Safety of 6′‐sialyllactose (6′‐SL) sodium salt produced by a derivative strain (Escherichia coli NEO6) of E. coli W (ATCC 9637) as a Novel Food pursuant to Regulation (EU) 2015/2283

Abstract

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on 6′‐sialyllactose (6′‐SL) sodium salt as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is mainly composed of the human‐identical milk oligosaccharide (HiMO) 6′‐SL (sodium salt), but it also contains sialic acid, d‐glucose, d‐lactose, 6′‐sialyllactulose sodium salt, 3′‐sialyllactose (3′‐SL) sodium salt and a small fraction of other related saccharides. The NF is produced by fermentation by a genetically modified strain (Escherichia coli NEO6) of E. coli W (ATCC 9637). The information provided on the identity, manufacturing process, composition and specifications of the NF does not raise safety concerns. The applicant intends to add the NF to a variety of foods, including infant formula and follow‐on formula, food for special medical purposes and food supplements (FS). The target population is the general population. The applicant applies for the same uses and use levels already assessed for 6′‐SL sodium salt produced by fermentation by a genetically modified strain of E. coli K‐12 DH1. Therefore, since the NF would be consumed at the same extent as the already assessed 6′‐SL sodium salt, no new estimates of the intake have been carried out. Similarly, FS are not intended to be used if other foods with added 6′‐SL or human milk are consumed on the same day. The Panel concludes that the NF is safe under the proposed conditions of use.

Introduction

Background and Terms of Reference as provided by the requestor

On 26 March 2021, the company Kyowa Hakko Bio Co., Ltd. submitted a request to the Commission in accordance with Article 10 of Regulation (EU) 2015/2283¹ to place on the EU market 6′-sialyllactose (6′-SL) sodium salt as a novel food (NF).

6′-SL sodium salt is intended to be used in a number of food categories.

The applicant has requested data protection under Article 26 of Regulation (EU) 2015/2283 for data in support of this request.

In accordance with Article 10(3) of Regulation (EU) 2015/2283, the European Commission (EC) asks the European Food Safety Authority (EFSA) to provide a scientific opinion on 6′-SL sodium salt as a NF.

In this opinion on 6′-SL sodium salt, EFSA should also document whether and to what extent the requirements of Article 26(2)(c) of Regulation (EU) 2015/2283 are fulfilled regarding the data for which the applicant is requesting data protection.

Additional information

The sodium salt of 6′-SL is included in the Union list of authorised NFs (Commission Implementing Regulation (EU) 2017/2470²) when produced by fermentation by a genetically modified strain of Escherichia coli K-12 DH1 (EFSA NDA Panel, 2020a). Moreover, the safety of the 6′-SL sodium salt produced by genetically modified strains of E. coli BL21 (DE3) has been assessed by EFSA with a positive outcome (EFSA NDA Panel, 2022a). Two sodium salts of 3′-sialyllactose (3′-SL), a constitutional isomer of 6′-SL, are also included in the Union list of authorised NFs when produced by genetically modified strains of E. coli K-12 DH1 or E. coli BL21 (DE3) (EFSA NDA Panel, 2020b, 2022b).

Since 2015, several scientific opinions with positive outcomes have been adopted by the EFSA NDA Panel on the safety of human-identical milk oligosaccharides (HiMOs) as NFs pursuant to Regulation (EC) No 258/97 or Regulation (EU) 2015/2283:

• Chemically synthetised 2′-fucosyllactose (2′-FL) (EFSA NDA Panel, 2015a) and 2′-FL produced by a genetically modified strain (APC199) of Corynebacterium glutamicum ATCC 13032 (EFSA NDA Panel, 2022c);
• Chemically synthetised lacto-N-neotetraose (LNnT) (EFSA NDA Panel, 2015b) and LNnT produced by genetically modified strains of E. coli BL21 (DE3) (EFSA NDA Panel, 2020c);
• Extension of use in food supplements (FS) for infants of chemically synthetised 2′-FL and LNnT (EFSA NDA Panel, 2015c) or 2′-FL and LNnT produced by genetically modified strains of E. coli K-12 DH1 (EFSA NDA Panel, 2022d);
• Chemically synthetised N-acetyl-d-neuraminic acid (NANA) (EFSA NDA Panel, 2017);
• 2′-FL/difucosyllactose (DFL) mixture produced by a genetically modified strain of E. coli K-12 DH1 (EFSA NDA Panel, 2019a);
• Lacto-N-tetraose (LNT) produced by genetically modified strains of E. coli K-12 DH1 (EFSA NDA Panel, 2019b) or E. coli BL21 (DE3) (EFSA NDA Panel, 2022e);
• Extension of use in FS for infants of 2′-FL/DFL mixture and LNT produced by genetically modified strains of E. coli K-12 DH1 (EFSA NDA Panel, 2022f);
• 6′-SL sodium salts produced by genetically modified strains of E. coli K-12 DH1 (EFSA NDA Panel, 2020a) or E. coli BL21 (DE3) (EFSA NDA Panel, 2022a);
• 3′-SL sodium salts produced by genetically modified strains of E. coli K-12 DH1 (EFSA NDA Panel, 2020b) or E. coli BL21 (DE3) (EFSA NDA Panel, 2022b);
• 3-fucosyllactose (3-FL) produced by genetically modified strains of E. coli K-12 MG1655 (EFSA NDA Panel, 2021) or E. coli BL21 (DE3) (EFSA NDA Panel, 2022g).

Data and Methodologies

Data

The safety assessment of this NF is based on data supplied in the application, information submitted by the applicant following an EFSA request for supplementary information and additional data identified by the Panel.

Administrative and scientific requirements for NF applications referred to in Article 10 of Regulation (EU) 2015/2283 are listed in Commission Implementing Regulation (EU) 2017/2469³.

A common and structured format on the presentation of NF applications is described in the EFSA guidance on the preparation and presentation of an NF application (EFSA NDA Panel, 2016). As indicated in this guidance, it is the duty of the applicant to provide all of the available (proprietary, confidential and published) scientific data (including both data in favour and not in favour) that are pertinent to the safety of the NF.

This NF application includes a request for protection of proprietary data in accordance with Article 26 of Regulation (EU) 2015/2283. The data requested by the applicant to be protected comprise: (i) identity of the NF; (ii) production process; (iii) information on the genetically modified production strain; (iv) composition and stability of the NF; (v) toxicological and allergenicity studies.

Methodologies

The assessment follows the methodology set out in the EFSA guidance on NF applications (EFSA NDA Panel, 2016) and the principles described in the relevant existing guidance documents from the EFSA Scientific Committee. The legal provisions for the assessment are laid down in Article 11 of Regulation (EU) 2015/2283 and in Article 7 of Commission Implementing Regulation (EU) 2017/2469. The legal provisions for the assessment of food intended for infants and young children, food for special medical purposes (FSMP) and total diet replacement for weight control are laid down in Regulation (EU) No 609/2013⁴ and, respectively, in Commission Delegated Regulation 2017/1798⁵ (total diet replacement for weight control), in Commission Delegated Regulation (EU) 2016/128⁶ (FSMP) and in Commission Delegated Regulation (EU) 2016/127⁷ (as regards the specific compositional and information requirements for infant formula (IF) and follow-on formula (FOF) and as regards requirements on information relating to infant and young child feeding).

This assessment concerns only the risks that might be associated with consumption of the NF under the proposed conditions of use and is not an assessment of the efficacy of the NF with regards to any claimed benefit. This assessment also is not an assessment on whether the NF is suitable as stipulated by Regulation (EU) No 609/2013.

Assessment

Introduction

The NF, which is the subject of the application, contains 6′-SL sodium salt as primary constituent (≥ 82% w/w dry matter (DM)). 6′-SL has been identified as a relevant component of the complex fraction of oligosaccharides naturally occurring in human milk, also denominated as human milk oligosaccharides (HMOs). 6′-SL is a sialylated (acidic) trisaccharide composed of d-glucose, d-galactose and NANA (hereinafter also referred to as ‘sialic acid’). 6′-SL is the predominant acidic HMO and one of the most abundant HMOs along with 2′-FL, lacto-N-fucopentaose I, LNT and LNnT (Thurl et al., 2010, 2017). The Panel notes that although the 6′-SL sodium salt is the major component of the NF, related substances, namely sialic acid, d-glucose, d-lactose, 6′-sialyllactulose sodium salt, 3′-SL sodium salt and a small fraction of other related saccharides, are also present. The NF is produced by fermentation by a genetically modified strain (E. coli NEO6) of E. coli W (ATCC 9637), and is isolated as a purified ingredient in the sodium salt form.

The applicant applies for the same uses and use levels (as a food ingredient, including FSMP, IF and FOF and the use in FS) already assessed for the 6′-SL sodium salt produced by fermentation by a genetically modified strain of E. coli K-12 DH1 (EFSA NDA Panel, 2020a).

The target population is the general population.

The sodium salt of 6′-SL produced by a genetically modified strain of E. coli K-12 DH1 (EFSA NDA Panel, 2020a) is already authorised as a NF in the European Union (Commission Implementing Regulation (EU) 2017/2470). Moreover, the safety of the 6′-SL sodium salt produced by genetically modified strains of E. coli BL21 (DE3) has been assessed by EFSA with a positive outcome (EFSA NDA Panel, 2022a). Two sodium salts of 3′-SL produced by genetically modified strains of E. coli K-12 DH1 or E. coli BL21 (DE3) (EFSA NDA Panel, 2020b, 2022b) are also included in the Union list of authorised NFs.

According to Article 3(2)(a) of Regulation (EU) 2015/2283, the NF falls under the following categories:

• ‘food with a new or intentionally modified molecular structure, where that structure was not used as, or in, a food within the Union before 15 May 1997’; and
• ‘food consisting of, isolated from or produced from microorganisms, fungi or algae’.

Identity of the NF

The NF is a powdered mixture mainly composed of 6′-SL sodium salt (≥ 82.0% w/w DM), but it also contains sialic acid (≤ 6.0% w/w DM), d-glucose (≤ 3.0% w/w DM), d-lactose (≤ 3.0% w/w DM), 6′-sialyllactulose and 3′-SL sodium salts (≤ 5.0% w/w DM, sum of both) and a small fraction of other related saccharides (sum of other carbohydrates ≤ 13.0% w/w DM). It is produced by fermentation by a genetically modified strain (E. coli NEO6) of E. coli W (ATCC 9637). The main component is the sodium salt of Neu5Ac-α-(2–6)-Gal-β-(1–4)-Glc (6′-SL), in which sodium N-acetyl-d-neuraminate is linked through an α-(2–6) bond to d-galactose, which is linked through a β-(1–4) bond to d-glucose, in its α- and β-anomeric forms (Table 1 and Figure 1). 6′-SL is a regioisomer of 3′-SL, which contains the same monosaccharide moieties as those present in 6′-SL but with the linkage between N-acetyl-d-neuraminic acid (Neu5Ac) and d-galactose being α-(2–3) instead of α-(2–6).

Table 1 Chemical identity of 6′-SL sodium salt

Chemical substance
Chemical (IUPAC) name	Sodium; (2R,4S,5R,6R)-5-acetamido-4-hydroxy-6-[(1R,2R)-1,2,3-trihydroxypropyl]-2-[[(2R,3R,4S,5R,6S)-3,4,5-trihydroxy-6-[(2R,3R,4R,5R)-1,2,4,5-tetrahydroxy-6-oxohexan-3-yl]oxyoxan-2-yl]methoxy]oxane-2-carboxylate
Common name	6′-Sialyllactose, sodium salt
Abbreviations	6′-SL, sodium salt
Alternative chemical names	N-Acetyl-α-d-neuraminyl-(2→6)-β-d-galactopyranosyl-(1→4)-d- glucopyranose, sodium salt 6′-SL sodium salt 6′-N-acetylneuraminyl-d-lactose sodium salt α-Neu5Ac-(2→6)-β-d-Gal-(1→4)-d-Glc sodium salt
CAS Number	157574-76-0 (sodium salt)/35890-39-2 (acid)
Molecular formula	C23H38NO19Na
Molecular mass	655.53 Da

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Several analyses were performed on the NF in order to confirm the structure of 6′-SL, the major constituent of the NF.

The structure of 6′-SL⁸ was determined by mono-dimensional (1D) nuclear magnetic resonance (NMR) spectroscopy, including ¹H and ¹³C spectra, and two-dimensional (2D) NMR spectroscopy, including COSY (correlation spectroscopy), TOCSY (total correlation spectroscopy), HETCOR (heteronuclear correlation) and HMBC (heteronuclear multiple bond correlation) spectra, by comparison to a commercially available authentic specimen⁹. The relevant coupling constants measured by ¹H NMR together with the correlations evidenced on the 2D NMR spectra confirmed: (i) the α-(2″-6′) glycosidic linkage between Neu5Ac (C-2″) and the d-galactose (Gal-C-6′) moiety of d-lactose; (ii) the β-(1′-4) link between the d-galactose (Gal-C-1′) and d-glucose (Glc-C-4) moieties of d-lactose; and (iii) the β configuration of the Gal unit.

The molecular structure of 6′-SL⁸ was corroborated by liquid chromatography – tandem mass spectrometry (LC–MS/MS) based on its retention factor (R_f) and fragmentation pattern, by comparison to a commercially available high-purity analytical standard, which allowed to differentiate between 6′-SL α-(2″-6′) and 3′-SL α-(2″-3′).

The identity of 6′-SL⁸ was also corroborated by high-performance liquid chromatography – charged aerosol detection (HPLC-CAD) by comparison to a commercially available high-purity analytical standard.

On the basis of the spectroscopic and chromatographic evidence, the Panel considers that the 6′-SL present in the NF produced by E. coli NEO6 is identical to the 6′-SL in human milk, and therefore, it is regarded as being a HiMO.

Production process

According to the information provided, the NF is produced in line with Good Manufacturing Practice (GMP) and Hazard Analysis Critical Control Points (HACCP) principles, in a facility that is FSSC (Food Safety System Certification) 22000 certified.

The NF is produced by fermentation by a genetically modified strain (E. coli NEO6) of E. coli W (ATCC 9637) using food-grade raw materials and processing aids. The production microorganism is cultured under sterile conditions in a chemically defined nutrient medium (without soy peptone for commercial production purposes) and uses glucose and lactose to synthesise 6′-SL, which is excreted into the medium. The production microorganism is removed from the culture medium by microfiltration at the end of the fermentation process. 6′-SL is isolated as the sodium salt and purified from the fermentation medium using a series of filtration and cationic and anionic exchange steps, followed by concentration and spray-drying to obtain the final 6′-SL sodium salt product in powder form.

The production strain E. coli NEO6 is a genetically modified derivative of the parental strain E. coli W (Waksman's strain), which is deposited at the American Type Culture Collection (ATCC) (commercially available under ATCC 9637). The strain E. coli W is well characterised and its genome has been sequenced, annotated and compared to other safe E. coli strains and phylogroup B1 commensal/pathogenic E. coli strains (Archer et al., 2011). Although E. coli W harbours genes that encode pathogenicity determinants, these have been mutationally inactivated or are missing key components required for pathogenicity, similarly to other safe strains (Archer et al., 2011). Genomic analyses also confirmed the lack of genes encoding toxins that can be secreted (Archer et al., 2011). Although the species E. coli is considered not suitable for qualified presumption of safety (QPS) status (EFSA BIOHAZ Panel, 2023), the strain E. coli W does not cause disease in healthy adult humans nor does it colonise the human gut (Bauer et al., 2008; NIH, 2019), and it is considered as a safe and non-pathogenic or toxigenic microorganism widely used for biotechnological applications.

The production strain has been deposited at the National Biological Resource Center (NBRC) culture collection. A detailed description of the genetic modification steps applied to the parental strain E. coli W to obtain the production strain E. coli NEO6 has been provided by the applicant. No residual DNA from the production strain was detected in the NF by a quantitative polymerase chain reaction (qPCR) assay using primers specific to the production strain. The absence of both DNA and viable cells from the production strain in the NF has been demonstrated in accordance with the EFSA Guidance on the characterisation of microorganisms used as feed additives or as production organisms (EFSA FEEDAP Panel, 2018).

The Panel considers that the production process is sufficiently described and does not raise safety concerns.

Compositional data

In order to confirm that the manufacturing process is reproducible and adequate to produce on a commercial scale a product with certain characteristics, the applicant provided analytical information for eight batches of the NF produced with (five batches) or without (three batches) soy peptone in the fermentation media, the latter representing the conditions for commercial production of the NF (Table 2). Information was provided on the accreditation of the laboratories that conducted the analyses presented in the application.

Table 2 Batch-to-batch analysis of the NF

Parameters	NF (produced with soy peptone in the fermentation media)	NF (produced without soy peptone in the fermentation media)	Method of analysis
#1	#2	#3	#4	#5	#6	#7	#8
Composition
6′-SL sodium salt (% w/w DM)	87.0	92.0	90.0	92.0	92.0	91.0	93.0	95.0	HPLC-CAD (validated internal method)
Sialic acid (% w/w DM)	5.1	3.5	4.9	4.3	5.4	1.8	0.1	0.2	HPLC-CAD(a) (validated internal method)
d-Glucose (% w/w DM)	< 0.02	< 0.02	< 0.02	< 0.02	< 0.02	< 0.05	< 0.05	< 0.05	HPLC-PAD(b) (validated internal method)
d-Lactose (% w/w DM)	≤ 0.05	≤ 0.05	≤ 0.05	≤ 0.05	≤ 0.05	< 0.05	< 0.05	< 0.05	HPLC-PAD(b) (validated internal method)
Sum of 6′-sialyllactulose and 3′-SL sodium salts (% w/w DM)	0.4	0.4	0.5	0.5	0.4	1.7	0.5	0.3	HPLC-CAD(a),(c) (validated internal method)
Sum of other carbohydrates (% w/w DM)	6.6	3.3	3.8	2.5	1.4	5.0	5.8	3.8	Calculation(d)
Water (% w/w)	5.3	5.0	5.4	5.6	5.0	7.9	8.4	8.6	JP 2.48(e) – Karl Fischer titration (volumetric/coulometric titration)
Ash (% w/w DM)	–	–	11.6	–	11.7	11.3	11.1	11.2	JP 2.44(e) (residue on ignition, gravimetry)
Protein (% w/w)	–	–	≤ 0.01	–	≤ 0.01	≤ 0.01	≤ 0.01	≤ 0.01	Bradford assay(f) (spectrophotometry)
Sodium (% w/w DM)	3.8	3.8	3.8	3.7	3.8	3.5	3.5	3.6	USP 233(g) (ICP-MS or ICP-OES)
pH (5% solution, 25°C)	6.4	6.5	6.5	6.5	6.5	6.1	5.9	5.8	JP 2.54(e) (potentiometry)
Contaminants
Arsenic (mg/kg)	≤ 0.05	≤ 0.05	≤ 0.05	≤ 0.05	≤ 0.05	< 0.01(i)	< 0.01(i)	< 0.03(i)	USP 233(g),(h) (ICP-MS) AOAC (2019) 999.10 and 2011.14(i) (AAS and ICP-OES)
Cadmium (mg/kg)	≤ 0.05	≤ 0.05	≤ 0.05	≤ 0.05	≤ 0.05	< 0.01(j)	< 0.01(j)	< 0.01(j)	USP 233(g),(h) (ICP-MS) AOAC (2019) 999.10 and 2011.14(j) (AAS and ICP-OES)
Lead (mg/kg)	≤ 0.05	≤ 0.05	≤ 0.05	≤ 0.05	≤ 0.05	< 0.02(k)	0.03(k)	< 0.02(k)	USP 233(g),(h) (ICP-MS) AOAC (2019) 999.10 and 2011.14(k) (AAS and ICP-OES)
Mercury (mg/kg)	≤ 0.05	≤ 0.05	≤ 0.05	≤ 0.05	≤ 0.05	< 0.004(l)	< 0.004(l)	< 0.004(l)	USP 233(g),(h) (ICP-MS) US EPA, February 2007, Method 7473(l) (AAS)
Aflatoxin M1 (μg/kg)	–	< 0.02	< 0.02	< 0.02	< 0.02	< 0.02	< 0.02	< 0.02	AOAC 2000.08(m) (HPLC)
Microbial parameters
Total plate count (CFU/g)	< 10	< 10	< 10	< 10	< 10	< 10	< 10	< 10	ISO 4833-1:2013(n) (colony count)
Yeasts and moulds (CFU/g)	< 100	< 100	< 100	< 100	< 100	< 10(o)	< 10(o)	< 10(o)	ISO 21527-2:2008(p) (colony count)
Enterobacteriaceae (in 10 g)	ND	ND	ND	ND	ND	ND	ND	ND	ISO 21528-1:2017 (detection or qualitative method)
Salmonella spp. (in 25 g)	–	–	ND	–	–	ND	ND	ND	ISO 6579-1:2017 (detection or qualitative method)
Cronobacter spp. (in 10 g)	ND	ND	ND	ND	ND	ND	ND	ND	ISO 22964:2017 (detection or qualitative method)
Listeria monocytogenes (in 25 g)	ND	ND	ND	ND	ND	ND	ND	ND	ISO 11290-1:2017 (detection or qualitative method)
Presumptive Bacillus cereus (CFU/g)	< 10	< 10	< 10	< 10	< 10	< 10	< 10	< 10	ISO 7932:2004(q) (colony count)
Endotoxins (EU/mg)	0.006	0.011	0.020	0.034	0.026	< 0.0002(r)	< 0.0002(r)	< 0.0002(r)	JP 4.01(e) (kinetic-turbidimetric method)

Batch-to-batch analyses showed that the NF consists of 6′-SL sodium salt as main component (90.6% w/w DM¹⁰ or 93.0% w/w DM¹¹ in batches produced with or without soy peptone in the fermentation media, respectively). The remaining constituents include sialic acid (4.6% w/w DM¹⁰/0.7% w/w DM¹¹), d-lactose (≤ 0.05% w/w DM^10,11), d-glucose (< 0.02% w/w DM¹⁰/< 0.05% w/w DM¹¹), 6′-sialyllactulose and 3′-SL sodium salts (0.4% w/w DM¹⁰/0.8% w/w DM¹¹ – sum of both carbohydrates), and a small fraction of other related saccharides (sum of other carbohydrates¹², 3.5% w/w DM¹⁰/4.9% w/w DM¹¹).

With regard to physico-chemical properties, the NF can be described as a white to off-white powder. The solubility in water was measured in two batches of the NF produced with soy peptone and three batches without soy peptone in the fermentation media, according to the EFSA Guidance on technical requirements for regulated food and feed product applications to establish the presence of small particles including nanoparticles (EFSA Scientific Committee, 2021), resulting in an average value of 451 and 444 g/L, respectively.

The Panel considers that the information provided on the composition is sufficient for characterising the NF.

Stability

Stability of the NF

The applicant provided interim results for a 3-year (real-time) stability study at 25°C and 60% relative humidity (RH) on one batch of the NF produced with soy peptone in the fermentation media, including 6′-SL sodium salt, carbohydrate, sodium and water content, and physico-chemical parameters up to 24 months, and water activity measurements after 6 and 12 months. In addition, a 6-month accelerated stability study at 40°C and 75% RH was conducted on five batches of the NF produced with soy peptone in the fermentation media, including the above-mentioned parameters (water activity measured after 6 months of storage). Microbial parameters were also monitored after 24 months under normal storage conditions.

No significant changes in 6′-SL sodium salt, carbohydrate, sodium and water content, physico-chemical parameters and water activity were observed over the storage period under normal and accelerated conditions. Microbial parameters were also below the respective limits of detection after 24 months of storage under normal conditions. The applicant proposed a 2-year shelf-life under ambient conditions for the NF.

The Panel considers that, since the presence of soy peptone in the fermentation medium does not change the composition substantially, the available data provided sufficient information with respect to the stability of the NF for 24 months.

Stability of the NF under the intended conditions of use

No stability data for the 6′-SL sodium salt in food matrices were provided.

The applicant referred to (i) the stability studies included in the GRAS (Generally Recognised As Safe) notification GRN (GRAS Notice) 766 (US FDA, 2019) on the 3′-SL sodium salt in powdered IF (at room temperature for 24 months), milk (at 4 and 25°C for 45 days) and yoghurt (at 4°C for 45 days), and (ii) the stability of related HiMOs, e.g., 2′-FL, 2′-FL/DFL mixture, LNnT and sialic acid, in IF, FOF, yoghurt, ready-to-drink flavoured milk, citrus fruit drinks and cereal bars (EFSA NDA Panel, 2015a,b, 2017, 2019a).

The Panel considers that the available information is sufficient with respect to the stability of the NF in the proposed food matrices.

Specifications

The specifications of the NF are indicated in Table 3.

Table 3 Specifications of the NF

Description: 6′-sialyllactose sodium salt is a white to off-white powder produced by microbial fermentation and further isolated, purified and concentrated.
Source: A genetically modified strain (Escherichia coli NEO6) of E. coli W (ATCC 9637).
Parameter	Specification
Composition
6′-SL sodium salt (% w/w DM)	≥ 82.0
Sialic acid (% w/w DM)	≤ 6.0
d-Lactose (% w/w DM)	≤ 3.0
d-Glucose (% w/w DM)	≤ 3.0
Sum of 6′-sialyllactulose and 3′-SL sodium salts(a) (% w/w DM)	≤ 5.0
Sum of other carbohydrates(b) (% w/w DM)	≤ 13.0
Water (% w/w)	≤ 10.5
Protein (% w/w)	≤ 0.01
pH (5% solution, 25°C)	4.5–7.5
Sodium (% w/w DM)	≤ 5.0
Contaminants
Arsenic (mg/kg)	≤ 0.2
Aflatoxin M1 (μg/kg)	≤ 0.025
Microbiological parameters
Total plate count (CFU/g)	≤ 1,000
Yeasts and moulds (CFU/g)	≤ 100
Enterobacteriaceae (in 10 g)	ND
Salmonella (in 25 g)	ND
Cronobacter spp. (in 10 g)	ND
Listeria monocytogenes (in 25 g)	ND
Presumptive Bacillus cereus (CFU/g)	≤ 50
Endotoxins (EU/mg)	≤ 10

The Panel considers that the information provided on the specifications of the NF is sufficient and does not raise safety concerns.

History of use of the NF and/or of its source

History of use of the NF

There is no history of use of the NF. However, 6′-SL sodium salt, which is the major constituent of the NF, has been authorised as a NF in the EU (Commission Implementing Regulation (EU) 2021/82¹³) to be added to IF and FOF, and to a variety of foods including FS. The authorised 6′-SL sodium salt is produced by fermentation by a genetically modified strain of E. coli K-12 DH1. Another 6′-SL sodium salt produced by genetically modified strains of E. coli BL21 (DE3) has been recently assessed with a positive outcome (EFSA NDA Panel, 2022a), but it is not yet included in the Union list of NFs.

6′-SL has also been detected in domestic farm animal milk, albeit at lower concentrations as compared to human milk. Oligosaccharides in bovine milk are 20 times less concentrated than in human milk. However, sialylated oligosaccharides account for approximately up to 80% of the total oligosaccharide pools. The amount of 6′-SL in bovine milk is estimated to range from 4 to 10 mg/L and up to 100 mg/L in colostrum (Aldredge et al., 2013; Urashima et al., 2013; Albrecht et al., 2014), which is much lower than human milk concentrations (between 0.40 and 0.74 g/L; Soyyılmaz et al., 2021; EFSA NDA Panel, 2022a).

Proposed uses and use levels and anticipated intake

The applicant applies for the same uses and use levels already assessed for 6′-SL sodium salt produced by fermentation by a genetically modified strain of E. coli K-12 DH1 (EFSA NDA Panel, 2020a). Therefore, since the NF would be consumed at the same extent as the already assessed 6′-SL sodium salt, no new estimates of the intake have been carried out.

Target population

The target population proposed by the applicant is the general population.

Proposed uses and use levels

The intended uses and use levels for the NF are the ones already assessed for 6′-SL sodium salt manufactured by fermentation by a genetically modified strain of E. coli K-12 DH1.

Anticipated intake of the NF

Considering that the same uses and use levels as per the assessed 6′-SL sodium salt are proposed (EFSA NDA Panel, 2020a), the Panel considers that a new assessment of the intake is not needed.

Absorption, distribution, metabolism and excretion (ADME)

No ADME data have been provided for the NF.

The applicant made reference to the assessment performed by the NDA Panel on a previously evaluated 6′-SL sodium salt (EFSA NDA Panel, 2020a) concluding that the NF does not undergo any significant digestion by human enzymes in the upper gastrointestinal tract and that only small amounts are expected to be absorbed. Milk oligosaccharides are then fermented in the colon by intestinal microbiota with a fraction excreted unchanged in the faeces and a small fraction found in the urine (EFSA NDA Panel, 2022a).

In addition, cross reference is made to a study conducted with the labelled isomer of 6′-SL (¹³C-3′-SL and also its component sialic acid) in the mouse (Galuska et al., 2020). The main goal of the study was to verify the ¹³C enrichment in different areas of the brain to study the role of SL in brain development and cognitive functions. The study also demonstrated that absorption after oral administration occurs and is followed by rapid urinary excretion.

Finally, there are no indications that the absorption of 6′-SL, or other structurally related mono- and oligosaccharides (e.g., sialic acid) from the NF, differs from that of similar components in human milk.

Nutritional information

The NF is mainly composed of the non-digestible oligosaccharide 6′-SL.

The NF contains other carbohydrates, individually present at low concentrations. Sialic acid is an endogenous human and ubiquitous monosaccharide (EFSA NDA Panel, 2017; Röhrig et al., 2017). d-Lactose is the most abundant component of human milk (~ 7%) and its monomers, d-glucose and d-galactose, are normal constituents of human milk. 3′-SL is a regioisomer of 6′-SL and also a normal constituent of human milk (see Section 3.4).

The Panel notes that the NF, being a sodium salt, may contribute to the daily sodium intake. Since the same uses and use levels as per the authorised 6′-SL sodium salt are proposed and the sodium content is comparable, similar considerations for the sodium intake apply (i.e., intake ranging from 5% to 38% of the safe and adequate intake¹⁴ in different population groups; EFSA NDA Panel, 2020a).

The Panel considers that, taking into account the composition of the NF and the proposed conditions of use, consumption of the NF is not nutritionally disadvantageous.

Toxicological information

The applicant provided four toxicological studies on the NF, which were conducted in compliance with OECD principles of GLP (Organisation for Economic Co-operation and Development principles of Good Laboratory Practices (OECD, 1998)) and in accordance with the OECD test guidelines TG No 471, 474, 487 and 408. These studies, which were claimed proprietary by the applicant, are listed in Table 4.

Table 4 List of toxicological studies with the NF

Reference	Type of study	Test system	Dose
Unpublished Study No. AG200051	Bacterial reverse mutation test (GLP, OECD TG 471 (1997))	Salmonella Typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA	Up to 5,000 μg 6′-SL/plate (absence and presence of S9 mix)
Unpublished Study No. 200059	Micronucleus study in bone marrow cells of mice (GLP, OECD TG 474 (2016))	Slc:ICR(ICR) mice	500, 1,000 and 2,000 mg 6′-SL/kg bw
Unpublished Study No. CG220004	In vitro micronucleus study in cultured mammalian cells (GLP, OECD TG 487 (2016))	CHL/IU cells	500, 1,000 and 2,000 μg 6′-SL/mL (absence and presence of S9 mix)
Unpublished Study No. 100603RG	90-day repeated dose oral toxicity study in rats (GLP, OECD TG 408 (2018))	SD rats	542, 1,084 and 2,168 mg 6′-SL/kg bw/day

The Panel also noted that in solution under acidic conditions, the NF will be hydrolysed to d-lactose and sialic acid (EFSA NDA Panel, 2020a, 2022a). The amount of these by-products produced during the digestion remains always lower than the intake estimated from human milk. Finally, the possible formation of 6′-sialyllactulose derived from 6′-SL by isomerisation of the terminal d-glucose moiety into d-fructose mainly under alkaline conditions during the production process (Zeng et al., 2018) is also considered not of concern (EFSA NDA Panel, 2022a).

Genotoxicity

The in vitro assessment of the mutagenic potential of the NF was evaluated in a bacterial reverse mutation test (Unpublished Study Report, 2020a). Two main tests, conducted as pre-incubation assays, were performed using S. Typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2 uvrA, which were exposed to water or five different concentrations of 6′-SL sodium salt (313, 625, 1,250, 2,500 or 5,000 μg/plate), either in the presence or absence of liver microsomal fractions (S9 mix). No reproducible or dose-related increases in revertant colony numbers over control counts were observed with any of the strains following exposure to the NF at any concentration (irrespective of the presence or absence of S9 mix). Neither growth inhibition nor precipitation of the test substance was observed. Based on the results of the study, it was concluded that the NF is non-mutagenic at concentrations up to 5,000 μg/plate in the absence or presence of metabolic activation.

The potential ability of the NF to induce chromosome aberration in bone marrow of ICR mice was also assessed in an in vivo micronucleus (MN) study (Unpublished Study Report, 2020b). After a dose-range finding study, the NF was administered twice (with a 24-h interval) by gavage at doses of 0 (water for injection), 500, 1,000 or 2,000 mg/kg body weight to 5 male ICR mice/group. Animals were euthanised about 24 h after the second administration and femoral bone marrow smears were prepared and analysed. No statistically significant differences were noted in the frequency of micronucleated immature erythrocytes between the NF and negative control groups. No significant difference in the proportion of immature erythrocytes among total erythrocytes among study groups was observed. It was concluded that the NF does not have the potential for induction of chromosomal aberrations under the experimental condition applied. However, the Panel noted that the use of animals for this experiment was unnecessary as no evidence of the exposure of the bone marrow to the test substance was provided. Therefore, the applicant was requested to perform an additional in vitro MN test.

To investigate the MN-inducing potential of the NF in cultured mammalian cells, a study was conducted in Chinese hamster lung cells (CHL/IU) by a short-term treatment (6-h method; with and without metabolic activation (S9 mix)) and long-term treatment (27-h method) (Unpublished Study Report, 2022). Concentrations of 500, 1,000 and 2,000 μg 6′-SL sodium salt/mL in physiological saline in the main test were used. No cytotoxicity (measurement of relative population doubling) was observed in any treatment method. No statistically significant increases in the number of binucleate cells containing micronuclei both after 6-h treatment in the presence of or absence of S9 mix or following 27-h treatment were noted and remained within the background range. Therefore, the Panel concludes that the NF did not show any evidence of clastogenicity or aneugenicity in the absence and presence of metabolic activation up to the highest concentration of 2,000 μg/mL.

Taking into account the results provided and considering the nature, source and production process of the NF, the Panel considers that there are no concerns regarding genotoxicity.

Subchronic toxicity

The applicant provided the technical report of a 90-day study where groups of 10 Crl:CD(SD) rats/sex were given distilled water, 542, 1,084 or 2,168 mg 6′-SL sodium salt/kg bw per day by oral gavage (Unpublished Study Report, 2021).

There were no deaths in the course of the study and no treatment-related changes in clinical signs, body weight, body weight gain and food consumption were observed in any rats. Also, functional observations, ophthalmological examination and oestrus cycle examination performed at the end or towards the end of the treatment period did not reveal any test item-related finding.

Some statistically significant changes (Appendix A) of small magnitude were noted in haematological parameters (change in platelets count in low-dose males) and in clinical chemistry: increased alanine aminotransferase (ALT) in high-dose males and increased sodium levels in high-dose females. At urinalysis, variations in a few electrolytes were recorded: increased sodium concentration at intermediate dose in both sexes and in high-dose males, decreased chloride concentration in high-dose males, increased sodium excretion in the intermediate and high-dosed males, decreased total excretion of potassium and chloride at high dose in both sexes. These observed changes were sporadic, of low magnitude and limited to only one sex and they are overall considered by the Panel as not toxicologically relevant.

No findings in treated rats were noted at gross pathology. Some statistically significant changes were noted in both absolute and relative (to the bw) organ weights: increased thyroid weight (low-dose males), decreased pituitary weight (low- and intermediate-dose females), decreased thyroid weight (low- and intermediate-dose females), decreased ovaries weight (intermediate dose, limited to absolute weight), which the Panel considers as incidental and not treatment related.

At histopathology, a few changes were observed in rats receiving the high dose. Changes included mononuclear cell infiltration in the ventricular wall of the heart (2M, 1F), focal fibrosis in the pancreas (1M) or atrophy of acinar cells (1M), tubular basophilic change (1M) and unilateral scar in the kidney (1F), lumen dilatation in the glandular stomach (2M, and 1M in controls), focal ectopic tissue in the mucosa of glandular stomach (1M), accessory adrenal gland (1F), unilateral retinal dysplasia (3M and 1M, 1F in controls). These findings occurred in single animals, in controls or historical controls, or were unilateral or mild. Therefore, they were considered as incidental.

The Panel considers that no adverse effects were observed in this study at the highest tested dose of 2,168 mg 6′-SL sodium salt/kg bw per day.

Human data

No human intervention studies with the NF were provided by the applicant.

The Panel noted that a recent publication (Kim et al., 2022) describes a randomised, controlled clinical trial conducted in 60 healthy adult participants with a 6′-SL with 98.8% purity produced with enzymes from ‘non-pathogenic E. coli K-12’. The oral dose administered was 6 g/day divided in two doses (after the morning and evening meals, approximate interval of 12 h) for 12 consecutive weeks. The placebo group received the same amount of maltodextrin powder according to the same schedule. Only minor adverse reactions (most of the symptoms were gastrointestinal troubles with abdominal discomfort and diarrhoea) were recorded without difference between the treated group and the control group. The Panel notes that the resulting intake is around 90 mg/kg bw per day and that the estimated intake from the proposed uses is ranging from 10 (in elderly) to 192 (in infants) mg/kg bw at the highest 95th percentile (EFSA NDA Panel, 2020a).

The Panel considers this information as supportive for the tolerability of 6′-SL in adults.

Allergenicity

The applicant did not identify an allergenic potential of introduced proteins as a result of the genetic modification of the E. coli W (ATCC 9637) parental strain, according to the ‘Scientific opinion on the assessment of allergenicity of GM plants and microorganisms and derived food and feed of the Scientific Panel on Genetically Modified Organisms’ (EFSA GMO Panel, 2010). The criterion used for identifying allergenic proteins was that of considering ‘higher than 35% identity in a sliding window of 80 amino acids’. The applicant also conducted an ELISA test on one batch of the NF for milk proteins, which were below the LOQ (1 μg/g).

The protein content in the NF is low (≤ 0.01% w/w) as indicated in the specifications (Table 3).

The Panel considers that, for these reasons, the likelihood of allergenic reactions to the NF is low.

Discussion

The NF is a powdered mixture mainly composed of 6′-SL sodium salt, but it also contains sialic acid, d-lactose, d-glucose, 6′-sialyllactulose sodium salt, 3′-SL sodium salt and a small fraction of other related saccharides. The NF is produced by fermentation by a genetically modified strain (E. coli NEO6) of E. coli W (ATCC 9637).

The target population proposed by the applicant is the general population. The applicant intends to add the NF to a variety of foods, including IF and FOF, FSMP and FS. The applicant applies for the same uses and use levels already assessed for the 6′-SL sodium salt produced by fermentation by a genetically modified strain of E. coli K-12 DH1. Therefore, since the NF produced with the new process has similar composition and would be consumed at the same extent as the already assessed 6′-SL in IF, FOF and other foods, no new estimates of the intake have been carried out. Similar considerations apply for the sodium intake. The applicant stated that FS containing the NF are not intended to be used if other foods with added NF or human milk are consumed on the same day. Additional sources for the oligosaccharides contained in the NF are cow milk and milk-derived products. However, the contribution from consumption of cow milk and milk-derived products is small.

The submitted toxicity studies did not raise safety concerns. No toxicologically relevant effects were observed in the subchronic toxicity study in rats at up to the highest dose tested of 2,168 mg 6′-SL/kg bw per day.

Taking into account the intrinsic nature of HMOs with their limited absorption, the absence of toxicologically relevant effects in the subchronic study and considering that infants are naturally exposed to these substances, the Panel considers that the consumption of the NF at the proposed uses and use levels does not raise safety concerns.

Conclusions

The Panel concludes that the NF, which is composed of 6′-SL and other structurally related mono- and oligosaccharides, is safe under the proposed conditions of use.

Protection of Proprietary data in accordance with Article 26 of Regulation (EU) 2015/2283

The Panel could not have reached the conclusion on the safety of the NF under the proposed conditions of use without the data claimed as proprietary by the applicant: (i) identity of the NF as confirmed by NMR spectroscopy, LC–MS/MS and HPLC-CAD; (ii) production process; (iii) information on the genetically modified production strain; (iv) composition and stability of the NF; (v) toxicological (Table 4) and allergenicity studies.

Steps taken by EFSA

• On 07 December 2021 EFSA received a letter from the European Commission with the request for a scientific opinion on the safety of 6′-sialyllactose (6′-SL) sodium salt as a novel food. Ref. Ares(2021)7548385.
• On 07 December 2021, a valid application on the safety of 6′-sialyllactose (6′-SL) sodium salt as a novel food, which was submitted by Kyowa Hakko Bio Co., Ltd, was made available to EFSA by the European Commission through the Commission e-submission portal (NF 2021/2458) and the scientific evaluation procedure was initiated.
• On 21 July 2022, EFSA requested the applicant to provide additional information to accompany the application and the scientific evaluation was suspended.
• On 14 April 2023, additional information was provided by the applicant through the Commission e-submission portal and the scientific evaluation was restarted.
• During its meeting on 27 April 2023, the NDA Panel, having evaluated the data, adopted a scientific opinion on the safety of 6′-sialyllactose (6′-SL) sodium salt as a novel food pursuant to Regulation (EU) 2015/2283.

Abbreviations

• 1D
• Mono-dimensional
• 2D
• Two-dimensional
• 2′-FL
• 2′-Fucosyllactose
• 3-FL
• 3-Fucosyllactose
• 3′-SL
• 3′-Sialyllactose
• 6′-SL
• 6′-Sialyllactose
• AAS
• Atomic absorption spectroscopy
• ADME
• Absorption, Distribution, Metabolism and Excretion
• ALT
• Alanine aminotransferase
• AOAC
• Association of Official Analytical Collaboration
• ATCC
• American Type Culture Collection
• BIOHAZ
• EFSA Panel on Biological Hazards
• bw
• Body weight
• CAS
• Chemical Abstracts Service
• CFU
• Colony forming units
• CHL/IU
• Chinese hamster lung cells
• COSY
• Correlation spectroscopy
• Crl:CD(SD) rats
• Charles River Laboratories: Caesarean-derived (Sprague Dawley) rats
• DFL
• Difucosyllactose
• DM
• Dry matter
• DNA
• Deoxyribonucleic acid
• Escherichia coli W
• Waksman's E. coli strain
• EU
• Endotoxin units
• FEEDAP
• EFSA Panel on Additives and Products or Substances used in Animal Feed
• FOF
• Follow-on formula
• FS
• Food supplements
• FSMP
• Food for special medical purposes
• FSSC 22000
• Food Safety System Certification 22000
• Gal
• Galactose
• Glc
• Glucose
• GLP
• Good Laboratory Practices
• GMO
• EFSA Panel on Genetically Modified Organisms
• GMP
• Good Manufacturing Practice
• GRAS
• Generally Recognised As Safe
• GRN
• GRAS Notice
• HACCP
• Hazard Analysis Critical Control Points
• HETCOR
• Heteronuclear correlation
• HiMO
• Human-identical milk oligosaccharide
• HMBC
• Heteronuclear multiple-bond correlation
• HMO
• Human milk oligosaccharide
• HPLC-CAD
• High-performance liquid chromatography – charged aerosol detection
• HPLC-PAD
• High performance liquid chromatography-pulsed amperometric detection
• ICP-MS
• Inductively coupled plasma – mass spectrometry
• ICP-OES
• Inductively coupled plasma – optical emission spectroscopy
• ICR
• Institute of Cancer Research
• IF
• Infant formula
• ISO
• International Organisation for Standardisation
• IU
• International Units
• IUPAC
• International Union of Pure and Applied Chemistry
• JP
• Japanese Pharmacopoeia
• LC
• Liquid chromatography
• LNnT
• Lacto-N-neotetraose
• LNT
• Lacto-N-tetraose
• LOD
• Limit of detection
• LOQ
• Limit of quantification
• MN
• micronucleus
• MS/MS
• Tandem mass spectrometry
• MW
• Molecular weight
• NANA, Neu5A
• N-acetyl-d-neuraminic acid, sialic acid
• NBRC
• National Biological Resource Center
• ND
• Not detected
• NDA
• EFSA Panel on Nutrition, Novel Foods and Food Allergens
• NF
• Novel food
• NIH
• National Institutes of Health
• NMR
• Nuclear magnetic resonance spectroscopy
• OECD
• Organisation for Economic Co-operation and Development
• qPCR
• Quantitative polymerase chain reaction
• QPS
• Qualified presumption of safety
• R_f
• Retention factor
• RH
• Relative humidity
• SD rats
• Sprague Dawley rats
• TG
• Test guidelines
• TOCSY
• Total correlation spectroscopy
• US
• United States
• US EPA
• US Environmental Protection Agency
• US FDA
• US Food and Drug Administration
• USP
• US Pharmacopoeia
• w/w
• weight per weight

Appendix A – Summary of the 90-day oral toxicity study in rats

Study title	GLP 90-day oral toxicity study on 6′-SL sodium salt in SD rats (Unpublished study report, 2021)
Key results
Parameters	Sex	Dose groups (expressed in mg 6′-SL sodium salt/kg bw per day)
0 (control, G1); mean ± SD	542 (low dose, G2); mean ± SD	1084 (intermediate dose, G3); mean ± SD	2168 (high dose, G4); mean ± SD
Haematology
Platelets (104/μL)	M	113.1 ± 11.9	133.5 ± 12.8**	117.0 ± 18.7	114.6 ± 7.8
F	116.5 ± 10.5	114.5 ± 10.4	109.6 ± 14.5	122.6 ± 19.3
Clinical chemistry
Alanine aminotransferase (ALT) IU/L	M	27 ± 4	31 ± 4	34 ± 9	33 ± 4##
F	28 ± 6	28 ± 6	32 ± 18	30 ± 5
Sodium (mmol/L)	M	145 ± 2	146 ± 1	145 ± 1	146 ± 1
F	143 ± 1	144 ± 1	144 ± 2	145 ± 1**
Urinalysis
Sodium (mmol/L)	M	78 ± 21	86 ± 22	133 ± 37##	160 ± 80##
F	99 ± 45	110 ± 40	158 ± 62*	153 ± 52
Sodium (mmol/24 h)	M	1.4 ± 0.9	1.5 ± 0.4	2.2 ± 0.6*	2.4 ± 0.8**
F	1.3 ± 0.5	1.6 ± 0.4	1.5 ± 0.4	1.7 ± 0.4
Potassium (mmol/24 h)	M	4.3 ± 2.3	3.4 ± 0.3	3.4 ± 0.5	2.6 ± 0.6#
F	3.3 ± 0.7	3.3 ± 0.9	2.7 ± 0.8	2.4 ± 0.7*
Chloride (mmol/L)	M	88 ± 21	64 ± 15	70 ± 31	52 ± 32*
F	118 ± 46	109 ± 46	122 ± 54	90 ± 34
Chloride (mmol/24 h)	M	1.6 ± 0.9	1.1 ± 0.2	1.2 ± 0.4	0.8 ± 0.3#
F	1.5 ± 0.5	1.6 ± 0.5	1.2 ± 0.4	1.0 ± 0.4*
Organ weight values – absolute
Pituitary (mg)	M	17.8 ± 1.8	18.8 ± 2.5	19.8 ± 2.8	16.5 ± 2.0
F	25.3 ± 4.6	18.2 ± 4.7**	17.6 ± 3.2**	23.8 ± 3.3
Thyroid (mg)	M	44.2 ± 6.8	53.6 ± 11.2*	43.8 ± 6.8	37.9 ± 4.5
F	40.1 ± 4.0	25.4 ± 3.1**	29.26 ± 4.7**	36.9 ± 3.7
Ovaries (mg)	M	–	–	–	–
F	112.7 ± 20.0	95.3 ± 18.2	85.5 ± 17.5*	94.5 ± 27.2
Organ weight values – relative to bw
Pituitary (mg/g bw)	M	0.0317 ± 0.0034	0.0329 ± 0.0029	0.0337 ± 0.0032	0.0292 ± 0.0035
F	0.0840 ± 0.0128	0.0611 ± 0.0133**	0.0584 ± 0.0066**	0.0790 ± 0.0129
Thyroid (mg)	M	0.0782 ± 0.0104	0.0937 ± 0.0198*	0.0750 ± 0.0130	0.0674 ± 0.0103
F	0.1343 ± 0.0171	0.0860 ± 0.0132**	0.0972 ± 0.0113**	0.1221 ± 0.0138

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