Safety and efficacy of a feed additive consisting of l‐isoleucine produced with Corynebacterium glutamicum CCTCC M 2022764 for all animal species (Innobio Europe B.V.)
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, Katerina Theodoridou
TL;DR
The paper evaluates the safety and effectiveness of l-isoleucine produced by a specific bacteria for use in animal feed.
Contribution
It confirms the safety of l-isoleucine for non-ruminant animals and highlights concerns about its use in drinking water.
Findings
The additive is safe for production and target species when used appropriately.
Concerns exist regarding its use in drinking water.
It is effective for non-ruminants but needs 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‐isoleucine produced by fermentation with a non‐genetically modified strain of Corynebacterium glutamicum (CCTCC M 2022764) as a nutritional feed additive for use in feed and water for drinking for all animal species. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the additive does not give rise to any safety concern regarding the production strain. The use of l‐isoleucine produced with C. glutamicum CCTCC M 2022764 is considered safe for the target species when supplemented in appropriate amounts to the diet according to the nutritional needs of the target species. The FEEDAP Panel has concerns on the use of l‐isoleucine in water for drinking. The use of l‐isoleucine produced with C. glutamicum CCTCC M…
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FIGURE 1|
| |
|
| ≥ 90 |
|
| ≤ 2 |
| Other amino acids (% DM) | ≤ 2 |
| Loss on drying (%) | ≤ 3 |
| Specific optical rotation (°) | +38.9° to +41.8° |
|
| [5] |
|
| 95.5 (95.4–95.6) |
|
| 1.11 (1.04–1.16) |
| Other amino acids (% DM) | 1.84 (1.81–1.87) |
| Loss on drying (%) | 1.26 (1.18–1.32) |
| Specific optical rotation (°) | 39.8 (39.4–40.3) |
|
| [3] |
| Lead (mg/kg) | < 0.01 |
| Mercury (mg/kg) | < 0.002 |
| Cadmium (mg/kg) | < 0.002 |
| Arsenic (mg/kg) | < 0.01–0.01 |
| Dioxins and furans (upper bound) | [3] |
| PCDD/Fs (ng WHO2005‐TEQ/kg) | 0.121 |
| PCDD/Fs + PCBs (ng WHO2005‐TEQ/kg) | 0.238 |
| nDL‐PCBs (μg/kg) | 3.0 |
| Mycotoxins (μg/kg) | [3] |
| Fumonisins (B1 + B2 + B3) | < 25 |
| Aflatoxins | 0.84–1.47 |
| Ochratoxin A | < 2.8 |
| Deoxynivalenol | < 134 |
| Citrinin | < 15 |
| Zearalenone | < 17 |
|
| [5] |
|
| Not detected |
|
| < 3 |
|
| Not detected |
| Yeast and moulds (CFU/g) | < 100 |
|
| |
| Physical form | Solid |
| Bulk density (kg/m3) | [5] |
| 550–580 | |
| Solubility (g/L) | 22.3 |
| Dusting potential (Stauber Heubach) (g/m3) | [3] |
| 53.7–62.3 | |
|
| |
|
| [3] |
| Room temperature, 6 months | 0–0.6 |
|
| [3] |
| Vitamin‐mineral premix, room temperature, 3 months | 0 |
|
| |
| Chickens for fattening | [3] |
| Mash – Room temperature, 3 months | 0 |
| Pelleted – Room temperature, 3 months | 0 |
|
| [3] |
| 20°C, 48 h | 5 |
|
| |
| Chickens for fattening | [1] |
| Pelleted feed, total isoleucine | 4.3 |
| Pelleted feed, free isoleucine | 23.1 |
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Taxonomy
TopicsAgricultural safety and regulations · Food Allergy and Anaphylaxis Research · Coccidia and coccidiosis research
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 Innobio Europe B.V.2 for the authorisation of the additive consisting of l‐isoleucine produced with Corynebacterium glutamicum CCTCC M 2022764, when used as a feed additive for all animal species (category: nutritional additive; 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 28 May 2024 and the general information and supporting documentation are available at https://open.efsa.europa.eu/questions/EFSA‐Q‐2024‐00316. The particulars and documents in support of the application were considered valid by EFSA as of 11 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 consisting of l‐isoleucine produced with C. glutamicum CCTCC M 2022764, when used under the proposed conditions of use (see Section 3.1.4).
Additional information
1.2
The additive consists of l‐isoleucine produced by fermentation with C. glutamicum CCTCC M 2022764. This additive is not currently authorised in the European Union. l‐isoleucine produced by fermentation with different production strains is currently authorised for use in feed for all animal species in the European Union.3
The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) issued a series of scientific opinions on the safety and efficacy of l‐isoleucine produced by fermentation using different production strains, when used in feed for all animal species as a nutritional additive (functional group: amino acids, their salts and analogues).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‐isoleucine produced by fermentation with C. glutamicum CCTCC M 2022764 as a feed additive.
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 21 March to 11 April 2025 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 16 October 2024 to 16 January 2025 for which the received comments were considered for the assessment.
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‐isoleucine in animal feed.8
Methodologies
2.2
The approach followed by the FEEDAP Panel to assess the safety and the efficacy of l‐isoleucine produced by fermentation with C. glutamicum CCTCC M 2022764 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), Guidance on the assessment of the safety of feed additives for the users (EFSA FEEDAP Panel, 2023), Guidance on the assessment of the efficacy of feed additives (EFSA FEEDAP Panel, 2024) and EFSA statement on the requirements for whole genome sequence analysis of microorganisms intentionally used in the food chain (EFSA, 2024).
ASSESSMENT
3
The product subject of this application is l‐isoleucine produced by fermentation with a non‐genetically modified strain of C. glutamicum (CCTCC M 2022764). It 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 microorganism is a non‐genetically modified strain of C. glutamicum, deposited in the China Center for Type Culture Collection (CCTCC) with the accession number CCTCC M 2022764.10 The production strain is derived from C. glutamicum CICC 21756 ■■■■■.11 The applicant provided whole genome sequence (WGS)‐based analyses to characterise the parental strain. Those included the confirmation of its taxonomic identification as C. glutamicum, absence of acquired antimicrobial resistance (AMR) genes ■■■■■.12
The identification of the production strain CCTCC M 2022764 was confirmed as C. glutamicum ■■■■■.13 ■■■■■ No plasmids were identified by screening the WGS data of the production strain.
The susceptibility of the production strain was tested against the battery of antibiotics recommended by the EFSA FEEDAP Panel (EFSA FEEDAP Panel, 2018).14 All the minimum inhibitory concentration (MIC) values were below the corresponding cut‐off values for ‘Corynebacterium and other Gram‐positive’, ■■■■■. Therefore, the production strain is considered susceptible to all relevant antibiotics ■■■■■.
The WGS data of the production strain was interrogated for the presence of AMR genes in the ■■■■■.15 The search resulted in one hit exceeding the EFSA thresholds (EFSA, 2024). Further analysis of this hit following EFSA criteria (EFSA BIOHAZ Panel, 2023a) showed that the hit is not an acquired AMR gene. Therefore, the FEEDAP Panel concludes that the strain harbours no acquired AMR genes.
■■■■■.16 Since no acquired AMR genes were found in the WGS data, this resistance does not raise safety concerns.
Manufacturing process
3.1.2
l‐Isoleucine is produced by fermentation with C. glutamicum CCTCC M 2022764.17 ■■■■■.
The applicant stated that no antimicrobial substances are used in the manufacturing process.18
Characterisation of the additive
3.1.3
l‐Isoleucine [International Union of Pure and Applied Chemistry (IUPAC) name: (2S,3S)‐2‐amino‐3‐methylpentanoic acid], a compound identified by Chemical Abstracts Service (CAS) No 73‐32‐5 and European Inventory of Existing Commercial Chemical Substances (EINECS) No 200‐798‐2, has a molecular weight of 131.17 g/mol; the molecular formula is C_6_H_13_NO_2_ and its structural formula is given in Figure 1.
Structural formula of l‐isoleucine.
The additive is specified to contain ≥ 90% l‐isoleucine on dry matter (DM) basis.19 It is also specified that the l‐leucine content should be ≤ 2% and water ≤ 3%.
The data provided by the applicant on the batch‐to‐batch variation,20 specific optical rotation,21 impurities22 and physicochemical and technological properties23 of the additive are reported in Table 1.
Based on the above analytical results, the FEEDAP Panel notes that 98.4% of the additive on DM basis has been characterised (sum of total amino acids).
The data provided showed compliance of the batches tested with the specifications proposed by the applicant. The FEEDAP Panel considers that the microbial contamination and the amounts of the detected impurities do not raise safety concerns.
The presence of viable cells of the production strain was investigated in three batches of the product, each batch tested in triplicate (1 g per sample).24 Methodology, incubation conditions and controls were performed according to the requirements set by the FEEDAP Panel (EFSA FEEDAP Panel, 2018). No viable cells of the production strain were detected.
The presence of DNA from the production strain was investigated by PCR in three batches of the product, each batch tested in triplicate (1 g per sample).25 ■■■■■ The limit of detection in samples spiked with total DNA of the production strain was 1 ng/g of additive. No DNA of the production strain was detected.
Conditions of use
3.1.4
The additive under assessment is proposed to be used in feeds to achieve the adequate amino acid profile and meet the requirements on l‐isoleucine for all animal species.26 It can be used for all species through direct addition to feed, through complementary feed, through premixtures or water, without special requirements, without maximum or minimum levels and without withdrawal period.
No inclusion levels are proposed, as the requirements in quantitative terms depend on the species, the physiological state of the animal, the performance level and the environmental conditions, as well as the amino acid composition of the unsupplemented diet.
Safety
3.2
Safety of the production microorganism
3.2.1
The production strain CCTCC 2022764 belongs to a species, C. glutamicum, that is included in the qualified presumption of safety (QPS) list when used for production purposes (EFSA BIOHAZ Panel, 2023b). The strain was unambiguously identified as C. glutamicum and was shown to be phenotypically susceptible to all relevant antibiotics, ■■■■■. Since no acquired AMR determinants for therapeutic antimicrobials were identified, this resistance is considered of no concern. Additionally, no viable cells nor DNA of the production strain were found in the final product. Therefore, it can be concluded that the additive does not pose any safety concern regarding the production strain.
Safety for the target species, consumers and the environment
3.2.2
The l‐isoleucine requirements of the target animal species and the safety of this essential amino acid in non‐ruminant and ruminant nutrition are well known by feed formulators and available in general publications on animal nutrition.
Concerns on the use of the additive would not derive from the l‐isoleucine, which is considered safe, but may arise from residues of the fermentation process/production strain remaining in the final product. The additive is produced by fermentation with a non‐genetically modified strain of C. glutamicum (CCTCC M 2022764) and no safety concerns were identified for the production strain (see Section 3.2.1), its residues/metabolites and the fermentation process. Moreover, the resulting product consists of ≥ 90% l‐isoleucine (95.4%–95.6%) and about 1.6% unidentified material on a DM basis.
l‐Isoleucine, produced with C. glutamicum CCTCC M 2022764, is safe for the target species when used to supplement the diet in appropriate amounts to satisfy the animal requirements. However, the FEEDAP Panel reiterates its statement on the safety of the use of amino acids in water for drinking (EFSA FEEDAP Panel, 2010), for hygienic reasons and for the risk of nutritional imbalances when amino acids are administered simultaneously in feed and in water for drinking.
The absorption and metabolic fate of l‐isoleucine in the organism are well known. The amino acid l‐isoleucine, supplemented to feed, will be incorporated into proteins of tissues and/or products of animal origin and any of its potential excess will be metabolised and excreted. Therefore, the composition of tissues and products of animal origin will not be affected using l‐isoleucine in animal nutrition. Therefore, the Panel considers that the use of the additive in animal nutrition is safe for the consumer.
The amino acid l‐isoleucine 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 the product l‐isoleucine in animal nutrition would not lead to any localised increase in the concentration in the environment. The use of the additive 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. It is concluded that the use of the product, l‐isoleucine produced by fermentation with C. glutamicum CCTCC M 2022764 as a feed additive does not represent a risk to the environment.
Safety for the user
3.2.3
No specific information was submitted.27 In the absence of data, the FEEDAP Panel is not in the position to conclude on the potential of the additive to be irritant to skin or eyes, or on its potential to be a dermal sensitiser.
Efficacy
3.3
Efficacy studies are not required for amino acids that occur naturally in plant and animal proteins. The nutritional role of the amino acid l‐isoleucine is well established in the scientific literature. l‐Isoleucine, produced by fermentation with C. glutamicum CCTCC M 2022764, is regarded as an efficacious source of the essential amino acid l‐isoleucine for non‐ruminant nutrition. For supplemental l‐isoleucine 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 Regulation28 and Good Manufacturing Practice.
CONCLUSIONS
4
The use of l‐isoleucine produced by fermentation with C. glutamicum CCTCC M 2022764 in feed is safe for the target species, when supplemented in appropriate amounts to the diet according to the nutritional needs of the target species. The FEEDAP Panel has concerns on the use of l‐isoleucine in water for drinking.
The use of l‐isoleucine produced by fermentation with C. glutamicum CCTCC M 2022764 in animal nutrition is considered safe for the consumer and for the environment.
Regarding the user safety, the FEEDAP Panel cannot conclude on the potential of the additive to be irritant to skin and/or eyes and to be a potential skin sensitiser.
The feed additive consisting of l‐isoleucine produced by fermentation with C. glutamicum CCTCC M 2022764 is regarded as an effective source of the amino acid l‐isoleucine for all non‐ruminant species. In order to be as efficacious in ruminants as in non‐ruminants, it should be protected from ruminal degradation.
ABBREVIATIONSAMRantimicrobial resistanceANIaverage nucleotide identityCFUcolony forming unitCVcoefficient of variationDL‐PCBsdioxin‐like polychlorinated biphenylsDMdry matterEURLEuropean Union Reference LaboratoryFEEDAPEFSA Scientific Panel on Additives and Products or Substances used in Animal FeedMICminimum inhibitory concentrationnDL‐PCBsnon‐dioxin‐like PCBsPCBspolychlorinated biphenylsPCDDspolychlorinated dibenzo‐p‐dioxinsPCDFspolychlorinated dibenzofuransQPSqualified presumption of safetyTEQtoxic equivalent factors for dioxins, furans and dioxin‐like PCBsWGSwhole genome sequence
REQUESTOR
European Commission
QUESTION NUMBER
EFSA‐Q‐2024‐00316
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 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.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
- 1EFSA (European Food Safety Authority) . (2024). EFSA statement on the requirements for whole genome sequence analysis of microorganisms intentionally used in the food chain. EFSA Journal, 22(8), 8912. 10.2903/j.efsa.2024.8912 PMC 1131780639135845 · doi ↗ · pubmed ↗
- 2EFSA BIOHAZ Panel (EFSA Panel on Biological Hazards) , Koutsoumanis, K. , Allende, A. , Alvarez‐Ordóñez, A. , Bolton, D. , Bover‐Cid, S. , Chemaly, M. , De Cesare, A. , Hilbert, F. , Lindqvist, R. , Nauta, M. , Nonno, R. , Peixe, L. , Ru, G. , Simmons, M. , Skandamis, P. , Suffredini, E. , Cocconcelli, P. S. , Suarez, J. E. , … Herman, L. (2023 a). Statement on how to interpret the QPS qualification on ‘acquired antimicrobial resistance genes’. EFSA Journal, 21(10), 1–13. 10.2 · doi ↗ · pubmed ↗
- 3EFSA BIOHAZ Panel (EFSA Panel on Biological Hazards) , Koutsoumanis, K. , Allende, A. , Álvarez‐Ordóñez, A. , Bolton, D. , Bover‐Cid, S. , Chemaly, M. , De Cesare, A. , Hilbert, F. , Lindqvist, R. , Nauta, M. , Peixe, L. , Ru, G. , Simmons, M. , Skandamis, P. , Suffredini, E. , Cocconcelli, P. S. , Fernández Escámez, P. S. , Prieto Maradona, M. , … Herman, L. (2023 b). Scientific opinion on the update of the list of qualified presumption of safety (QPS) recommended microorgani · doi ↗
- 4EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances Used in Animal Feed) , Aquilina, G. , Bories, G. , Brantom, P. , Chesson, A. , Cocconcelli, P. S. , de Knecht, J., Dierick, N. A. , Gralak, M. A. , Gropp, J. , Halle, I. , Kroker, R. , Leng, L. , Haldorsen, A. K. L. , Mantovani, A. , Mézes, M. , Renshaw, D. , & Saarela, M. (2010). Scientific Opinion on the use of feed additives authorised/applied for use in feed when supplied via water. EFSA Journal, 8(12), 1 · doi ↗
- 5EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed) , Rychen, G. , Aquilina, G. , Azimonti, G. , Bampidis, V. , Bastos, M. d. L. , Bories, G. , Chesson, A. , Cocconcelli, P. S. , Flachowsky, G. , Gropp, J. , Kolar, B. , Kouba, M. , López‐Alonso, M. , López Puente, S. , Mantovani, A. , Mayo, B. , Ramos, F. , & Innocenti, M. L. (2017 a). Guidance on the assessment of the safety of feed additives for the consumer. EFSA Journal, 15(10), 5022. · doi ↗
- 6EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed) , Rychen, G. , Aquilina, G. , Azimonti, G. , Bampidis, V. , Bastos, M. d. L. , Bories, G. , Chesson, A. , Cocconcelli, P. S. , Flachowsky, G. , Gropp, J. , Kolar, B. , Kouba, M. , López‐Alonso, M. , López Puente, S. , Mantovani, A. , Mayo, B. , Ramos, F. , Saarela, M. , … Innocenti, M. L. (2017 b). Guidance on the identity, characterisation and conditions of use of feed additives. EFSA J · doi ↗ · pubmed ↗
- 7EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed) , Rychen, G. , Aquilina, G. , Azimonti, G. , Bampidis, V. , Bastos, M. d. L. , Bories, G. , Chesson, A. , Cocconcelli, P. S. , Flachowsky, G. , Gropp, J. , Kolar, B. , Kouba, M. , López‐Alonso, M. , López Puente, S. , Mantovani, A. , Mayo, B. , Ramos, F. , Saarela, M. , … Martino, L. (2017 c). Guidance on the assessment of the safety of feed additives for the target species. EFSA Journal · doi ↗ · pubmed ↗
- 8EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed) , Rychen, G. , Aquilina, G. , Azimonti, G. , Bampidis, V. , Bastos, M. d. L. , Bories, G. , Chesson, A. , Cocconcelli, P. S. , Flachowsky, G. , Gropp, J. , Kolar, B. , Kouba, M. , López‐Alonso, M. , López Puente, S. , Mantovani, A. , Mayo, B. , Ramos, F. , Saarela, M. , … Galobart, J. (2018). Guidance on the characterisation of microorganisms used as feed additives or as production orga · doi ↗ · pubmed ↗