This is a joint FSA and FSS publication.
1. Introduction
In accordance with assimilated Regulation 2283/2015 on novel foods, the application RP2119 for the use of 2’-Fucosyllactose derived from genetically modified Corynebacterium glutamicum APC199 as a novel food has been submitted for authorisation in each nation of Great Britain (GB).
Whilst it was a Member State of the EU, the UK accepted the risk assessments of the European Food Safety Authority (EFSA) in respect of authorisations for regulated food and feed products. When GB left the EU, it retained the same regulations for food and feed regulated products; the FSA and FSS also adopted equivalent technical guidance and quality assurance processes (EFSA NDA, 2016) to be able to undertake GB risk assessments for regulated product applications.
To ensure our regulatory systems are risk proportionate and resources are used effectively, the FSA and FSS have used the evidence submitted by the applicant and other information in the public domain, including the EFSA risk assessment opinion, to provide a summary assessment of the evidence of safety presented in this report.
Specifically, in reviewing the risk assessment that EFSA have recently completed, the reviewers have verified that the standard approach taken, when compared to the relevant guidance applied in GB, has been followed and the conclusions made are consistent with the data summarised in the opinion. Consideration has been given to the processes undertaken to ensure the EFSA opinion is robust and whether there are any aspects that would require further review, such as specific issues for the countries of GB. The result of the assessment is that there is sufficient evidence of safety to conclude without requiring further risk assessment at this time.
This safety assessment represents the opinion of the FSA and FSS.
2. Details of other regulators opinions
The applicant, Advanced Protein Technologies Corp., is seeking authorisation for 2’-FL as novel food derived from genetically modified Corynebacterium glutamicum APC199.
2’-FL, a trisaccharide consisting of L-fucose, D-galactose, and D-glucose produced by either chemical synthesis or microbial fermentation using derivate strains of Escherichia coli K-12 (DH1) or Escherichia coli BL21 (DE3) is already authorised to be used in a number of food products including food supplements (FS) as defined in Directive 2002/46/EC. 2’-FL is included in the Union list of authorised Novel Foods (Commission Implementing Regulation (EU) 2017/2470) and has since been authorised to be produced in a number of production organisms. However, this novel food is being produced by another production organism not yet authorised and therefore assessment under the novel food regulations is required.
A 2’-FL/difucosyllactose (DFL), a mixture produced by fermentation with a genetically modified strain of E. coli K-12 and 3-fucosyllactose (3-FL), a constitutional isomer of 2’-FL produced by fermentation with a derivative strain of E. coli K-12 (MG1655), are also included in the Union list of authorised Novel Foods (NFs). The extension of use in FS for infants of 2’-FL and 2’-FL/DFL mixture, both produced with derivative strains of E. coli K-12 DH1, and the safety of 3-FL produced with a derivative strain of E. coli BL21 (DE3), have recently been assessed by EFSA with positive outcomes (EFSA NDA Panel, 2022 a,b,c).
Several scientific opinions have been adopted by the EFSA NDA Panel since 2015, on the safety of human-identical milk oligosaccharides (HiMOs) as NFs pursuant to Regulation (EC) No 258/97 or Regulation (EU) 2015/2283.
The primary difference in the specifications for 2’-FL from different microbial sources is limited to the carbohydrate components, specifically the levels of difucosyllactose (DFL), lactose, fucose, 3-fucosyllactose, fucosylgalactose, 2’-fucosyl-D-lactulose, glucose, and galactose. These differences are not considered to be a safety concern, as they relate to innocuous and structurally related carbohydrate components.
APTech’s 2’-FL derived from genetically modified Corynebacterium glutamicum APC199 has a Generally Recognized as Safe (GRAS) notice re-submitted in April 2020 after data gaps were raised (GRN 932). The Food and Drug Agency issued a “no questions” letter in February of 2021 (U.S. FDA, 2021). A number of other 2’-FL ingredients produced by chemical synthesis or microbial fermentation have made a GRAS notification for use in infant formula (GRNs 546, 571, 650), as well as other food products such as baby foods (GRN 650 – U.S. 2015a, b, 2016).
In Israel, 2’-FL obtained from microbial fermentation of a strain derived from E. coli BL21 (DE3) is authorised as a novel food for use in milk-based infant formulas (ages 0 to 6 months) and follow-on formulas (ages 6 to 12 months) of the ready-to-feed product (Israel MOH, 2019). There are no authorisations for 2’FL produced using the microorganism in this application.
In December 2022, the safety of 2’-FL derived from Corynebacterium glutamicum APC199 as a novel food manufactured by Advanced Protein Technologies Corp. (“APTech”) was assessed by EFSA and received a positive opinion (EFSA NDA Panel, 2022). This opinion has been reviewed by FSA/FSS risk assessors to inform the assessment of the novel food.
2.1. Methodology applied in the EFSA Opinion
The EFSA Assessment was in accordance with the procedure as lined out in the EFSA scientific opinion ‘Guidance on the preparation and presentation of an application for authorisation of a novel food in the context of Regulation EU (2015/2283)’ (EFSA NDA Panel, 2016) and assimilated Commission Implementing Regulation (EU) 2017/2469.
2.1.1. Identity of the novel food
The Novel food is a powdered mixture of 2’-FL (97%) with trace levels of other carbohydrates such as lactose, DFL, glucose, and galactose. The Novel food is demonstrated by 1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy to be chemically and structurally identical to 2’-FL that is produced synthetically and is naturally present in human breast milk. 2’-FL is a trisaccharide consisting of L-fucose linked via an a-(1-2′) bond to the D-galactose moiety of D-lactose and is characterised by the following information:
The identity of 2’-FL was also confirmed by high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC–PAD) by comparison with a high purity standard. The molecular structure of 2’-FL was determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS) based on the retention time, accurate mass and fragmentation pattern, which allowed to differentiate between 2’-FL characterised by a 1″-2′ bond and 3’-FL characterised by a 1″-3’ bond as well as being by comparison with a high purity standard.
2.1.2. Production Process
The Novel Food 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.
2’-FL is produced by fermentation using a genetically modified strain derived from the host strain C. glutamicum ATCC 13032. The production strain C. glutamicum APC199 has been modified to effectively synthesise 2’-FL. Glucose and lactose are used as carbon source and substrate, respectively, by C. glutamicum APC199 to produce 2’-FL, which is released into the fermentation media. At the end of the fermentation process, the bacterial biomass is heat inactivated and removed by microfiltration. The isolation, purification and concentration of the product involve a series of filtration steps and drying to obtain purified 2’-FL in powder form.
The production strain C. glutamicum APC199 is a genetically modified derivative of the host strain C. glutamicum ATCC 13032. C. glutamicum has been recommended for the qualified presumption (QPS) status but only for production purposes (EFSA BIOHAZ Panel, 2022; EFSA Scientific Committee, 2007) suggesting the absence of viable cells of the production microorganism in the final product should be a consideration in establishing safety.
The production organism is considered a Category 1 under EFSA’s guidance on the risk assessment of genetically modified microorganisms and their products intended for food and feed use (EFSA GMO Panel, 2010). This refers to the organism being used for the production of another substance. The focus for managing risk is to ensure the organism and any recombinant DNA are not present in the final food.
Four antimicrobial resistance genes either introduced during the genetic modification or identified in the whole genome sequence (WGS) analysis of the production strain, had been previously identified in the bioinformatic analysis performed in accordance with the EFSA statement on WGS analysis (EFSA, 2021).
No residual DNA from the production strain was detected in the 2’FL by quantitative polymerase chain reaction (qPCR) amplification of 10 target genes. In accordance with the EFSA Guidance on the characterisation of microorganisms used as feed additives or as production organisms (EFSA FEEDAP Panel, 2018) the absence of both DNA and viable cells from the production strain provides reassurance that there are not risks from contamination of the novel food from the production organism.
2.1.3. Compositional information and Specification
Batch-to-batch analyses confirmed that the novel food consists of 2’-FL (>97% w/w DM) as the main component. The remaining constituents include D-lactose (< 0.14% w/w DM), L-fucose (< 0.07% w/w DM), 3-FL (< 0.32% w/w DM), DFL (< 0.28% w/w DM), D -glucose (< 0.28% w/w DM) and D-galactose 0.18% w/w DM).
The novel food is a white to off-white/ivory powder. The solubility in water was measured in five batches of the novel food according to the Organisation for Economic Cooperation and Development (OECD) test guideline (TG) 105 (OECD, 1995), resulting in an average value of 716.6 g/L at 20°C.
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 five batches of 2’-FL. Information was provided on the accreditation of the laboratories that conducted the analyses was presented in the application. Specification of the novel food is provided in Table 1.
Shelf-life stability studies were carried out on five batches of the novel food under normal storage conditions (25oC, 60% relative humidity) and under accelerated conditions (40oC, 75% relative humidity). The results from these studies support the stability of the novel food for up to 24-months.
Information on the production process, composition, stability and the specification of the novel food does not raise safety concerns.
2.1.4. History of Use
2’FL is a human milk Oligosaccharide found naturally in breast milk. 2’-FL is the most represented oligosaccharide in human milk. The level of 2’FL in breastmilk is considered safe for infants and young children.
There is no history of use for 2’-FL produced from genetically modified Corynebacterium glutamicum APC199 in GB but currently has a positive opinion (EFSA NDA Panel, 2022) for the same intended food categories (assimilated Commission Implementing Regulation (EU) 2023/950).
2’-FL is already included in the GB list of NFs when manufactured by chemical synthesis or fermentation with derivative strains of E. coli K-12 DH1 or E. coli BL21 (DE3). It is authorised to be added to a variety of food categories including foods for special groups such as infant formula and follow-on formula, and food supplements (FS), excluding FS for infants (intended for individuals above 1 year of age).
2.1.5. Proposed Use and Intake
The applicant does not intend to amend the uses and use levels already authorised for 2’-FL manufactured by chemical synthesis or fermentation with derivative strains of E. coli K-12 DH1 or E. coli BL21 (DE3). The intended food categories and maximum use levels therefore remain the same as those listed in assimilated Commission Implementing Regulation (EU) 2023/950 (Table 2).
2.1.6. Absorption, Distribution, Metabolism and Excretion (ADME)
As reported in previous EFSA opinions (EFSA NDA Panel, 2014; 2019a, 2019b, 2020b, 2020a, 2021, 2022a, 2022b) Human Milk Oligosaccharides (HMOs), including fucosyllactoses, are considered ‘non-digestible oligosaccharides’ (EFSA NDA Panel, 2014) since they do not undergo any significant digestion in the upper gastrointestinal tract (Brand-Miller et al., 1995, 1998; Chaturvedi et al., 2001; Engfer et al., 2000; Gnoth et al., 2000, 2001; Rudloff & Kunz, 2012).
Brand-Miller et al. (1995, 1998) reported that HMOs, consumed as a load (a purified oligosaccharide fraction from human milk), are fermented in the colon by the intestinal microbiota. Chaturvedi et al. (2001) and Coppa et al. (2001) reported that 97% and 40–50%, respectively, of the ingested HMOs are excreted unchanged in faeces of breastfed infants. Furthermore, approximately 1–2% of the ingested amounts of HMOs is excreted unchanged in the infants’ urine (EFSA NDA Panel, 2019b; Goehring et al., 2014; Kunz et al., 2017; Rudloff et al., 1996, 2006; Vazquez et al., 2017).
Based on information available on HMOs, it is considered that the NF does not undergo any significant digestion in the gastrointestinal tract and that only small amounts are expected to be absorbed. Moreover, there are no indications that the absorption of 2’-FL, which is the main constituent of the NF, or other structurally related mono- and oligosaccharides (e.g. D-lactose), differs from that of similar components found in human milk.
The ADME of human milk oligosaccharides are well understood and the information provided does not suggest HMOs produced by bio-fermentation would perform differently in the gut. As such the ADME information does not indicate any further areas of concern.
2.1.7. Nutritional Information
The novel food is mainly composed of the non-digestible oligosaccharide 2’-FL. The NF contains other carbohydrates individually present at low concentrations. Taking into account the composition of the NF and the proposed conditions of use, consumption of the NF is not considered to be nutritionally disadvantageous.
2.1.8. Toxicological Information
Toxicological proprietary studies were provided. The studies were conducted in accordance with Organisation for Economic Co-operation and Development (OECD) principles of Good Laboratory Practice (GLP) to support the safety assessment of the novel food. No human intervention studies with the NF were provided by the applicant and no reference to human data was made.
2.1.8.1. Genotoxicity
The potential genotoxicity of the NF was investigated in a bacterial reverse mutation test, an in vitro mammalian cell micronucleus test, an in vitro chromosomal aberration test in human lymphocytes and also in an in vivo micronucleus test in mice. The results of the studies are summarised in Table 3. As such the novel food was not considered genotoxic.
2.1.8.2. Sub-chronic toxicity study
A 90-day oral toxicity study was performed according to the OECD Test Guideline 408 following GLP conditions, where groups of 10 neonatal Sprague–Dawley rats/sex/group (7-day old) were administered daily doses of 2,500, 5,000 and 7,500 mg per kg bw of 2’-FL in water by oral gavage. Additional rats (5 sex/dose) for the control and high dose groups were observed over a 4-week recovery period. Following the studies, the author of the study proposed the high dose level of 7,500 mg 2’-FL/kg bw as the no observed adverse effects level (NOAEL).
However, it was noted by the EFSA Panel that even in the absence of changes in bodyweight, food consumption and gross or histological changes, the highest dose of 7,500 mg 2’-FL/kg bw induced soft stool and diarrhoea in almost all animals of both sexes in the second and third month of treatment. The Panel therefore identified the intermediate dose of 5,000 mg 2’-FL/kg bw per day as the NOAEL (EFSA NDA Panel, 2022).
2.1.9. Allergenicity
The applicant did not identify any allergenic potential of introduced proteins as a result of the genetic modification of the C. glutamicum ATCC 13032 host strain according to the relevant opinion on the assessment of allergenicity (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 protein content in the NF is low (≤ 0.005%) as indicated in the specifications.
The likelihood that the novel food will trigger allergic reactions in the target population under the proposed conditions of use was therefore considered to be low.
3. Other Regulators Opinions and Conclusions
EFSA (2022) found that this product, 2’-FL derived from genetically modified Corynebacterium glutamicum was safe under the proposed conditions of use.
2’-FL produced by either chemical synthesis or microbial fermentation using derivate strains of Escherichia coli K-12 (DH1) or Escherichia coli BL21 (DE3) is already authorised to be used in a number of food products including food supplements, with consumption expected to be safe.
The genotoxicity studies did not indicate any safety concerns. The sub-chronic toxicology studies indicated some effects on gastrointestinal transit at the highest doses of 7,500 mg 2’-FL/kg bw in the 90-Day Oral Toxicity Study in Rodents, the NOAEL of an intermediate dose of 5,000 mg 2’-FL/kg bw per day. Given humans are exposed to 2’-FL through breast milk and the novel food is identical to 2’-FL from other sources, the safe upper intake was set on the basis of infant exposure through breast milk.
Consumption of the novel food under the proposed conditions of use is not expected to be a safety concern as the NF would be consumed at the same extent as the already authorised 2’-FL. It was noted that breastfed infants are naturally exposed to these substances at the levels sought in the proposed uses.
4. Caveats and Uncertainties
The FSA and FSS noted that no specific uncertainties were flagged in the assessment.
The FSA and FSS could not have completed the safety assessment of the novel food under the proposed conditions of use without the following data claimed as proprietary by the applicant:
(i) identity of the NF as confirmed by NMR spectroscopy.
(ii) information on the genetically modified production strain (DNA analysis and absence of viable cells).
5. FSA-FSS conclusion for GB safety assessment
The application has been assessed in line with the applicable guidance. The conclusions of the EFSA opinion have been reviewed in detail by the FSA and FSS and are considered appropriate and consistent within the identified caveats and uncertainties identified in the opinion and would be applicable to GB.
6. Outcome of the assessment
The FSA and FSS has reviewed the applicant’s application, supporting documentation, and other regulators risk assessments, most notably the EFSA risk assessment opinion (EFSA NDA Panel, 2022) and consider sufficient evidence has been demonstrated to conclude without further questions or risk assessment.
The FSA and FSS conclude that 2’-FL was safe under the proposed conditions of use. The anticipated intake levels and the proposed use in food and food supplements was not considered to be nutritionally disadvantageous.
In making this assessment, the following principles have been applied:
1) There is not a legal duty to perform a separate risk assessment for GB, there was sufficient scientific evidence to make a conclusion on safety with no further questions to the applicant, and therefore no further risk assessment activities are necessary.
2) Sufficient evidence was available in the literature, for example, where other National food safety authorities had positively assessed the application using the same risk assessment guidance in principle and legal requirements in GB with the exception to changes in the General Food Law.
3) Applicants provided sufficient relevant information as requested by FSA/FSS.
4) The FSA and FSS review did not find any issues of divergence from guidance or mutual approaches or new scientific issues for consideration.
5) There were no other specific issues that would require an assessment for the UK or the nations of the UK.