Safety of the fungal biomass from Fusarium species strain flavolapis as a novel food pursuant to Article 10 of 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 the safety of the fungal biomass from Fusarium species strain flavolapis as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF as the frozen form of the Fusarium sp. str. flavolapis (named by the applicant), which is proposed by the applicant to be used as an ingredient in several food categories, is sufficiently characterised. The information provided on the production process, composition, stability and specifications of the NF is sufficient and does not raise safety concerns. Based on the findings of a repeated dose 90‐day oral toxicity study in rats conducted with the dehydrated NF, and considering the evidence for thyroidal effects along with the lack of robustness of other findings at the low dose tested, the Panel considers the lowest dose tested as the no observed adverse effect level (NOAEL), i.e. 2744 mg/kg bw per day of the NF in a dehydrated form, corresponding to approximately 10,400 mg/kg bw per day NF. Under the proposed conditions of use, the highest intake estimates of the NF range from 189 to 556 mg/kg bw per day. Applying a default uncertainty factor of 200 to the NOAEL, the Panel considers that the margins of exposure (i.e. 55 in adolescents and 19 in infants) between the intake of the NF at the proposed use and use levels and the NOAEL from the 90‐day study are insufficient. The Panel considers that it is likely that the NF may trigger allergic reactions in allergic subjects. The Panel concludes that, based on the available data, the safety of the NF, i.e. biomass of Fusarium strain flavolapis (F. sp. strain flavolapis) fungus, cannot be established.

1. INTRODUCTION

1.1. Background and Terms of Reference as provided by the requestor

On 20 July 2021, the company Nature's Fynd submitted an application to the European Commission in accordance with Article 10 of Regulation (EU) 2015/2283 to authorise the placing on the Union market of fungi protein from Fusarium strain flavolapis as a novel food. The applicant requests to authorise the use of fungi protein from Fusarium strain flavolapis as a novel food ingredient in several products intended for the general population. The applicant has also requested data protection under Article 26 of Regulation (EU) 2015/2283.

In accordance with Article 29(l)(a) of Regulation (EC) No 178/2002, the European Commission asks EFSA to provide a scientific opinion on fungi protein from Fusarium strain flavolapis as a novel food in accordance with Article 10(3) of Regulation (EU) 2015/2283. The Commission also asks EFSA to evaluate and inform the Commission as to whether and if so, to what extent, the requirements of Article 26(2)(c) of Regulation (EU) 2015/2283 are fulfilled in elaborating its opinion on fungi protein from Fusarium sp. str. flavolapis as a novel food regarding the proprietary data for which the applicant is requesting data protection.

2. DATA AND METHODOLOGIES

2.1. Data

The safety assessment of this NF is based on data supplied in the application and information submitted by the applicant following EFSA requests for supplementary information.

Administrative and scientific requirements for NF applications referred to in Article 10 of Regulation (EU) 2015/2283 are listed in Commission Implementing Regulation (EU) 2017/2469. ¹

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, 2021). 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.

The applicant has submitted a confidential and a non‐confidential version of a dossier following the ‘EFSA guidelines on the preparation and presentation of a NF application’ (EFSA NDA Panel, 2021) and the ‘Administrative guidance for the preparation of applications on novel foods pursuant to Article 10 of Regulation (EU) 2015/2283’ (EFSA, 2021).

In accordance with Article 38 of Regulation (EC) No 178/2002 ² 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 ³ , the non‐confidential version of the dossier has been published on Open.EFSA ⁴ .

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 25 July 2023 to 15 August 2023, for which no comments were received.

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) production process; (ii) composition and stability of the NF; (iii) toxicological information; and (iv) allergenicity.

2.2. Methodologies

The assessment follows the methodology set out in the EFSA guidance on NF applications (EFSA NDA Panel, 2021) 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.

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 regard to any claimed benefit.

3. ASSESSMENT

3.1. Introduction

The NF which is the subject of the application is Fusarium sp. strain flavolapis (F. sp. str. flavolapis) fungus biomass.

The NF falls under Article 3(2)(a)(ii) of Regulation (EU) 2015/2283, i.e. ‘food consisting of, isolated from or produced from microorganisms, fungi or algae’.

The NF is produced by fermentative growth of the filamentous fungus Fusarium sp. strain flavolapis and consists mainly of water, protein, carbohydrates and fat. The NF is proposed to be used as an ingredient in a variety of food products at concentrations up to 17.5% (by weight). The target population is the general population.

3.2. Identity of the NF

The NF under assessment is the frozen form of heat‐treated biomass obtained by fermentative growth of the filamentous fungus Fusarium sp. str. flavolapis, which belongs to the family Nectriaceae.

The specific strain used by the applicant for the production of the NF is the F. oxysporum strain MK7,which was originally isolated from an acidic geothermal spring in the Yellowstone National Park. Upon EFSA's request to clarify the nomenclature and taxonomic assignation at species level, the applicant proposed the naming as Fusarium sp. str. flavolapis, which is an internal name assigned by the applicant and does not refer to a published designated species name in the international standardised repositories.

The strain was deposited in the American Type Culture Collection (ATCC) under the code PTA‐10698 as Fusarium oxysporum strain MK7. ATCC is a depository body acting in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms.

The taxonomic classification of the fungus is defined as follows: Kingdom: Fungi, Phylum: Ascomycota, Class: Sordariomycetes, Order: Hypocreales, Family: Nectriaceae, Genus: Fusarium, Species: Fusarium strain flavolapis.

The identity of the fungal isolate known as MK7 was first identified at genus level as a species of Fusarium via macroconidial microscopy and further by using 18S rRNA sequencing that indicated that the sequence was close to Fusarium oxysporum MK7, using a Basic Local Alignment Search Tool (BLAST) search and the GenBank database. The applicant further addressed the identity of the isolated F. sp. strain flavolapis by using whole genome sequencing (WGS) in accordance with the ‘EFSA statement on the requirements for whole genome sequence analysis of microorganisms intentionally used in the food chain’ (EFSA, 2021). The sequence is currently available under the BioProject accession number PRJNA665233 from the National Center for Biotechnology Information (NCBI). The genome was sequenced using Illumina and PacBio sequencing technologies and the assembly was annotated using eggNOG Mapper (Version 2.19).

The taxonomic identification was additionally established by several phylogenetic analyses including closely related strains, by using 18S/ITS DNA sequences, a set of single copy orthologues and housekeeping genes, cultural (colony morphology), macroscopic and microscopic characteristics of the strain (hyphae, arrangement of macro, microconidia) that indicated that the strain Fusarium sp. str. flavolapis clustered with other strains belonging to Fusarium proliferatum and is included in the F. fujikuroi complex.

To characterise the capacity of the strain to express genes of potential concern, i.e. of clinical relevance, toxigenicity and pathogenicity traits, the applicant performed several analyses on the genome of the strain. An analysis to determine the presence of genes encoding proteins involved in mycotoxin pathways was performed using AntiSMASH. The analysis identified that Fusarium sp. str. flavolapis has genes with high homology to all 16 genes for fumonisin biosynthesis, where all the genes encode proteins that have over 94% identity to either F. fujikuroi or F. proliferatum FUM clusters (Wiemann et al., 2013). These results suggest that the NF strain has the potential to produce the mycotoxin fumonisin.

The genome was further interrogated for the presence of AMR genes using standard thresholds (≥ 80% identity and ≥ 70% coverage). No hits were identified.

Upon EFSA's request, the applicant investigated the potential presence of mycotoxins (fumonisins B1, B2, B3 and total fumonisins) in three supernatant samples taken at the end of the fermentation process. All mycotoxins analysed were below the limit of quantification of the analytical method employed (LOQ < 10 ppb) and in line with the performance criteria specified in EU Regulation 2023/2782. ⁵ However, no secondary metabolites were investigated in the supernatant samples.

Upon EFSA's request, the applicant further investigated the antifungal resistance by completing a query of the genome against the ResFungi Database and antifungal susceptibility using CLSI Method 38. The interrogation identified several genes in F. str. flavolapis that are involved in antifungal resistance pathways. To further test the relevance of these genes in antifungal resistance, the applicant tested the susceptibility of the strain to antifungal agents of clinical relevance (voriconazole, amphotericin B, natamycin, terbinafine). As a control test strain, the yeast Pichia kudriavzevii was used. The results showed that F. str flavolapis was susceptible to at least two commonly used antifungal compounds of clinical relevance for which published cut‐off values are available (voriconazole, amphotericin B).

Lastly, the presence of protein toxins or virulence factors was assessed through a Protein BLAST (BLASTP) search against manually curated venom proteins and toxins. No evidence of human pathogenicity was found in the literature for the identified virulence factors, except for a putative vacuolar ATP synthase subunit B, which shared significant sequence homology with a V‐type proton ATPase subunit B from Candida albicans. However, since virulence traits are elicited when the full multisubunit complex is present (Jiang et al., 2025; Li et al., 2018; Rane et al., 2014) and as no other components were identified in F. sp. str. flavolapis, this presumed virulence factor is unlikely to pose a safety concern. The Panel considers that the NF is sufficiently characterised.

3.3. Production process

According to the applicant, the NF is produced in line with the good manufacturing practice (GMP) and Hazard Analysis Critical Control Points (HACCP) principles. The process flow diagram together with a detailed description of the methods involved at each step and a complete list of the culture media and processing aids plus the respective certificates of analysis were provided (confidential information).

The production process refers to an automated closed system which can be divided into two main parts, i.e. the upstream and downstream processes.

The upstream process includes preparation of raw materials (carbon, nitrogen and mineral sources), and the media used during the liquid‐surface fermentation step. An inoculum of F. sp. str. flavolapis is initially generated under sterile and controlled conditions and is subsequently used as the working cells in the main fermenter where they grow at a specific temperature and pH (confidential information) while being stirred.

The downstream process comprises harvesting of the biomass, heat treatment, size reduction, washing, mechanical pressing (moisture content 70%–75%), separation of residual water, packing in plastic bags and storage under freezing conditions. The absence of 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.

3.4. Compositional data

In order to confirm that the manufacturing process is reproducible and adequate for producing a product with the required characteristics on a commercial scale, the applicant provided analytical information for a number of different batches of the NF (Table 1). The Panel notes that not all the analyses have been performed on the independently produced batches of the NF which were used for the proximate analysis.

TABLE 1.

Batch‐ to‐batch analysis of the NF (wet basis).

Parameter analysis	Batch #1	Batch #2	Batch #3	Batch #4	Batch #5	Method of analysis
Composition
Moisture (%)	75.8	76.8	74.7	75.1	75.3	AOAC 925.09
Protein (%)	11.3	11.8	13.4	12.2	11.4	AOAC 990.03; AOAC 992.15
Total carbohydrates (%)	10.5	8.7	8.9	10.1	10.2	Calculated
Total sugars a (%)	< 0.4	< 0.4	< 0.4	< 0.4	< 0.4	AOAC 982.14 (modified)
Total dietary fibre 6 (%)	8.5	8.7	8.6	9.1	9.2	AOAC 991.43
Total fat (%)	1.2	1.3	1.4	1.4	1.6	AOAC 954.02
Ash (%)	1.2	1.5	1.7	1.2	1.5	AOAC 942.05

Minerals
Batches	Batch #6	Batch #7	Batch #8	Batch #9	Batch #10
Magnesium (mg/100 g)	8	12	9	13	10	AOAC 984.27, 927.02, 985.01, 965.17 mod
Calcium (mg/100 g)	25	60	32	64	38	AOAC 984.27, 927.02, 985.01, 965.17 mod
Potassium (mg/100 g)	0.15	0.17	0.14	0.11	0.12	AOAC 984.27, 927.02, 985.01, 965.17 mod
Chloride (%)	< 0.06	< 0.06	< 0.06	< 0.06	< 0.06	AOAC 969.10 mod
Phosphorus (mg/100 g)	0.19	0.23	0.18	0.17	0.18	AOAC 984.27, 927.02, 985.01, 965.17 mod
Sodium (mg/100 g)	2	<2	6	<2	<2	AOAC 984.27, 927.02, 985.01, 965.17 mod
Sulfites (SO2) (mg/100 g)	< 1	< 1	< 1	< 1	< 1	AOAC 990.28
Sulfates (%)	< 0.02	< 0.02	< 0.02	0.04	< 0.02	AOAC 920.46

Batches	Batch #11	Batch #12	Batch #13	Batch #14	Batch #15
Copper (mg/100 g)	< 0.1	< 0.1	< 0.1	< 0.1	< 0.1	AOAC 984.27 mod,927.02 mod,985.01 mod,965.17 mod
Molybdenum (mg/100 g)	< 0.005	< 0.005	0.006	< 0.001	0.119	AOAC 2011.19 mod.
Iron (mg/100 g)	< 0.2	< 0.2	< 0.2	< 0.2	< 0.2	AOAC 984.27 mod,927.02 mod,985.01 mod,965.17 mod
Manganese (mg/100 g)	0.11	0.13	0.13	0.17	0.09	AOAC 984.27 mod,927.02 mod,985.01 mod,965.17 mod
Zinc (mg/100 g)	0.15	0.2	0.2	0.13	0.13	AOAC 984.27 mod,927.02 mod,985.01 mod,965.17 mod
Boron (mg/100 g)	< 0.25	< 0.29	< 0.25	< 0.24	< 0.24	AOAC 984.27 (mod), 985.01 (mod), 2011.14 (mod)

Contaminants
	Batch #1	Batch #2	Batch #3	Batch #4	Batch #5
Arsenic (mg/kg)	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	J. AOAC vol. 90:844–856 (modified) Mwd‐ICP‐MS
Cadmium (mg/kg)	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	J. AOAC vol. 90:844–856 (modified) Mwd‐ICP‐MS
Lead (mg/kg)	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	J. AOAC vol. 90:844–856 (modified) Mwd‐ICP‐MS
Mercury (mg/kg)	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	J. AOAC vol. 90:844–856 (modified) Mwd‐ICP‐MS
Nitrates (mg/kg)	29	< 10	13	28	16	Internal Method, IC, based on EN 12014–2
Total fumonisins (mg/kg)	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	US‐Multitoxin LC–MS–MS 45‐2‐LWI

	Batch #6	Batch #7	Batch #8	Batch #9	Batch #10
Beauvericin (mg/kg)	0.04	0.01	0.01	0.02	0.03	Internal method, LC–MS/MS

Microbial parameters
	Batch #1	Batch #2	Batch #3	Batch #4	Batch #5
Total aerobic bacteria (CFU/g)	110	380	< 10	< 10	100	APHA CMMEF CHP 8/U.S. Pharmacopoeia Chapter 61
Enterobacteriaceae (CFU/g)	< 10	< 10	< 10	< 10	< 10	CMMEF CHP 9 (AOAC 2003.01)
Yeasts (CFU/g)	< 10	< 10	20	< 10	< 10	AOAC 2014.05
Moulds (CFU/g)	< 10	< 10	< 10	< 10	< 10	AOAC 2014.05
Escherichia coli (CFU/g)	< 10	< 10	< 10	< 10	< 10	AOAC 991.14/U.S. Pharmacopoeia Chapter 62
Salmonella (in 25 g)	ND	ND	ND	ND	ND	AOAC 2011.03/AOAC 2003.09
Lactobacillus (CFU/g)	< 10	< 10	< 10	< 10	< 10	APHA CMMEF CHP 19/ISO 15214
Listeria spp. (in 25 g)	ND	ND	ND	ND	ND	AOAC 2004.06/AOAC‐RI 050903

Abbreviations: AOAC, Association of Official Analytical Chemists; APHA, American Public Health Association; CFU, colony forming units; CHP, chapter; CMMEF, Compendium Methods Microbiological Examination Foods; EN, Comité Européen de Normalisation; IC, Ion chromatography; ISO, International Organization for Standardization; LC–MS/MS, liquid‐chromatography coupled with mass spectrometry; ND, not detected; U.S., United States.

^a Sum of monosaccharides and disaccharides.

Certificates of accreditation for the laboratories that conducted the analyses were provided by the applicant. Analytical data were produced using methods validated for other types of matrices. Whenever in‐house methods were employed, a full description of the method as well as the results of the respective validation procedures were provided.

The NF consists predominantly of water, protein, carbohydrates and fat. The appearance of the NF is that of a light‐tan semi‐solid and frozen mass.

The applicant also provided data on five batches of the NF for vitamins as reported in Table 2.

TABLE 2.

Analytical data on vitamins and minerals in the NF (wet basis).

Parameter	Batches					Analytical method
	Batch #6	Batch #7	Batch #8	Batch #9	Batch #10
Vitamins (wet basis)
Vitamin B1 (mg/100 g)	0.0944	0.0167	0.0185	0.0152	0.0164	AOAC 942.23 mod.
Vitamin B5 (mg/100 g)	0.296	0.117	0.0852	0.0974	< 0.055	AOAC 945.74 (mod.)
Vitamin B6 (mg/100 g)	0.0270	0.0130	0.0200	0.0190	0.0240	AOAC 88, 30–37
Vitamin B12 (μg/100 g)	< 0.44	< 0.44	0.50	< 0.44	< 0.44	AOAC 952.20 mod
Vitamin C (mg/100 g)	< 0.44	< 0.44	< 0.44	0.93	< 0.44	AOAC 967.22 mod
Vitamin D (μg/100 g)	< 0.0025	< 0.0025	< 0.0025	< 0.0025	< 0.0025	Huang et al., Rapid Commun. Mass Spectrum 2014, 28 LC–MS/MS
Vitamin E (mg/100 g)	< 0.200	< 0.200	< 0.200	< 0.200	< 0.200	AOAC 971.30 with HPLC quantification mod.

Abbreviations: AOAC, Association of Official Analytical Chemists; LC–MS/MS, liquid‐chromatography coupled with mass spectrometry.

Moreover, amino acid batch‐to‐batch analyses are reported in Appendix A. Nutritional parameters are assessed in the section ‘3.9. Nutritional information’.

The applicant also provided analytical data for mycotoxins (five independently produced batches), specifically for aflatoxins B1, ⁷ B2, G1, G2, deoxynivalenol, ⁸ ochratoxin A ⁹ , fumonisins B1, B2, B3, ¹⁰ T‐2 toxin and HT‐2 toxin ¹¹ and zearalenone. ¹² Upon EFSA's request, the applicant provided analytical data on the levels of beauvericin (Table 1). The values reported for all mycotoxins analysed are below the limit of detection (LOD) of the analytical methods implemented (Internal method). The LOD values are lower than the maximum levels (MLs) set for foodstuffs considered in Regulation (EC) No 2023/915. The Panel notes that, in the current EU legislation, no MLs of mycotoxins are set for biomass derived from microorganisms.

Due to the nature of the NF, the applicant was requested to further elaborate on the reliability and proficiency of the method for the fungal matrix and for the extraction/analysis of fumonisins and the potential presence of hidden/non‐covalently bound fumonisins in the NF. The applicant provided experimental data for five batches of the NF on the hydrolysed fumonisins by using an analytical method adapted from Dall'Asta et al. (2009). The Panel notes that the analytical method employed fulfils the performance criteria concerning methods of sampling and analysis for the control of the levels of mycotoxins as set out in Regulation (EU) No 2023/2782. The results of the batches analysed were below the LOQ (100 μg/kg) for the analytical method employed.

The level of contaminants was also monitored in the NF. Analytical information on chemical and microbiological parameters for different batches of the NF was provided (Table 1). Concentrations of heavy metals in the NF analysed by ICP‐MS are reported in Table 1. The applicant compared the values to the MLs set for foodstuffs considered in Regulation (EC) No 2023/915. ¹³ The Panel notes that the concentrations of heavy metals reported for the NF do not exceed the maximum levels set for other foods. Analytical data on the pesticide residue levels of five independently produced batches of the NF have been provided. The results showed that all the analysed pesticide residues were not detected in the NF using the QuEChERS method (GC‐MS/MS; LC‐MS/MS) in accordance with AOAC 2007.01 (below the MRL of 0.01 mg/kg for all pesticides, except for fipronil: 0.05 mg/kg).

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

3.4.1. Stability

The applicant performed stability tests with five independently produced batches of the NF under real‐time storage conditions. The tests were carried out at −20°C and analysed at time points 0, 12 weeks or 0, 14 weeks and 0, 26 weeks for microbial contaminants, physicochemical and organoleptic characteristics, respectively (Table 3).

TABLE 3.

Stability of the NF (wet basis).

Parameters	Batch #11		Batch #12		Batch #13		Batch #14		Batch #15		Analytical method
Time points (weeks)	0	12	0	12	0	14	0	26	0	26
Appearance	Stringy; strands are frozen into a solid block
Protein (%)	12	11	10	12	14	13	12	12	12	12	AOAC 990.03; AOAC 992.15
Moisture (%)	75	76	79	76	73	75	77	77	76	76
Aerobic plate count (CFU/g)	40	220	< 10	< 10	< 10	< 10	< 10	100	60	390	APHA CMMEF CHP 8
Yeasts and moulds (CFU/g)	< 10	< 10	< 10	< 10	< 10	< 10	< 10	< 10	< 10	< 10	AOAC 2014.05
Escherichia coli (CFU/g)	< 10	< 10	< 10	< 10	< 10	< 10	< 10	< 10	< 10	< 10	AOAC 991.14
Salmonella spp. (CFU/g)	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	AOAC 2011.03

Abbreviations: AOAC, Association of Official Analytical Chemists; APHA, American Public Health Association; CFU, colony forming units; CHP 8, Chapter 8; CMMEF, Compendium Methods Microbiological Examination Foods; N.D., not detected.

All batches after storage appeared to be solid in the form of frozen strands. The analysed microbial parameters (yeasts, moulds, total aerobic microbial count (TAMC), Salmonella spp., E. coli) remained within the limits as set in the specifications. Upon EFSA's request to provide the certificates of analysis for the batches analysed in the stability study, the applicant submitted another stability study on different batches, tested on 6‐week intervals from week 0 to week 24. The analysed microbial and physicochemical parameters were within the limits set in specifications. However, the Panel notes that the certificates of analysis were not provided for all analysed batches. Based on these results, the applicant proposed a shelf‐life of 12 weeks stored at –20°C.

Given that the NF is proposed to be used as an ingredient in a number of food categories (e.g. cereal bars, dairy analogues), the applicant was requested to investigate the stability of the NF when used as an ingredient in the intended‐for‐use matrices. The applicant provided the requested analytical data on the microbial stability of the NF as an ingredient (per wet biomass) in the intended‐for‐use matrices. The analysed parameters were within the limits set in specifications. The stability data on microbial contamination did not raise safety concerns. Provided that the specifications are met also at the end of the shelf‐life, and that products containing the NF are compliant with respective legislative limits on processing contaminants, the Panels consider that the stability data do not raise safety concerns.

3.5. Specifications

The specifications of the NF are indicated in Table 4.

TABLE 4.

Specifications of the NF.

Description: Frozen form of heat‐treated biomass obtained by fermentative growth of the filamentous fungus Fusarium strain flavolapis
Description	The NF is derived from the filamentous fungus Fusarium strain flavolapis
Appearance	Off‐white to tan
Parameter	Specification
Moisture (%)	< 73–77
Crude protein (%)	11–15
Total carbohydrates (%)	< 11.5
Total sugars a (%)	< 0.5
Total dietary fibre (%)	< 10
Total fat (%)	< 2.5
Ash (%)	< 2.5
Impurities
Nitrate (mg/kg)	< 135
Total fumonisins (mg/kg)	< 0.075
Heavy metals
Lead (mg/kg)	< 0.01
Cadmium (mg/kg)	< 0.01
Mercury (mg/kg)	< 0.01
Arsenic (mg/kg)	< 0.01
Microbiological
TAMC (CFU/g)	< 103
TYMC (CFU/g)	< 100
Escherichia coli	Not detected in 1 g
Salmonella spp.	Not detected in 25 g
Listeria monocytogenes	Not detected in 25 g
Fusarium spp.	Not detected in 25 g

Abbreviations: AOAC, Association of Official Analytical Chemists Sum of monosaccharides and disaccharides; APHA, American Public Health Association; CFU, colony forming units; TAMC, total aerobic microbial count; TYMC, total yeast and mould count.

^a Sum of monosaccharides and disaccharides.

The applicant proposed a specification limit for heavy metals (lead, cadmium, mercury and arsenic) of < 0.075 mg/kg. The Panel notes that considering the production process and compositional analyses of five batches (below 0.01 mg/kg), a lower specification limit could be met. Therefore, the Panel proposes a specification limit of < 0.01 mg/kg for each of the heavy metals analysed.

The Panel considers that the information provided on the specifications of the NF is sufficient.

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

According to the applicant, a fungal ingredient from F. str. flavolapis is authorised and consumed as a food ingredient in the United States since 2018 with generally recognised as safe (GRAS) status for use in several food and beverage products (GRN 904). During the risk assessment, the applicant also provided information about recent authorisations for such fungal ingredient in Singapore, Canada and India.

3.7. Proposed uses and use levels and anticipated intake

3.7.1. Target population

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

3.7.2. Proposed uses and use levels

The NF is proposed to be used as an ingredient in several food products. These food products defined using the FoodEx2 ¹⁴ hierarchy and the maximum use levels are reported in Table 5.

TABLE 5.

Food categories and maximum use levels intended by the applicant.

Proposed food category & use	Food category	FoodEx2 level	FoodEx2 code	Maximum use level (g NF/100 g)
Cereal bars	Cereal bars	L3	A00EY	15
Meat imitates	Meat imitates	L3	A03TE	17.5
Dairy analogues, including beverage whiteners	Imitation cheese	L5	A03TY	15
	Imitation yogurt, non‐soy	L5	A03TZ	15
	Milk imitates	L4	A03TH	10
Processed fish products	Structured/textured fish meat products or fish paste	L4	A0EYV	10
Protein product	Whey powder 15	L4	A02PN	15

3.7.3. Anticipated intake of the NF

EFSA performed an intake assessment of the anticipated daily intake of the NF based on the applicant's proposed uses and maximum proposed use levels (Table 6), using the EFSA Dietary Exposure (DietEx) Tool ¹⁶ , which is based on individual data from the EFSA Comprehensive European Food Consumption Database (EFSA, 2011). The lowest and highest mean and 95th percentile estimated daily intakes of the NF (on a mg/kg body weight (bw) basis), among the EU dietary surveys, are presented in Table 6.

TABLE 6.

Intake estimate of the NF resulting from its use as an ingredient in the intended food categories at the maximum proposed use levels.

Population group	Age, years	Mean intake (mg/kg bw per day)		P95 intake (mg/kg bw per day)
		Lowest a	Highest a	Lowest b	Highest b
Infants	< 1	1.02	76	0.00 e	556
Young children c	1–< 3	2.94	117	0.00	524
Other children	3–< 10	3.37	86	0.00	471
Adolescents	10–< 18	2.85	29	0.00	189
Adults d	≥ 18	1.46	31	0.00	270

^a Intakes are assessed for all EU dietary surveys available in the food comprehensive database on 16/06/2025. The lowest and the highest averages observed among all EU surveys are reported in these columns.

^b Intakes are assessed for all EU dietary surveys available in the food comprehensive database on 16/06/2025. The lowest and the highest P95 observed among all EU surveys are reported in these columns (P95 based on less than 60 individuals are not considered).

^c Referred as ‘toddlers’ in the EFSA food consumption comprehensive database (EFSA, 2011).

^d Includes elderly, very elderly, pregnant and lactating women.

^e P95 = 0.00 mg/kg bw per day: Lowest estimated intake level is very low or not existent for the vast majority of individuals in that population group (95% of the population).

The estimated daily intake of the NF for each population group from each EU dietary survey is available in the Excel file annexed to this scientific opinion (under supporting information).

3.8. Absorption, distribution, metabolism and excretion (ADME)

No ADME data have been provided for the NF.

3.9. Nutritional information

The applicant provided a nutritional analysis of the NF, which consisted mainly of moisture, protein and carbohydrates (see Section 3.4 Compositional data). Analytical data on vitamins and minerals in the NF have also been provided for five independently produced batches of the NF (see Section 3.4 Compositional data). Based on the P95 intake estimate (Table 5), EFSA calculated the exposure to vitamins and minerals for all population groups. The maximum values reported for the analysed batches were used. The Panel considers that the consumption of the NF under the proposed uses and use levels does not contribute substantially to the overall dietary intake of the analysed substances.

The NF is intended to be used as an ingredient in various food products (Section 3.7.2 Proposed uses and use levels). The applicant provided data on the amino acid profile for five non‐consecutive batches of the NF (Appendix A), indicating that the amino acid profile of the NF is consistent across the analysed batches. The applicant addressed protein quality of the NF by deriving a Protein Digestibility Corrected Amino Acid Score (PDCAAS). For this, faecal digestibility of true protein was investigated in 12 Sprague‐Dawley male rats. Four of the rats were assigned to each group including a casein control (containing 10% protein, Nx6.25), endogenous control (protein free) and the NF Protein test group. The animals were fed with their respective diets for 9 days and diet consumption was measured daily. The faeces were collected daily for the last 5 days of the study and analysed for nitrogen content using the Kjeldahl procedure (AOAC 2001.11). The true protein digestibility was 89.08 calculated using AOAC Method 991.29. Using the recommended amino acid scoring patterns as reference values (FAO, 2013), the resulting PDCAAS value for the NF was 0.91 as compared to 1.23 for the casein control. The applicant provided a comparison of the recommended amino acid scoring patterns for infants (birth to 6 months), children (6 months to 3 years) and older children, adolescents and adults (> 3 years) (FAO, 2013) with the amino acid profile of the NF. The Panel notes that, for infants, the amino acid scores of the NF protein for leucine (0.76), isoleucine (0.8), methionine and cysteine (0.75), phenylalanine and tyrosine (0.68) and tryptophan (0.88) are below the respective amino acid scores recommended by FAO (2013) for this age group. For the population group of children 6 months to 3 years, methionine and cysteine, for which the amino acid score is 0.93 compared to the recommended value by FAO (2013), are the limiting amino acids in the NF protein. With regard to the age group older children, adolescents and adults, the amino acid scores meet or exceed the recommended values by the FAO (2013).

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

3.10. Toxicological information

The applicant provided toxicological studies on the NF, which were conducted in compliance with OECD principles of GLP (OECD, 1998a) and in accordance with the test guidelines No 471, 487 and 408 from OECD (OECD, 1997, 1998b, 2010).

These studies, which were claimed proprietary by the applicant, are listed in Table 7.

TABLE 7.

List of toxicological studies with the NF.

Reference	Type of study	Test system	Dose
Unpublished (2019a)	Bacterial reverse mutation test (GLP, OECD TG 471)	S. typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2 uvrA	Up to 5000 μg/plate (absence and presence of S9 mix)
Unpublished (2019b)	In vitro mammalian cell micronucleus test (GLP, OECD 487)	Human lymphocytes	12.5, 25, or 50 μg/mL with and without S9 for 4 h (Experiment I) or without S9 for 44 h (Experiment II)
Unpublished (2019c)	14‐day dose range‐finding study	CRL Sprague‐Dawley CD® IGS rats	0, 4167, 8333, 12,500 mg/kg per day (% in diet 0, 5, 10, 15)
Unpublished (2020)	90‐day repeated dose oral toxicity study (GLP, OECD TG 408)	Sprague Dawley rats	50,000, 100,000 and 150,000 mg/kg (calculated to be at least 2744.3, 5518.9, 8115.9 mg/kg/day in males and 3341.2, 6996.0 and 9891.7 mg/kg bw per day in females)

3.10.1. Genotoxicity

The applicant investigated the mutagenicity of the NF in a dehydrated form with a bacterial reverse mutation test according to OECD TG 471 (Unpublished report, 2019a) and an in vitro mammalian cell micronucleus test according to OECD TG 487 (Unpublished report, 2019b).

The NF was tested using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2 uvrA at concentrations of 0 (sterile water, solvent control), 1.58, 5.0, 15.8, 50, 158, 500, 1580 and 5000 μg/plate in the absence and presence of metabolic activation via the plate incorporation method (main test) or pre‐incubation method (confirmatory test). No mutagenicity from the NF was observed in the absence or presence of metabolic activation. No cytotoxicity was observed, but precipitation occurred in all tests at concentrations of 1580 μg/plate or more, which according to the applicant did not affect counts of revertant colonies. Individual plate contamination was noted in strain TA98 at a concentration of 5000 μg/plate in the absence of metabolic activation in the main test.

The applicant further investigated the genotoxicity of the NF with an in vitro mammalian cell micronucleus test according to OECD TG 487 (Unpublished report, 2019b). NF samples in dehydrated form were tested using human lymphocytes at concentrations of 12.5, 25 or 50 μg/mL with and without S9 for 4 h (Experiment I) or without S9 for 44 h (Experiment II). During a preliminary test, precipitation of the NF test material had been observed at concentrations of 100 μg/mL and above without metabolic activation, and at 50 μg/mL and above with metabolic activation. In the main experiments (I and II), precipitation was observed at the highest concentration (50 μg/mL) with and without metabolic activation. The culture medium was used as a negative control while DMSO (1% v/v in culture medium) was used as a solvent control. No cytotoxic effects and no genotoxicity were observed up to 50 μg/mL.

Taking into account the test results provided, the Panel considers that there are no concerns regarding genotoxicity.

3.10.2. Subchronic toxicity

In a 90‐day repeated dose oral toxicity study conducted according to OECD TG 408 (OECD, 1998a, 1998b; Unpublished report, 2020), Sprague Dawley rats (n = 10 males and 10 females per group) were exposed to the NF in a dehydrated form as 0% diet (0 mg/kg), 5% diet (50,000 mg/kg), 10% diet (100,000 mg/kg) or 15% diet (150,000 mg/kg), corresponding to mean overall daily intake levels for males and females of 0, 2744.3, 5518.9 or 8115.9 mg/kg bw per day and 0, 3341.2, 6996.0 and 9891.7 mg/kg bw per day, respectively. The applicant indicated that the dehydration was conducted to dose the animals ‘at a quantity and nutrient levels that would not cause any nutritional deficiencies’. Diets were formulated using the supplied basal diets to which the test substance was added to achieve the target doses and to ensure comparable fat, protein and carbohydrate content across dose groups.

There were no mortalities or clinical observations attributable to the test item. Body weight, body weight gain and food consumption remained unchanged in treated animals as compared to the control group.

There were no statistically significant changes in haematology parameters and urinalysis. Clinical chemistry parameters remained unchanged in females. In males, statistically significant non‐dose dependent decreases in LDL, HDL and total cholesterol levels were observed. Additionally, statistically significant non‐dose dependent changes in sorbitol dehydrogenase (SDH) were noted at all doses tested (−28%, +35% and +22% at the low, middle and high doses, respectively).

A dose‐dependent increase in thyroid‐stimulating hormone (TSH) levels was observed in both males and females, with statistical significance starting from the middle dose (+5%, +108% and +118% in males, +14%, +137% and +145% in females at the low, middle and high doses, respectively). These changes were substantially higher than the mean values, but within the range of historical control data. These changes were not accompanied by statistically significant alterations of triiodothyronine (T3) and thyroxine (T4) levels, except for an increase in T3 levels in females at the middle dose (+37%). There were no statistically significant changes in thyroid weights or histopathology as compared with the control group.

Several statistically significant changes were noted in males at the lowest dose tested, including a decrease in absolute thymus weight (−23%), an increase in pituitary weight relative to body weight (+33%) and an increase in seminal vesicles and coagulating gland weights (both absolute and relative to body weight, +42%). The Panel notes that pituitary, seminal vesicle and coagulating gland weights are subject to high variation and that historical control data were not provided for these endpoints. There were no histopathological changes in these organs as compared to the control. The Panel considers these findings as incidental.

Macroscopic observations at necropsy were considered incidental. At the microscopic level, a unilateral, acute, mild, segmental necrosis of the hippocampus was observed in one male from the high‐dose group (i.e. 8115.9 mg/kg bw per day).

Considering the evidence for thyroidal effects at the middle and high doses, and the lack of robustness of other findings at the low dose tested, the Panel considers the lowest dose tested in males as the no observed adverse effect level (NOAEL), i.e. 2744 mg/kg bw per day of the NF in a dehydrated form. The NF is proposed as a frozen biomass, and this corresponds to approximately 10,400 mg/kg bw per day ¹⁷ of the NF.

3.10.3. Human data

No human studies conducted with the NF were provided.

3.11. Allergenicity

The NF contains protein (> 11%). The applicant performed a comparison of all proteins predicted on the basis of the entire genome of the Fusarium. Using IgenBio's ERGO Bioinformatics platform, the annotated data were converted to a FASTA file that represented the expected full proteome of the hypothetical proteins of Fusarium str. flavolapis. This FASTA file was searched against the AllergenOnline.org database. No homologies to high potency allergenic proteins that are common in the food supply chain (FARRP, 2021) ¹⁸ have been identified. There were no matches to 2S albumins of peanut or tree nuts, and no matches to lipid transfer proteins from peach or related fruits. There were no significant matches to vicilins or tropomyosins that are major sources of severe reactions from peanut, tree nut, shrimp, cockroach or house dust mites. From the bioinformatic assessment, the applicant concluded on the Fusarium str. flavolapis on the basis of the structural homology with proteins from F. venenatum that the allergenicity potential of the NF would be no greater than the risk of food allergy from mycoprotein in Fusarium venenatum products. In addition, the applicant provided an allergenicity literature search on Fusarium sp. proteins and concluded that allergic reactions from consumption of foods containing proteins from various Fusarium sp. are rare, even if there are case reports, including one on a fatal incidence, of allergic reactions following the consumption of F. venenatum available in the literature (Hoff et al., 2003; Katona & Kaminski, 2002; Tee et al., 1993).

The Panel considers that, given the protein content of the NF, allergic reactions may occur. However, the risk of allergic reactions is considered as comparable to the consumption of other food products produced from Fusarium fungi.

3.12. Discussion

The NF which is the subject of the application is the frozen form of heat‐treated biomass of Fusarium strain flavolapis (F. sp. strain flavolapis) fungus.

The Panel notes that the NF strain, Fusarium strain flavolapis, has the potential to produce the mycotoxins fumonisins; however, the analytical data on mycotoxins showed that the levels in the NF were below the limits of detection (LODs) of the analytical methods used.

The NF is intended to be used as an ingredient in several food products and the target population is the general population. The Panel considers that, taking into account the composition of the NF and the proposed conditions of use, the NF is not nutritionally disadvantageous.

Considering the lack of robustness of findings at the low dose and the evidence for thyroidal effects at the middle and high dose in the subchronic toxicity study provided by the applicant with the NF in a dehydrated form, the Panel considers the lowest dose tested as the NOAEL, i.e. 2744 mg/kg bw per day. The NF being proposed as a frozen biomass with 75% moisture, this corresponds to approximately 10,400 mg/kg bw per day of the NF.

Under the proposed conditions of use, the highest intake estimates of the NF (at the 95th percentile) range from 189 mg/kg bw per day for adolescents to 556 mg/kg bw per day for infants.

Applying a default uncertainty factor of 200 to the NOAEL, the Panel considers that the margins of exposure (i.e. 55 in adolescents and 19 in infants) between the intake of the NF at the proposed use and use levels and the identified NOAEL are insufficient.

The Panel considers that it is likely that the NF may trigger allergic reactions in fungi (e.g. Aspergillus sp., Penicillium sp. or Fusarium sp.) allergic subjects.

3.13. Conclusions

The Panel concludes that, based on the available data, the safety of the NF, i.e. biomass of Fusarium strain flavolapis (F. sp. strain flavolapis) fungus cannot be established under the proposed conditions of use.

ABBREVIATIONS

ADME

Absorption, distribution, metabolism and excretion

AOAC

Association of Official Analytical Collaboration

ATCC

American Type Culture Collection

BLAST

Basic Local Alignment Search Tool

bw

body weight

CFU

colony forming units

GMP

Good Manufacturing Practice

GRAS

generally recognised as safe

HACCP

Hazard Analysis Critical Control Points

ICP‐MS

inductively coupled plasma mass spectrometry

ITS

internal transcribed spacer

LOD

limit of detection

LOQ

limit of quantification

MLs

maximum levels

NDA

Panel on Nutrition, Novel Foods and Food Allergens

NF

novel food

OECD

Organisation for Economic Co‐operation and Development

PDCAAS

Protein Digestibility Corrected Amino Acid Score

NOAEL

no observed adverse effect level

TG

Test Guideline

TAMC

total aerobic microbial count

WGS

whole genome sequence

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2021‐00519

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PANEL MEMBERS

Dominique Turck, Torsten Bohn, Montaña Cámara, Jacqueline Castenmiller, Stefaan De Henauw, Karen Ildico Hirsch‐Ernst, Ángeles Jos, Alexandre Maciuk, Inge Mangelsdorf, Breige Mcnulty, Androniki Naska, Kristina Pentieva, Alfonso Siani, and Frank Thies.

Supporting information

ANNEX A Dietary exposure estimates to the novel food for each population group from each EU dietary survey

APPENDIX A. Batch‐to‐batch amino acid analysis

Amino acid (% amino acid, wet weight)	Batch #6	Batch #7	Batch #8	Batch #9	Batch #10
Cysteine	0.09	0.11	0.10	0.09	0.09
Methionine	0.20	0.24	0.21	0.20	0.21
Tryptophan	0.20	0.24	0.20	0.19	0.20
Threonine	0.62	0.71	0.60	0.58	0.59
Valine	1.28	1.42	1.23	1.13	1.09
Isoleucine	0.54	0.65	0.55	0.53	0.54
Leucine	0.91	1.06	0.90	0.87	0.88
Tyrosine	0.36	0.47	0.41	0.38	0.38
Phenylalanine	0.50	0.6	0.51	0.48	0.49
Lysine	0.95	1.08	0.92	0.88	0.97
Histidine	0.28	0.32	0.27	0.26	0.27
Aspartic acid	1.12	1.28	1.09	1.04	1.07
Serine	0.57	0.65	0.55	0.52	0.54
Glutamic acid	1.31	1.53	1.30	1.25	1.26
Proline	0.52	0.6	0.52	0.49	0.52
Glycine	0.55	0.63	0.54	0.51	0.53
Alanine	0.71	0.83	0.7	0.67	0.69
Arginine	0.68	0.83	0.71	0.66	0.69

ANNEX A. Dietary exposure estimates to the novel food for each population group from each EU dietary survey

Information provided in this Annex is shown in an Excel file (downloadable at https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9536#support‐information‐section).

EFSA NDA Panel (EFSA Panel on Nutrition, Novel Foods and Food Allergens) , Turck, D. , Bohn, T. , Cámara, M. , Castenmiller, J. , De Henauw, S. , Jos, Á. , Maciuk, A. , Mangelsdorf, I. , McArdle, H. J. , Mcnulty, B. , Moldeus, P. , Naska, A. , Pentieva, K. , Siani, A. , Thies, F. , Aguilera‐Gómez, M. , Cubadda, F. , Frenzel, T. , … Hirsch‐Ernst, K. I. (2025). Safety of the fungal biomass from Fusarium species strain flavolapis as a novel food pursuant to Article 10 of Regulation (EU) 2015/2283. EFSA Journal, 23(9), e9536. 10.2903/j.efsa.2025.9536