Safety and efficacy of a feed additive consisting of l‐lysine monohydrochloride produced with Corynebacterium glutamicum CGMCC 7.453 for all animal species (Eppen Europa SAS)

TL;DR

This paper evaluates the safety and effectiveness of a genetically modified feed additive for all animal species, finding it safe for most uses but with some concerns.

Contribution

The paper provides a scientific evaluation of a genetically modified l-lysine monohydrochloride feed additive for animal species.

Findings

The additive is safe for non-ruminant animals when administered via feed.

The additive is considered irritant to skin, eyes, and respiratory tract.

l-Lysine HCl is an efficacious source of l-lysine for non-ruminant species but requires protection for ruminants.

Abstract

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of l‐lysine monohydrochloride produced by fermentation with a genetically modified strain of Corynebacterium glutamicum (CGMCC 7.453) as nutritional feed additive for all animal species. Neither viable cells nor recombinant DNA of the production strain were detected in the final product. Therefore, the FEEDAP Panel concluded that the additive does not pose any safety concern regarding the production strain. The FEEDAP Panel concluded that the use of l‐lysine HCl produced by fermentation with the strain C. glutamicum CGMCC 7.453 is safe for the target species when administered via feed. However, the FEEDAP Panel expressed concerns on the use of l‐lysine HCl in water for drinking. The Panel concluded that the use of l‐lysine HCl produced by fermentation with C. glutamicum CGMCC 7.453 in animal nutrition is considered safe for the consumers and for the environment. With regards user safety, the additive should be considered irritant to skin, eyes and the respiratory tract. Any exposure to the additive is a risk. l‐Lysine HCl is considered as efficacious source of the essential amino acid l‐lysine for non‐ruminant animal species. For the supplemental l‐lysine to be as efficacious in ruminants as in non‐ruminant species, it would require protection against degradation in the rumen.

Figures

Molecular structure of l‐lysine monohydrochloride.

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 feed additive shall submit an application in accordance with Article 7.

The European Commission received a request from Eppen Europe SAS2 for the authorisation of the additive consisting of l‐lysine monohydrochloride produced by fermentation with Corynebacterium glutamicum CGMCC 7.453, when used as a feed additive for all animal species (category: nutritional additives; functional group: amino acids, their salts and analogues).

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 14 November 2023 and the general information and supporting documentation are available at https://open.efsa.europa.eu/questions/EFSA‐Q‐2023‐00723. The particulars and documents in support of the application were considered valid by EFSA as of 29 January 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 consisting of L‐lysine monohydrochloride produced by fermentation with Corynebacterium glutamicum CGMCC 7.453, when used under the proposed conditions of use (see Section 3.1.6).

Additional information

1.2

l‐Lysine monohydrochloride (minimum 78% lysine) produced by fermentation with C. glutamicum CGMCC 7.453 is currently not authorised in the European Union (EU). l‐Lysine produced by fermentation with different microbial strains is currently authorised for its use in animal species as nutritional additive and as sensory additive.3

The Scientific Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) has published several opinions on the safety and efficacy of l‐lysine and/or its salts produced by different production strains for all animal species.4

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 l‐lysine monohydrochloride produced by fermentation with C. glutamicum CGMCC 7.453 as a feed additive.

The confidential version of the technical dossier was subject to a target consultation of the interested Member States from 30 January 2024 to 30 April 2024; 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 from 30 May to 20 June 2024 for which no comments were received.

The FEEDAP Panel used the data provided by the applicant together with data from other sources, such as previous risk assessments by EFSA or other expert bodies, peer‐reviewed scientific papers, other scientific reports and experts' knowledge, to deliver the present output.

EFSA has verified the European Union Reference Laboratory (EURL) report as it relates to the methods used for the control of the l‐lysine monohydrochloride in animal feed.8

Methodologies

2.2

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of l‐lysine monohydrochloride produced by fermentation with C. glutamicum CGMCC 7.453 is in line with the principles laid down in Regulation (EC) No 429/20089 and the relevant guidance documents: Guidance on the assessment of the safety of feed additives for the consumer (EFSA FEEDAP Panel, 2017a), Guidance on the identity, characterisation and conditions of use of feed additives (EFSA FEEDAP Panel, 2017b), Guidance on the assessment of the safety of feed additives for the target species (EFSA FEEDAP Panel, 2017c), Guidance on the characterisation of microorganisms used as feed additives or as production organisms (EFSA FEEDAP Panel, 2018), Guidance on the assessment of the safety of feed additives for the environment (EFSA FEEDAP Panel, 2019) and Guidance on the assessment of the safety of feed additives for the users (EFSA FEEDAP Panel, 2023), EFSA statement on the requirements for whole genome sequence analysis of microorganisms intentionally used in the food chain (EFSA, 2024) and Guidance on the assessment of the efficacy of feed additives (EFSA FEEDAP Panel, 2024).

ASSESSMENT

3

The additive l‐lysine monohydrochloride (l‐lysine HCl) produced with C. glutamicum CGMCC 7.453 is intended to be used as a nutritional additive (functional group: amino acids, their salts and analogues) in feed and water for drinking for all animal species.

Characterisation

3.1

Characterisation of the production microorganism

3.1.1

The production strain is a genetically modified strain of C. glutamicum that is deposited in the China General Microbiological Culture Collection Centre (CGMCC) with accession number CGMCC 7.453.10

The taxonomic identification of the production strain CGMCC 7.453 as C. glutamicum was confirmed by ■■■■■ whole genome sequence (WGS) data, ■■■■■.11 ■■■■■. ■■■■■.

The susceptibility of the production strain to relevant antibiotics was tested against the list of antimicrobials described for ‘Corynebacterium and other Gram‐positive’ in the Guidance on the characterisation of microorganisms used as feed additives or as production organisms (EFSA FEEDAP Panel, , 2018).12 All the minimum inhibitory concentration (MIC) values were below or equal to the corresponding cut‐off specified in this guidance. Therefore, the production strain is considered susceptible to all relevant antibiotics.

The WGS data of the production strain were interrogated for the presence of antimicrobial resistance (AMR) genes ■■■■■.13 ■■■■■. Therefore, the FEEDAP Panel concludes that the strain harbours no acquired AMR genes and raises no safety concerns.

Information regarding the genetically modified microorganism

14

3.1.1.1

■■■■■.

Description of the genetic modification

■■■■■

• ■■■■■■■■■■
• ■■■■■■■■■■
• ■■■■■■■■■■

■■■■■15 ■■■■■. No safety concerns were identified in the production strain.

Manufacturing process

3.1.2

l‐Lysine HCl is produced by fermentation with C. glutamicum CGMCC 7.453. ■■■■■.16

The applicant states that no antibiotics are used in the manufacturing process.17

Characterisation of the active substance/additive

3.1.3

l‐Lysine monohydrochloride (IUPAC name: (2S)‐2,6‐diaminohexanoic acid monohydrochloride, synonym l‐lysine hydrochloride, a compound identified with the CAS No. 657‐27‐2 and the EC number 211‐519‐9), has a molecular weight of 182.65 g/mol. The theoretical content of lysine in lysine monohydrochloride is 80%. The molecular formula is C_6_H_14_N_2_O_2_·HCl and the molecular structure is given in Figure 1.

Molecular structure of l‐lysine monohydrochloride.

The product is specified to contain ≥ 78% l‐lysine on a dry matter (DM) basis, and maximum 1% moisture. Compliance with the specification was shown in five batches in which lysine was on average 79.1% (78.8%–79.3%) on as is basis.18 The average water content was 0.44% (0.38%–0.51%).19 The average content of lysine on DM basis corresponds to 79.5% (79.2%–79.7%). The average content of chloride was on average 19.3% (19.2%–19.4%).20 The amount of identified material on DM basis was on average 98.7% (98.5%–98.9%).

The specific optical rotation of five batches of l‐lysine HCl was analysed (United States Pharmacopoeia [USP] method) and ranged from +20.4 to +20.7°,21 which is within the USP reference range of +20.3 to +21.5°, confirming the presence of the l‐enantiomer.

Three batches of the additive were analysed for impurities and the concentrations of arsenic, cadmium and lead were below the limit of detection (LOD) of the analytical methods. Mercury concentrations ranged below the LOD to 0.0023 mg/kg.22 Polychlorinated dibenzo‐p‐dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and dioxin‐like polychlorinated biphenyls (DL‐PCBs) were analysed in three batches. The calculated upper bound (UB) concentrations for the sum of PCDD/Fs were below 0.50 ng world health organisation (WHO) (PCDD/F)‐TEQ 88% DM/kg, and between 0.55 and 0.62 ng WHO (PCDD/F/PCB)‐TEQ 88% DM/kg the sum of PCDD/Fs and DL‐PCBs. The UB for the sum of non‐DL‐PCBs was 3 μg/kg.23

The analysis of mycotoxins, including aflatoxins (not specified), zearalenone, ochratoxin A, fumonisins (B1 + B2 + B3), deoxynivalenol and citrinin showed values below the LOD except for total aflatoxins (1.5–3 μg/kg), fumonisins (58.7–61.2 μg/kg) and citrinin (34.4–90.7 μg/kg).24

Microbiological contamination was analysed by determination of Enterobacteriaceae, Salmonella spp., yeasts and moulds and Escherichia coli with no detection in 25 g samples.25

The FEEDAP Panel considers that the amounts of the detected impurities do not raise safety concerns.

The presence of viable cells of the production strain was tested in three batches of the final product l‐lysine HCl, each batch tested in triplicate.26 ■■■■■ No viable cells of the production strain were found in the samples tested.

The presence of recombinant DNA from the production strain was analysed ■■■■■.27 ■■■■■ No DNA from the production strain was detected.

Physical properties of the additive

3.1.4

Solubility in water is reported to be 649.5 g/L.28

The dusting potential of 3 batches of the additive was determined using the Stauber‐Heubach method and showed values in the range 3400–3700 mg/m^3^.

Stability and homogeneity

3.1.5

No information is available on the shelf‐life, stability and capacity of the additive to distribute homogeneously in feed. The applicant provided information on the shelf‐life, stability in premixtures, feed and water for drinking, and on the capacity to distribute homogeneously in feed of a l‐lysine HCl of the same characteristics produced by fermentation with a different production strain (EFSA FEEDAP Panel, 2020). The producer of both l‐lysine HCl products and the manufacturing process is the same. The FEEDAP Panel considers that the outcome of the shelf‐life, stability and homogeneity studies of the l‐lysine HCl reported in the previous opinion are applicable to the shelf‐life, stability and capacity to distribute homogeneously of the product under assessment.

Conditions of use

3.1.6

The additive is intended to be used in feed for all species and can be added directly in compound feed, through complementary feed or via premixtures. It is also intended for use in water for drinking in all animal species. No proposed inclusion levels are provided, as the optimal daily allowance in quantitative terms depends on the nutrient composition, in particular the amino acid composition of the unsupplemented diet, the species, the animal's age, the physiological state of the animal, the performance level of the animal and the environmental conditions.

Safety

3.2

Safety of the production organism

3.2.1

The production strain C. glutamicum CGMCC 7.453 is a genetically modified strain developed to increase the production of l‐lysine. The production strain belongs to a species, C. glutamicum that is suitable for the qualified presumption of safety (QPS) approach to safety assessment when used for production purposes (EFSA BIOHAZ panel, 2023). The taxonomic identification of the production strain was unequivocally established, it does not carry acquired AMR genes and the genetic modification does not raise safety concerns. The production strain and its DNA were not detected in the final product. Therefore, the final product does not pose any safety concern as regards the genetically modified production strain.

Safety for the target species, consumers and the environment

3.2.2

Safety concerns on the use of the additive would not derive from the l‐lysine, which is considered safe but may arise from residues of the fermentation process/production strain remaining in the final product. The final product is highly purified (≥ 78% lysine and an average of 98.7% identified material on a DM basis). The additive is produced by fermentation using a strain identified as C. glutamicum which qualifies for the QPS safety assessment approach and no safety concerns were identified for the production strain (see Section 3.2.1), the fermentation process and its residues/metabolites. The ingredients of the fermentation medium do not raise safety concerns. Consequently, no safety concerns for target animals, consumers and the environment are expected from the additive concerning potential fermentation residues that may be present in the final additive.

The l‐lysine requirements of different non‐ruminant species and animal categories, the absorption and metabolic fate of l‐lysine, the tolerance to l‐lysine excess and the lysine to arginine antagonism are well known and described in the literature. The Panel considers that no safety concerns for ruminants would arise from ruminal lysine metabolism. The use of the amino acid ‘per se’ will not raise safety concerns for the target animals provided it is supplemented in appropriate amounts to the diets. However, due to the risk of nutritional imbalances and hygienic reasons, associated to the use of amino acids via water for drinking (EFSA FEEDAP Panel, 2010), the FEEDAP Panel has concerns on the safety of the use of the amino acid via water for drinking.

The absorption, distribution, metabolism and excretion of l‐lysine are well described in the scientific literature. The use of the additive under assessment in animal nutrition is considered safe for consumers.

The amino acid l‐lysine is a physiological and natural component of animals and plants. It is not excreted as such (but as urea/uric acid and carbon dioxide). The use of amino acids in water for drinking, when given in addition to complete diets with a well‐balanced amino acid profile, would disturb the nitrogen balance and increase nitrogen excretion via urine. The use of l‐lysine in animal nutrition would not lead to any localised increase in the concentration of l‐lysine or its metabolites in the environment.

Conclusions on the safety for the target species, consumers and the environment

3.2.2.1

The FEEDAP Panel concludes that l‐lysine HCl produced by fermentation with C. glutamicum CGMCC 7.453 is safe for the target species, for the consumer and the environment. The FEEDAP Panel has concerns on the use of l‐lysine HCl in water for drinking.

Safety for the user

3.2.3

No specific studies were submitted to support the safety for the user.

Based on the highest dusting potential measured value (3700 mg/m^3^), the FEEDAP Panel considers that the exposure of users through inhalation is likely.

Based on the information present in the safety data sheet,29 the additive is to be considered as an irritant to the skin, eyes and the respiratory tract, and therefore, any exposure is a risk.

Efficacy

3.3

Efficacy studies are not required for amino acids naturally occurring in proteins of plants and animals. The nutritional role of the amino acid l‐lysine is well established in the scientific literature. In general, the product l‐lysine HCl is considered as an efficacious source of the essential amino acid l‐lysine for non‐ruminant animal species. For the supplemental l‐lysine to be as efficacious in ruminants as in non‐ruminant species, it would require protection against degradation in the rumen.

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 Regulation30 and Good Manufacturing Practice.

CONCLUSIONS

4

The production strain C. glutamicum CGMCC 7.453 does not pose any safety concern as regards the genetic modifications. No viable cells or DNA of the production strain were detected in the final product. Therefore, the FEEDAP Panel concludes that the additive does not pose any safety concern regarding the production strain.

l‐Lysine HCl produced with C. glutamicum CGMCC 7.453 is considered safe for the target species when administered via feed. FEEDAP Panel has concerns on the use of l‐lysine HCl in water for drinking.

The use of l‐lysine HCl produced by fermentation with C. glutamicum CGMCC 7.453 in animal nutrition is considered safe for the consumers and for the environment.

With regards user safety, the additive should be considered irritant to skin, eyes and the respiratory tract. Any exposure to the additive is a risk.

l‐Lysine HCl is considered an efficacious source of the essential amino acid l‐lysine for non‐ruminant animal species. For the supplemental l‐lysine to be as efficacious in ruminants as in non‐ruminant species, it would require protection against degradation in the rumen.

ABBREVIATIONSAMRantimicrobial resistanceANIaverage nucleotide identityCASChemical Abstracts ServiceCFUcolony forming unitCVcoefficient of variationDMdry matterDL‐PCBsdioxin‐like polychlorinated biphenylsEINECSEuropean Inventory of Existing Chemical SubstancesEURLEuropean Union Reference LaboratoryFEEDAPEFSA Scientific Panel on Additives and Products or Substances used in Animal FeedIUPACInternational Union of Pure and Applied ChemistryLODlimit of detectionLOQlimit of quantificationMCEmixed cellulose estersMICminimum inhibitory concentrationOECDOrganisation for Economic Co‐operation and DevelopmentPCDDspolychlorinated dibenzo‐p‐dioxinsPCDFspolychlorinated dibenzofuransQPSqualified presumption of safetyUBupper boundUSPUnited States PharmacopoeiaWGSwhole genome sequence

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00723

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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, and Katerina Theodoridou.

LEGAL NOTICE

Relevant information or parts of this scientific output have been blackened in accordance with the European Commission decision on the confidentiality requests formulated by the applicant and further confidentiality requests formulated by the applicant for which a decision by the European Commission is pending. The blackened text will be subject to review once the full decision on the confidentiality requests is adopted by the European Commission. The full output was shared with the European Commission, EU Member States and the applicant.