an impact on the efficacy of the additive. Therefore, there is no need for assessing the efficacy of the additive in the context of the renewal of the authorisation.

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 Regulation29 and Good Manufacturing Practice.

CONCLUSIONS

4

The applicant has provided evidence that the additive currently on the market complies with the existing terms of authorisation.

The Panel concludes that Oralin® remains safe for all target animal species, consumers and the environment under the authorised conditions of use.

Regarding user safety, the additive should be considered as a skin and respiratory sensitiser. Exposure by dermal and respiratory routes is considered a risk and should be minimised. The powder (Oralin® 350 P), granulated (Oralin® 350 G) and liquid (Oralin® 100 S) preparations of the additive are not irritant to eyes but no conclusion can be reached for the coated form (Oralin® 200 C).

There is no need for assessing the efficacy of the additive in the context of the renewal of the authorisation.

RECOMMENDATIONS

5

The Panel notes that no proof of deposit of the active agent in the British National Collection of Industrial and Marine Bacteria (NCIMB) has been provided in the current application. Therefore, the deposit number that is to be retained is DSM 10663.

ABBREVIATIONSAMRantimicrobial resistanceANIaverage nucleotide identityCFUcolony forming unitdDDHdigital DNA‐DNA hybridisationDMdry matterDSMZDeutsche Sammlung von Mikroorganismen und ZellkulturenDSMZGerman Collection of Microorganisms and Cell Cultures GmbHEURLEuropean Union Reference LaboratoryFEEDAPEFSA Scientific Panel on Additives and Products or Substances used in Animal FeedLOQlimit of quantificationMICminimum inhibitory concentrationNCIMBNational Collection of Industrial and Marine BacterianDL‐PCBsnon‐dioxin‐like PCBsOELoccupational exposure limitPCBspolychlorinated biphenylsPCDDspolychlorinated dibenzo‐p‐dioxinsPCDFspolychlorinated dibenzofuransTEQtoxic equivalent factors for dioxins, furans and dioxin‐like PCBsWGSwhole genome sequence

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00476

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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, 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.