Safety and efficacy of a feed additive consisting of l‐lysine sulfate produced using Corynebacterium glutamicum KCCM 80368 for all animal species (CJ Europe GmbH)

TL;DR

The paper evaluates the safety and effectiveness of l-lysine sulfate produced using a genetically modified bacterium for use in animal feed.

Contribution

It confirms the safety of the genetically modified strain and its use in feed for all animal species.

Findings

The additive is safe for target species, consumers, and the environment.

The additive is a safe and effective source of l-lysine for non-ruminant animals.

It should be protected from ruminal degradation to be effective in 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 sulfate produced by fermentation using the genetically modified strain of Corynebacterium glutamicum, KCCM 80368, as a nutritional additive in feed for all animal species and categories. In the manufacturing process used to obtain the product under assessment, the cells from the production strain are inactivated but not removed from the final product. The FEEDAP Panel concluded that the additive does not give rise to any safety concern regarding the genetic modification of the strain. DNA from the strain is present in the final product, but its presence does not raise any safety issue since the strain does not harbour any gene of concern. No viable cells of the strain were detected in the final product. The use of l‐lysine sulfate produced using C. glutamicum KCCM 80368 in feed is safe for the target species when supplemented in appropriate amounts to the diet according to the nutritional needs of the species. The use of the additive in animal nutrition is considered safe for the consumers of products from animals receiving the additive, and to the environment. With regard to the safety for the user, the additive is not irritant to the skin, but no conclusions could be reached on the eye irritation potential. The additive should be considered as a skin and respiratory sensitiser. Exposure by inhalation and dermal routes is considered a risk. The additive l‐lysine sulfate produced using C. glutamicum KCCM 80368 is regarded as an effective source of the amino acid l‐lysine for all non‐ruminant species. To be as efficacious in ruminants as in non‐ruminants, it should be protected from ruminal degradation.

Figures

Molecular structure of l‐lysine sulfate.

Tables

TABLE 1. Results of analysis to determine the composition of the additive (% as is, five batches).

	Average	Range
Lysine (free, %)	55.4	53.4–56.1
Sulfate (%)	20.4	19.7–21.5
Lysine sulfate (calculated, %)	76.2	75.8–77.0
Moisture (%)	2.4	2.3–2.7
Ash (%)	2.8	2.6–3.6
Nitrogen (%)	10.8	10.6–11
Crude fat (%)	0.4	0.4–0.5
Crude fibre (%)	< 0.5
Starch (%)	3.7	3.4–4.3
Total sugars 1 (%)	1.6	1.0–2.1
Ammonium nitrogen (mg/kg)	5798	2580–11,100

TABLE 2. Results of total amino acid profile, mineral content and trace elements of the additive (% as is, five batches).

	Average	Range
Amino acids
Lysine 1	55.4	55.0–55.7
Tryptophan	0.08	0.06–0.09
Methionine	0.09	0.05–0.12
Cystine	0.06	0.05 2 – 0.07
Threonine	0.26	0.19–0.33
Serine	0.21	0.15–0.27
Glutamic acid	1.12	0.90–1.28
Proline	0.24	0.19–0.31
Glycine	0.34	0.28–0.40
Alanine	0.79	0.75–0.83
Valine	0.41	0.31–0.51
Isoleucine	0.30	0.24–0.36
Leucine	0.52	0.40–0.63
Tyrosine	0.15	0.11–0.18
Phenylalanine	0.25	0.20–0.29
Histidine	0.15	0.11–0.18
Arginine	0.38	0.29–0.45
Organic acids (%)
Citric acid	0.05	0.04–0.07
Malic acid	0.17	0.14–0.26
Succinic acid	0.18	0.12–0.28
Lactic acid	1.44	1.37–1.53
Formic acid	< 0.006
Acetic acid	0.04	0.02–0.05
Minerals and trace elements 3
Chloride	0.23	0.22–0.24
Calcium	0.08	0.05–0.13
Phosphorous	0.17	0.16–0.18
Phosphate 4	0.53	0.50–0.56
Sodium	0.18	0.15–0.20
Potassium	0.65	0.60–0.74
Sulfur	6.48	6.01–6.99
Copper		0.000064–0.000095
Iron		0.003044–0.003314
Zinc		0.000196–0.000711
Manganese		0.059922–0.112257
Magnesium		0.0010415–0.0013629

TABLE 3. Data on the impurities and physical properties of l‐lysine sulfate produced using C. glutamicum KCCM 80368. The data presented are ranges for all parameters.

Parameter	Range	No. of batches
Impurities
Lead (mg/kg)	< 0.015–0.019	3
Mercury (mg/kg)	< 0.010	3
Cadmium (mg/kg)	< 0.010	3
Arsenic (mg/kg)	< 0.04	3
Dioxins and furans (upper bound) 1
PCDD/Fs + PCBs (ng WHO2005‐TEQ/kg)	0.0702	3
Mycotoxins (μg/kg)
Aflatoxins B1, B2, G1, G2	< 0.2	3
Ochratoxin A	< 0.5	3
Deoxynivalenol	< 10.0	3
Zearalenone	< 5.0	3
Toxins HT‐2, T‐2	< 2.0	3
■■■■■
■■■■■	■■■■■	4
■■■■■	■■■■■	4
■■■■■	■■■■■	4
■■■■■	■■■■■	4
■■■■■	■■■■■	4
■■■■■
■■■■■	■■■■■	4
■■■■■	■■■■■	4
■■■■■	■■■■■	4
■■■■■	■■■■■	4
■■■■■	■■■■■	4
■■■■■	■■■■■	4
■■■■■	■■■■■	4
■■■■■	■■■■■	4
■■■■■	< ■■■■■ 2	3
Microbial contamination
Salmonella spp. (per 25 g)	Not detected	3
Enterobacteriaceae (per 10 g)	Not detected	3
Escherichia coli (per 10 g)	Not detected	3
Yeast and moulds (CFU/g)	< 100	3
Physical properties
Physical form	Light to dark brown granules
Bulk density (kg/m3)	552–574	3
Dusting potential (Stauber Heubach) (mg/m3)	115–320	3
Particle size distribution (laser diffraction) (% of particles below, v/v) 100 μm	0.0	3
Shelf‐life (% losses)
25°C/60% RH 6 months	0.0	3
40°C/75% RH 6 months	0.0	3
Stability in premixtures (% losses)
Chickens for fattening premix, 25°C/60% RH, 6 months	0.0–6.4	3
Stability in feed (% losses)
Chicken for fattening, mash, 25°C/60% RH, 3 months	4.1–9.5	3
Chicken for fattening, pellet, 25°C/60% RH, 3 months	0.0–12.6	3
Pelleting 70–75°C	0.0–11.8
Homogeneity (coefficient of variation, %)
Chickens for fattening premix	7	1
Chicken for fattening mash feed	9	1
Chicken for fattening pelleted feed	4	1

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 CJ Europe GmbH2 for the authorisation of the additive consisting of l‐lysine sulfate produced using the genetically modified strain of Corynebacterium glutamicum, KCCM 80368, 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 11 July 2023 and the general information and supporting documentation are available at https://open.efsa.europa.eu/questions/EFSA‐Q‐2023‐00484. The particulars and documents in support of the application were considered valid by EFSA as of 27 November 2023.

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 sulfate produced using C. glutamicum KCCM 80368, when used under the proposed conditions of use (see Section 3.1.4).

Additional information

1.2

The additive consists of a l‐lysine sulfate produced using the genetically modified C. glutamicum KCCM 80368, intended to be used as a nutritional additive (functional group: amino acids, their salts and analogues) in feed for all animal species. It has not been previously authorised as a feed additive in the European Union.

DATA AND METHODOLOGIES

2

Data

2.1

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 l‐lysine sulfate produced using C. glutamicum KCCM 80368 as a feed additive.

In accordance with Article 38 of the Regulation (EC) No 178/20024 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,5 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 13 May to 3 June 2024 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 27 November 2023 to 27 February 2024; the comments received 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‐lysine sulfate produced using C. glutamicum KCCM 80368 in animal feed.6

Methodologies

2.2

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of l‐lysine sulfate produced using C. glutamicum KCCM 80368 is in line with the principles laid down in Regulation (EC) No 429/20087 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), 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 subject of the assessment is a l‐lysine sulfate produced using the genetically modified C. glutamicum KCCM 80368. In the manufacturing process used to obtain the product under assessment, the cells of the production strain are inactivated but not removed from the final product. The additive is intended to be used as a nutritional additive (functional group: amino acids, their salts and analogues) in feed for all animal species.

Characterisation

3.1

Manufacturing process

3.1.1

l‐Lysine sulfate is produced by fermentation using C. glutamicum KCCM 80368.8 ■■■■■.9 ■■■■■ ■■■■■.

The applicant declared that no antimicrobials are used in the manufacturing process.10

Characterisation of the strain C. glutamicum

KCCM 80368

3.1.2

l‐Lysine sulfate is produced using a genetically modified strain of C. glutamicum, which is deposited at the Korean Culture Collection of Microorganisms (KCCM) with the accession number KCCM 80368.11

The taxonomic identification of KCCM 80368 was confirmed by ■■■■■ analysis of the whole genome sequence (WGS) ■■■■■.12 The results were further confirmed ■■■■■ which showed that the closest genomes belong to the C. glutamicum species.

■■■■■.13

The susceptibility of KCCM 80368 to relevant antibiotics was tested against the list of antimicrobials described for ‘Corynebacterium and other Gram‐positive’ recommended by the FEEDAP Panel (EFSA FEEDAP Panel, 2018).14 All measured minimum inhibitory concentrations were equal to or fell below the cut off values and, therefore, the strain is considered susceptible to the relevant antibiotics.

The WGS data of the strain were searched for the presence of antimicrobial resistance (AMR) genes ■■■■■. The search resulted in ■■■■■ the EFSA thresholds (EFSA, 2024), ■■■■■.15 Further analysis16 ■■■■■ following EFSA's criteria (EFSA BIOHAZ Panel, 2023; EFSA BIOHAZ Panel, 2023) ■■■■■ and therefore, it can be concluded that no acquired AMR genes were identified and the strain raises no concerns.

Information related to the genetically modified microorganism

3.1.2.1

Characterisation of the recipient or parental microorganism

■■■■■.17

Description of the genetic modification

■■■■■18.

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

■■■■■. All the genetic modifications, including the intended and unintended ones, were reported and no concerns were identified.19

■■■■■ (see Section 3.1.2).

In the context of this application, C. glutamicum KCCM 80368 is used for the production of a L‐lysine sulfate for which the cells of the production strain are inactivated but not removed from the final product. The FEEDAP Panel considers that the stability of the genetic traits as well as their rate and level of expression are not relevant for a product with non‐viable cells. The level of expression of the new genetic trait is only relevant with respect to the l‐lysine concentration in the final product, as described in Section 3.1.3.

Characterisation of the additive

3.1.3

l‐Lysine sulfate (International Union of Pure and Applied Chemistry [IUPAC] name: (2S)‐2,6‐diaminohexanoic acid;sulfuric acid), a compound identified by Chemical Abstracts Service [CAS] No 60343‐69‐3, has a molecular weight of 390.4 g/mol; the molecular formula is C_12_H_28_N_4_O_4_‐H_2_SO_4_ and the molecular structure is given in Figure 1. The theoretical content of lysine in the pure lysine sulfate is 75%.

Molecular structure of l‐lysine sulfate.

The additive is a l‐lysine sulfate produced using C. glutamicum KCCM 80368, for which the cells of the production strain are inactivated but not removed from the final product, and it is specified to contain ≥ 70% l‐lysine sulfate, ≥ 55% l‐lysine and ≤ 6.0% moisture on dry matter (DM) basis.20 Table 1 shows the results of the different compositional analysis, and Table 2 shows the amino acid profile, organic acids, mineral content and trace elements in five independent batches of the additive.21

The results showed an average of 78.1% l‐lysine sulfate (77.6%–78.8%) and an average of 57.2% l‐lysine (56.8%–57.4%) on DM basis.

The chiral purity of l‐lysine sulfate was analysed ■■■■■22

The data provided showed compliance with the specifications set by the applicant. Based on the above analytical results, the FEEDAP Panel notes that 93.2% (as is basis) of the additive has been characterised (sum of total amino acids, sulfate, moisture, ash, fat, fibre, starch and total sugars).

The data provided by the applicant on the impurities23 and physical properties24 of the additive are reported in Table 3. Magnesium and the trace elements (copper, iron, zinc, manganese) used in the manufacturing were also analysed (Table 2).

The FEEDAP Panel notes the high levels of ■■■■■, which deserve attention/monitoring during the manufacturing process. The FEEDAP Panel considers that the microbial contamination and the amounts of the detected impurities do not raise safety concerns. This includes also the residues of the ■■■■■, for which, taking into account its nature (and high limit of detection of the analytical method), a separate assessment is done in Appendix A.

The presence of viable cells of the strain was investigated in three batches of the final product, each batch tested in triplicate.25 ■■■■■. Therefore, no viable cells of the strain were detected.

The presence of DNA of the strain was investigated in three batches of the final product tested in triplicate.26 ■■■■■. DNA from the strain was detected in ■■■■■ samples tested.

Conditions of use

3.1.4

l‐Lysine sulfate produced using C. glutamicum KCCM 80368 is intended to be used in feed for all animal species, directly or through complementary feed or premixtures. No inclusion levels have been proposed, as the requirements of l‐lysine, in quantitative terms, depend on the nutrient composition, particularly the amino acid composition of the un‐supplemented diet, the species, the age of the animal, the physiological state of the animal, the performance level and the environmental conditions.

Safety

3.2

Safety of C. glutamicum

KCCM 80368

3.2.1

C. glutamicum KCCM 80368 is a genetically modified strain developed to increase the production of l‐lysine. The strain 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, 2023a, 2023b). For the product under assessment, the qualifications are the safety of the genetic modification, the absence of acquired AMR genes and the absence of viable cells of the strain in the final product. The taxonomic identification of the strain KCCM 80368 was unequivocally established, the genetic modifications do not raise safety concerns and no acquired AMR genes were found. Additionally, in the current assessment, viable cells were not detected in the final product. Therefore, the strain fulfils the requirements for the QPS approach. DNA from the strain is present in the final product, but its presence does not raise any safety issue since the strain does not harbour any gene of concern. Consequently, the FEEDAP Panel concludes that the additive does not pose any safety concern regarding the genetically modified strain C. glutamicum KCCM 80368.

Toxicological studies

3.2.2

The applicant provided toxicological studies with the product under assessment.

Genotoxicity studies, including mutagenicity

3.2.2.1

Bacterial reverse mutation test

3.2.2.1.1

In order to investigate the potential of l‐lysine sulfate produced using C. glutamicum KCCM 80368 to induce gene mutations in bacteria, an Ames test was performed according to the OECD Testing Guideline (TG) 471, and claimed Good Laboratory Practice (GLP) compliant.27 The experiments were done using strains of Salmonella Typhimurium (TA98, TA100, TA1535, TA1537) and Escherichia coli (WP2 uvrA). Two independent experiments were conducted applying the plate incorporation and pre‐incubation methods, both in the presence and absence of metabolic activation. Based on the results of a preliminary experiment, the examined test concentrations were 5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate in first and second tests, plus the concentration of 1.581 μg/plate in the second experiment. The test item was dissolved in deionised water. No precipitation nor cytotoxicity were observed. No significant increase in the number of revertant colonies was induced by the test item in any experimental condition.

The FEEDAP Panel concludes that the test item did not induce gene mutations in bacteria under the experimental conditions employed in this test.

In vitro mammalian cell micronucleus test

3.2.2.1.2

To investigate the potential of l‐lysine sulfate produced using C. glutamicum KCCM 80368 to induce chromosomal damage, an in vitro micronucleus test was performed in mouse lymphoma L5178Y TK+/− 3.7.2C cell line following the OECD TG 487 and claimed GLP compliant.28 Based on the results of a preliminary cytotoxicity experiment, the additive was tested at (i) 125, 250 and 500 μg applying a short treatment (3 + 21 h of recovery) with metabolic activation; (ii) 500, 1000 and 2000 μg/mL in a short treatment (3 + 21 h of recovery) without metabolic activation and; (iii) 187.5, 375, 1000 μg/mL in a continuous treatment (24 + 0 h recovery) without metabolic activation. Cytotoxicity up to 65%, 53% and 64%, evaluated by the analysis of the Relative Increase in Cell Counts (RICC), was observed at the top concentrations tested after short treatment in the presence of metabolic activation, short and continuous treatments without metabolic activation, respectively. No statistically significant increase in the frequency of micronucleated cells was induced by the test item compared to concurrent vehicle controls.

The FEEDAP Panel concludes that the test item did not induce chromosomal damage in vitro in mammalian cells under the experimental conditions employed in this test.

Repeated dose toxicity studies

3.2.2.2

In a 90‐day toxicity study, four groups of 10 animals/sex/group of Wistar Han IGS rats were administered l‐lysine sulfate produced using C. glutamicum KCCM 80368 by oral gavage in distilled water at dose levels of 0, 500, 1000 and 2000 mg/kg body weight (bw) per day. The highest dose corresponds to 22,222.2 mg/kg complete feed (EFSA Scientific Committee, 2012). Additional groups of five animals/sex were allocated in the control and high dose group to investigate reversibility of findings for 4 weeks. The study was conducted according to the OECD TG 408 and claimed to be GLP compliant.29

No test item‐related mortality was observed during the study. Clinical observations were limited to a dose related decrease of urine pH in males and females at 1000 and 2000 mg/kg bw per day. They were not noted thereafter during the recovery period. In the testis of a single male, at the highest dose (2000 mg/kg bw per day), spermatid retention (minimal) was observed, but considered unrelated to treatment. There were no other treatment‐related changes in any of the measurements or observations made during the study. Based upon the results of this study, the no observed adverse effect level (NOAEL) was the maximum dose tested of 2000 mg additive/kg bw per day.

Conclusions on toxicology

3.2.2.3

The FEEDAP Panel concludes that the additive does not raise concerns for genotoxicity. Moreover, the results obtained in a sub‐chronic oral toxicity study raised no concerns regarding the product.

Safety for the target species, the consumer and the environment

3.2.3

The Panel considers that safety concerns from the use of this product would not derive from the amino acids present in the additive, provided that they are supplemented at appropriate amounts to satisfy the nutritional needs of the animals. Concerns may arise from the unidentified part of the additive, which amounts to approximately 7%, and may contain residues from the fermentation. The impurities present, including those from manufacturing, were of no concern (see Section 3.1.3). The strain and its genetic modification raised no concerns (see Section 3.2.1), and the toxicological studies performed with the additive under assessment identified no concerns (see Section 3.2.2).

The l‐lysine requirements of the target animal species and the safety of this essential amino acid in non‐ruminant and ruminant nutrition are well known to feed formulators and available in general publications on animal nutrition. 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. With regard to the high intrinsic content of sulfate in l‐lysine sulfate, the FEEDAP Panel considers that the formulation of the complete feed should carefully take into account the maximum tolerable level of total sulfur (S), as established by NRC (2005), and set in ruminant diets at 3 g S/kg DM (diet rich in concentrate) or 5 g S/kg DM (diet rich in roughage); and in non‐ruminant diets at 4 g S/kg DM. Also, the contribution of sulfur/sulfate present in water for drinking to the total sulfur intake should be considered. Consequently, no negative effects are expected for the target species provided that the total sulfur intake complies with the recommendations established by scientific bodies.

The absorption and metabolic fate of l‐lysine in the organism, the tolerance to l‐lysine excess and the lysine to arginine antagonism are well known and described in scientific literature. The amino acid l‐lysine 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 by the use of supplemental l‐lysine 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‐lysine is a physiological and natural component of animals and plants. When consumed, it will be absorbed and the non‐absorbed fraction will be incorporated into the intestinal microbial mass and excreted as such. Therefore, the use of the product l‐lysine sulfate produced using C. glutamicum KCCM 80368 in animal nutrition would not lead to any localised increase in concentration in the environment. Moreover, sulfate is widely present in the terrestrial and aquatic environments (Forum of the European Geological Surveys [FOREGS] database, Salminen et al., 2005). It is a macronutrient in the marine environment, and the use of the additive will not substantially increase the natural background concentrations of sulfate in the environment.

Environmental risk assessment of horizontal gene transfer for the additive, for which the cells of the production strain are inactivated but not removed from the final product, is only needed in case genes of concern (e.g. AMR genes) are present in the strain under assessment due to potential environmental selection of the encoded trait(s) after transfer. No genes of concern (e.g. AMR genes) are present in C. glutamicum KCCM 80368; therefore, no further environmental risk assessment is needed. Therefore, the use of l‐lysine sulfate produced using C. glutamicum KCCM 80368 as a feed additive does not represent a risk to the environment.

Safety for the user

3.2.4

An acute inhalation study was performed following the OECD TG 403.30 The lethal concentration 50 (LC_50_) is more than 2.34 mg/L.

The skin irritation potential of the additive was tested in a skin irritation study performed according to OECD TG 439,31 which showed that the additive is not a skin irritant (UN GHS ‘No Category’).

The eye irritation potential of the additive was tested in a study performed according to OECD TG 437.32 According to the results of the study, an in vitro irritancy score (IVIS) of 8.5 was determined. Since the IVIS was between 3 and 55 no standalone prediction can be made; therefore, the FEEDAP Panel cannot conclude on the eye irritation properties of the additive.

The skin sensitisation potential of the additive was tested in a study performed according to OECD TG 429,33 which showed that the the additive is not a skin sensitiser (UN GHS ‘No Category’). However, 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 microbial‐based products are available. Therefore, considering the nature of the additive, it should be considered as a skin and respiratory sensitiser.

Conclusions on safety for the user

3.2.4.1

On the basis of the studies submitted, the additive, l‐lysine sulfate produced using C. glutamicum KCCM 80368 is not irritant to skin and no conclusions could be reached on the eye irritation potential. The additive should be considered as a skin and respiratory sensitiser. Exposure by inhalation and dermal routes is considered a risk.

Efficacy

3.3

Efficacy studies are not required for amino acids that occur naturally in plant and animal proteins. The nutritional role of the essential amino acid l‐lysine is well established in the scientific literature. The l‐lysine sulfate produced using C. glutamicum KCCM 80368 is regarded as an efficacious source of that amino acid 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 Regulation34 and Good Manufacturing Practice.

CONCLUSIONS

4

The additive l‐lysine sulfate is produced using the genetically modified strain C. glutamicum KCCM 80368. In the manufacturing process used to obtain the product under assessment, the cells of the production strain are inactivated but not removed from the final product. The additive was characterised up to 93.2%; therefore, the following conclusions apply only to the product obtained with the manufacturing process described in the application.

Viable cells were not detected in the additive. DNA from the strain is present in the additive and is not considered of concern. Consequently, the additive does not give rise to any safety concern regarding the genetically modified strain and the additive resulting from its fermentation.

The use of the additive in animal nutrition is considered safe for the target animals, the consumer and the environment.

With regard to the safety of users, the additive is not irritant to the skin, and no conclusions could be reached on the eye irritation potential. It should be considered a respiratory and skin sensitiser. Exposure by inhalation and dermal routes is considered a risk.

The additive is regarded as an effective source of the essential amino acid L‐lysine for all animal species. For the supplemental l‐lysine to be as efficacious in ruminants as in non‐ruminants, it should be protected from ruminal degradation.

ABBREVIATIONSAMRantimicrobial resistanceANIaverage nucleotide identityBWbody weightCASChemical Abstracts ServiceCFUcolony forming unitDMdry matterEURLEuropean Union Reference LaboratoryFEEDAPEFSA Scientific Panel on Additives and Products or Substances used in Animal FeedFOREGSForum of the European Geological SurveysGLPGood Laboratory PracticeIUPACInternational Union of Pure and Applied ChemistryIVISin vitro irritancy scoreKCCMKorean Culture Collection of MicroorganismsLC50lethal concentration 50LODlimit of detectionLOQlimit of quantificationNOAELno observed adverse effect levelNRCNational Research CouncilOECDOrganisation for Economic Co‐operation and DevelopmentPCBspolychlorinated biphenylsPCDDspolychlorinated dibenzo‐p‐dioxinsPCDFspolychlorinated dibenzofurans■■■■■■■■■■QPSqualified presumption of safetyRHrelative humidityRICCRelative Increase in Cell CountsRPDreplicative population doublingTEQstoxic equivalent factors for dioxins, furans and dioxin‐like PCBsTGTesting GuidelineUN GHSUnited Nations' Globally Harmonised System of Classification and Labelling of ChemicalsWGSwhole genome sequenceWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00484

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.