Safety of Clostridium Butyricum TO-A as a Novel Food Pursuantto Regulation (EU) 2015/2283

EFSA Journal. 2025;23:e9371. | 1 of 15 https://doi.org/10.2903/j.efsa.2025.9371 efsa.onlinelibrary.wiley.com/journal/1831-4732 SCIENTIFIC OPINION Safety of Clostridium butyricum TOA as a novel food pursuant to Regulation (EU) 2015/2283 EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) | Dominique Turck | Torsten Bohn | Montaña Cámara | Jacqueline Castenmiller | Stefaan DeHenauw | Ángeles Jos | Alexandre Maciuk | Inge Mangelsdorf | Breige McNulty | Androniki Naska | Kristina Pentieva | Alfonso Siani | Frank Thies | Margarita AguileraGómez | Francesco Cubadda | Thomas Frenzel | Marina Heinonen | Helle Katrine Knutsen | Monika NeuhäuserBerthold | Morten Poulsen | Miguel PrietoMaradona | Josef Rudolf Schlatter | Alexandros Siskos | Henk van Loveren | Reinhard Ackerl | Dafni Maria Kagkli | Karen Ildico HirschErnst Adopted: 24 March 2025 DOI: 10.2903/j.efsa.2025.9371 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 Clostridium butyricum TOA as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF, which is proposed by the applicant to be used as a food supplement, 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 90day oral toxicity study in rats, and considering an uncertainty factor of 200, the Panel estimated a safe dose in humans of 4.5 × 106 colony forming unit (CFU)/kg body weight (bw) per day. However, considering that appropriate initial bacteria colonisation of the gastrointestinal tract in humans, in particular during the first 3 years of life, profoundly affects health during infancy and childhood, that disruptions to the microbiota early in life can have lasting health effects into adulthood, and taking into account that the 90day oral toxicity study was conducted in adult rats, the Panel considers that the target population for the NF should be restricted to children above 3 years of age, adolescents and adults, excluding pregnant and lactating women. The Panel concludes that the NF, C. butyricum TOA, is safe at 1.0 × 108 CFU/day for other children (3 to < 10 years), 2.0 × 108 CFU/day for adolescents from 10 to < 14 years, 2.8 × 108 CFU/day for adolescents from 14 to < 18 years and 3.2 × 108 CFU/day for adults, excluding pregnant and lactating women. KEYWORDS Clostridium butyricum, food supplement, microorganism, novel foods, safety This is an open access article under the terms of the Creative Commons Attribution-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited and no modifications or adaptations are made. © 2025 European Food Safety Authority. EFSA Journal published by Wiley-VCH GmbH on behalf of European Food Safety Authority. Correspondence: [email protected] The declarations of interest of all scientific experts active in EFSA's work are available at https:// open. efsa. europa. eu/ experts 2 of 15 | SAFETY OF C. BUTYRICUM TOA AS A NF CONTENTS Abstract................................................................................................................................................................................................................................1 1. Introduction ..............................................................................................................................................................................................................3 1.1. Background and Terms of Reference as provided by the requestor .........................................................................................3 1.2. Additional information ..............................................................................................................................................................................3 2. Data and Methodologies ......................................................................................................................................................................................3 2.1. Data ...................................................................................................................................................................................................................3 2.2. Methodologies..............................................................................................................................................................................................4 3. Assessment ................................................................................................................................................................................................................4 3.1. Introduction ...................................................................................................................................................................................................4 3.2. Identity of the NF .........................................................................................................................................................................................4 3.3. Production process .....................................................................................................................................................................................5 3.4. Compositional data .....................................................................................................................................................................................6 3.4.1. Stability ............................................................................................................................................................................................7 3.5. Specifications ................................................................................................................................................................................................7 3.6. History of use of the NF and/or of its source ......................................................................................................................................7 3.6.1. History of use of the source ......................................................................................................................................................7 3.6.2. History of use of the NF ..............................................................................................................................................................7 3.7. Proposed uses and use levels ..................................................................................................................................................................8 3.7.1. Target population .........................................................................................................................................................................8 3.7.2. Proposed uses and use levels ..................................................................................................................................................8 3.8. Absorption, distribution, metabolism and excretion (ADME) .....................................................................................................8 3.9. Nutritional information ..............................................................................................................................................................................8 3.10. Toxicological information .........................................................................................................................................................................8 3.10.1. Genotoxicity ...................................................................................................................................................................................9 3.10.2. Subacute toxicity ........................................................................................................................................................................11 3.10.3. Subchronic toxicity ....................................................................................................................................................................11 3.10.4. Human data ..................................................................................................................................................................................11 3.11. Allergenicity ................................................................................................................................................................................................ 12 4. Discussion................................................................................................................................................................................................................ 12 5. Conclusions ............................................................................................................................................................................................................. 12 5.1. Protection of proprietary data in accordance with Article 26 of Regulation (EU) 2015/2283........................................ 12 Abbreviations ................................................................................................................................................................................................................. 12 Requestor ......................................................................................................................................................................................................................... 13 Question number .......................................................................................................................................................................................................... 13 Copyright for nonEFSA content.............................................................................................................................................................................. 13 Panel members .............................................................................................................................................................................................................. 13 References........................................................................................................................................................................................................................ 13 18314732, 2025, 5, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9371 by Readcube (Labtiva Inc.), Wiley Online Library on [05/08/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 3 of 15 SAFETY OF C. BUTYRICUM TOA AS A NF 1 | INTRODUCTION 1.1 | Background and Terms of Reference as provided by the requestor On 10 November 2021, the company ‘TOA Biopharma Co. Ltd.’ 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 Clostridium butyricum TOA as a novel food (NF). The applicant requests to authorise the use of Clostridium butyricum TOA as a NF in food supplements as defined in Directive 2002/46/EC, excluding infants below 3 months of age. The applicant has requested data protection under Article 26 of Regulation (EU) 2015/2283. In accordance with Article 10(3) of Regulation (EU) 2015/2283, the European Commission asks EFSA to provide a scientific opinion on Clostridium butyricum TOA. In this opinion on Clostridium butyricum TOA, 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. 1.2 | Additional information In 2020, Clostridium (C.) butyricum was notified to the EFSA Panel on Biological Hazards (BIOHAZ) for its assessment of qualified presumption of safety (QPS). The BIOHAZ Panel did not recommend C. butyricum for QPS status because some strains contain pathogenicity factors. Thus, the BIOHAZ Panel excluded this species for further QPS evaluation (EFSA BIOHAZ Panel,2020). This conclusion was confirmed by the BIOHAZ Panel in 2022 (EFSA BIOHAZ Panel,2022). In 2022, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) assessed the feed additive BIOTHREE®, which is based on viable cells/spores of a combination of three bacteria including the NF, i.e. Clostridium butyricum TOA (FERM BP10866), plus Bacillus subtilis FERM BP07462 and Enterococcus lactis FERM BP10867. The FEEDAP Panel concluded that BIOTHREE® is safe for the target species (chickens, turkeys, all avian species) under the proposed conditions of use. The Panel also concluded that the additive is safe for the consumers of products derived from animals receiving the additive (EFSA FEEDAP Panel, 2022). Following that assessment, the feed additive was authorised by Commission Implementing Regulation (EU) 2024/2185.1 In 2014, another C. butyricum strain, i.e. C. butyricum (CBM 588) (also denominated as C. butyricum MIYAIRI 588 (CBM 588)), was authorised to be placed on the market in food supplements at 1.35 × 108 CFU/day in the Union as a NF by Commission Implementing Decision 2014/907/EU.2 Consequently, C. butyricum (CBM 588) was also included in the Union list of novel foods.3 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.4 Administrative and scientific requirements for NF applications referred to in Article 10 of Regulation (EU) 2015/2283 are listed in the Commission Implementing Regulation (EU) 2017/2469.5 A common and structured format on the presentation of NF applications is described in the EFSA guidance on the preparation and presentation of a NF application (EFSA NDA Panel,2021). As indicated in this guidance, it is the duty of the applicant to provide all 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. 1Commission Implementing Regulation (EU) 2024/2185 of 3 September 2024 concerning the authorisation of a preparation of Bacillus subtilis FERM BP07462, Enterococcus lactis FERM BP10867 and Clostridium butyricum FERM BP10866 as a feed additive for all poultry species for fattening, all poultry species reared for laying or breeding and ornamental birds (holder of authorisation: Toa Biopharma Co., Ltd.). C/2024/5963. OJ L, 2024/2185. 2Commission Implementing Decision of 11 December 2014 authorising the placing on the market of Clostridium butyricum (CBM 588) as a novel food ingredient under Regulation (EC) No 258/97 of the European Parliament and of the Council (notified under document C(2014) 9345). OJ L 359, 16.12.2014, pp. 153–154. 3Commission Implementing Regulation (EU) 2017/2470 of 20 December 2017 establishing the Union list of novel foods in accordance with Regulation (EU) 2015/2283 of the European Parliament and of the Council on novel foods. OJ L 351, 30.12.2017, pp. 72–201. 4https:// open. efsa. europa. eu/ quest ions/ EFSAQ202200010 . 5Commission Implementing Regulation (EU) 2017/2469 of 20 December 2017 laying down administrative and scientific requirements for applications referred to in Article 10 of Regulation (EU) 2015/2283 of the European Parliament and of the Council on novel foods. OJ L 351, 30.12.2017, pp. 64–71. 18314732, 2025, 5, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9371 by Readcube (Labtiva Inc.), Wiley Online Library on [05/08/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 4 of 15 | SAFETY OF C. BUTYRICUM TOA AS A NF The applicant submitted a confidential and a nonconfidential version of a dossier following the EFSA guidance 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 Art. 38 of Regulation (EC) No 178/20026 and taking into account the protection of confidential information and of personal data in accordance with Articles 39 to 39e of the same Regulation, and of the Decision of EFSA's Executive Director laying down practical arrangements concerning transparency and confidentiality,7 the nonconfidential version of the dossier has been published on Open.EFSA.8 According to Art. 32c(2) of Regulation (EC) No 178/2002 and to the Decision of EFSA's Executive Director laying down the practical arrangements on presubmission phase and public consultations, EFSA carried out a public consultation (PC0430)9 on the nonconfidential version of the technical dossier from 05/04/2023 to 26/04/2023 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: • Annex_I_2_1_1_Manufacturing_process. • Annex_l_3_1_1_Nutritional_analysis (part 1–5). • Annex_l_3_1_2_C butyricum_analysis (part 1–5). • Annex_l_3_1_3_Purity (part 1–5). • Annex_l_9_2_1_AMES. • Annex_l_9_2_2_Micronucleous. • Annex_l_9_2_3_Ames_supernatant. • Annex_l_9_2_4_Micronucleus_supernatant. • Annex_l_9_2_5_in_vivo_micronucleus_comet_supernatant. • Annex_l_9_2_6_Lysis_efficiency_French_Press. • Annex_l_9_2_6_Appendix1_SEM_images. • Annex_l_9_2_7_Ames_Lysate. • Annex_l_9 _2_8_Micronucleus_Lysate. • Annex_l_9_3_Subchronic_toxicity. 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 the consumption of the NF under the proposed conditions of use, and it 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 Clostridium butyricum TOA. The NF is produced by anaerobic fermentation. The biomass is separated by centrifugation, dried and mixed with potato starch. The NF is proposed to be used in food supplements (as defined in Directive 2002/46/EC). The target population proposed by the applicant is the general population excluding infants below 3 months of age. The NF falls under Article 3(2)(a) (ii) food consisting of, isolated from or produced from microorganisms, fungi or algae, as defined in Regulation (EU) 2015/2283. 3.2 | Identity of the NF The NF is dried C. butyricum TOA, which is a sporeformer, strictly anaerobic and belongs to the Grampositive Clostridium genus. 6Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety. OJ L 31, 1.2.2002, pp. 1–48. 7Decision available at: https://www.efsa.europa.eu/en/corporatepubs/transparencyregulationpracticalarrangements. 8The nonconfidential version of the dossier has been published on Open.EFSA and is available at the following link: https:// open. efsa. europa. eu/ dossi er/ NF20211068. 9https:// conne ct. efsa. europa. eu/ RM/s/ consu ltati ons/ publi ccons ultat ion2/ a0l09 00000 6r2l7/ pc0430. 18314732, 2025, 5, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9371 by Readcube (Labtiva Inc.), Wiley Online Library on [05/08/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 5 of 15 SAFETY OF C. BUTYRICUM TOA AS A NF C. butyricum TOA was originally isolated from the gut of healthy humans. When grown anaerobically at 37°C in iron sulfite agar for 24 h, circular black colonies of C. butyricum TOA appear. Gramstaining reveals straight or slightly curved rodshaped cells and oval spores. The applicant was requested to clarify the nature of the NF, and in particular, the respective percentages of cells and spores. In reply, the applicant provided analyses of cells and spores for five batches of the NF, which indicated that the NF is a mixture of spores and vegetative cells with about 85% (range: 74%–89%) of spores. The full taxonomic classification of the employed strain is the following: Domain: Bacteria; Phylum: Firmicutes; Class: Clostridia; Order: Clostridiales; Family: Clostridialaceae; Genus: Clostridium; Species: Clostridium butyricum; Strain: Clostridium butyricum TOA. The applicant indicated as inhouse identifiers of C. butyricum TOA the following ones: Clostridium, Clostridium TOA, CB TOA, Clostridium butyricum, CB, CBT. The strain is deposited under number FERM BP10866 at the Japanese National Institute of Technology and Evaluation, which follows the Budapest Treaty on the International Recognition of the Deposit of Microorganisms. The certificate of deposit was provided by the applicant. The complete genome of C. butyricum TOA and its two plasmids pCBTOA1 and pCBTOA2 were sequenced, and the whole genome sequence (WGS) was provided. The overall organisation of the genome was determined based on the WGS alignment with assembled C. butyricum genomes in the NCBI GenBank. The provided sequence produced a nearly complete genome that belonged to this bacterial species. The taxonomic identification of the strain TOA as C. butyricum was established by alignment and nonalignment tools for the WGS data. The 16S rRNA gene analysis and the alignmentbased calculation of average nucleotide identity (ANI) assigned the strain to the Clostridium butyricum species, with an ANI of 99.99 with the type strain C. butyricum KNUL09T. Likewise, the phylogenetic tree based on the core gene alignment grouped the test strain together with C. butyricum KNUL09T. Based on these results, the taxonomic identification of the strain was confirmed as Clostridium butyricum. The WGS data allowed the assessment of the presence of antimicrobial resistance genes and potential toxin and virulence factors. The screening of the strain genome revealed that no putative antimicrobial resistance genes above 80% identity and 70% coverage were present (NCBI Bacterial Antimicrobial Resistance Reference Gene database and ResFinder database). The phenotypic resistance of the test strain was assessed according to the EFSA cutoff values for Grampositive bacteria (EFSA FEEDAP Panel,2018). The phenotypic testing of the antimicrobial susceptibility indicated that the strain is resistant to the aminoglycosides gentamicin, kanamycin and streptomycin. The minimum inhibitory concentration for kanamycin and streptomycin was 128 mg/L as compared to the cutoff values defined by the EFSA FEEDAP Panel in 2018 (16 mg/L for kanamycin and 8 mg/L for streptomycin, respectively). However, this phenotypic resistance is intrinsic to these species and anaerobes in general (EFSA FEEDAP Panel,2009, 2014; Isa etal.,2016) and no horizontal gene transfer is expected. The major virulence factors of pathogenic Clostridium spp. (i.e. C. botulinum, C. difficile, C. tetani and C. perfringens) include α, β and ε toxins and highly potent neurotoxins (BONT/AG) and they could be found in other Clostridium species. However, according to interrogations performed with the WGS of C. butyricum TOA against the virulence factor database (VFDB; full set, accessed on 14/07/2021), genes for these specific toxins or other (neuro)toxins are not present. Three factors were found in the database (with different percentages of identity), i.e. chaperonin GroEL (VFG012102: WP_011967678), RNA methyltransferase (VFG012183: WP_011969031.1) and urease accessory protein UreG (VFG019533: WP_000238754.1), which are all genes that are related to normal cellular functions in bacteria. The Panel considers that the NF is sufficiently characterised and does not raise safety concerns. 3.3 | 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. Flow charts of the production process and detailed information of the culture conditions and media together with the quality control methods involved at each step were provided (confidential information). The first step of the process consists in the cultivation and harvesting of C. butyricum. Initially, the C. butyricum TOA inoculum is prepared from a culture aliquot stored at −80°C. Successive subculture steps in liquid media of increasing volumes are performed in order to prepare the inoculation of the main fermenter. The main fermentation is conducted under strict anaerobic conditions at a certain temperature and pH (confidential information) and its microscopic morphology is confirmed. Biomass separation is achieved by centrifugation at a certain speed (confidential information). The cells are spraydried to bulk powder and mixed with potato starch to achieve the desired concentration of C. butyricum TOA. Cell counting, microbiological and purity tests as well as qualitative visual inspection of the powder are performed. The final product is packed to scale and stored under certain conditions (confidential information). The applicant was requested to provide more information on the potato starch, e.g. if there is some kind of particular potato starch used (e.g. resistant starch), which would favour survival of the C. butyricum TOA spores/cells in the gastrointestinal tract, or if some kind of encapsulation is applied. In reply, the applicant stated that neither resistant starch nor any form of encapsulation is used. According to the applicant, the main purpose of the potato starch is ‘to stabilise the spores by keeping a dry environment’. The Panel considers that the production process is sufficiently described and does not raise safety concerns. 18314732, 2025, 5, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9371 by Readcube (Labtiva Inc.), Wiley Online Library on [05/08/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6 of 15 | SAFETY OF C. BUTYRICUM TOA AS A NF 3.4 | Compositional data The NF, dried C. butyricum TOA, including the potato starch, consists of carbohydrates (~96%), moisture (~1.7%), dietary fibre (~1.5%), proteins (~0.6%) and minor amounts of fat (< 0.2%). 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 independent lots of the NF (Table1). In addition to the proximate analyses, the applicant provided detailed analyses of the fatty acids, which were all below the limit of quantification (LOQ = 0.01 g/100 g). Analyses of pesticides, mycotoxins and aflatoxins, dioxins and PCBs in the NF were also provided. No residues of these pesticides, mycotoxins and aflatoxins were detected above their respective LOQ or limit of detection as demonstrated by the provided analytical results. TABLE 1 Batch to batch analysis of the NF. Parameter (unit) Batch number Method of analysis#1 #2 #3 #4 #5 Species identification Conforms Conforms Conforms Conforms Conforms WGS and 16S rRNA; Gramstaining Sulphitereducing (Clostridium) counts (CFU/g) 5.2 × 1094.0 × 1093.9 × 1094.9 × 1095.3 × 109ISO 15213: 2003 Moisture & volatile matters (%) 1.81 1.64 1.59 2.09 1.52 Gravimetry Crude protein (%) 0.60 0.57 0.63 0.72 0.63 Kjeldahl, Regulation (EC) no 152/2009 Crude fat (%) < 0.20 < 0.20 < 0.20 < 0.20 < 0.20 Gravimetry Regulation (EC) no 152/2009 ‘Dietary fibre’ (%)a1.91 1.22 1.80 0.96 1.51 Gravimetry Regulation (EC) no 152/2009 Ash (%) 0.43 0.48 0.46 0.42 0.34 Gravimetry Regulation (EC) no 152/2009 Sodium (Na) (%) 0.078 0.076 0.076 0.076 0.077 PNTMF860/PNTMF859 (ICPOES) Carbohydrates (%) 95.25 96.09 95.52 95.81 96.00 PNTM605 (Calculated) Fructose (%) < 0.50 < 0.50 < 0.50 < 0.50 < 0.50 PNTMF144 (HPLCRefractive index. AOAC 984.22) Glucose (%) < 0.50 < 0.50 < 0.50 < 0.50 < 0.50 PNTMF144 (HPLCRefractive index. AOAC 984.22) Saccharose (%) < 0.50 < 0.50 < 0.50 < 0.50 < 0.50 PNTMF144 (HPLCRefractive index. AOAC 984.22) Maltose (%) < 0.50 < 0.50 < 0.50 < 0.50 < 0.50 PNTMF144 (HPLCRefractive index. AOAC 984.22) Lactose monohydrate (%) < 0.50 < 0.50 < 0.50 < 0.50 < 0.50 PNTMF144 (HPLCRefractive index. AOAC 984.22) Heavy metals Cadmium (mg/kg) < 0.20 < 0.20 < 0.20 < 0.20 < 0.20 EN 15510:2007 Lead (mg/kg) < 2.50 < 2.50 < 2.50 < 2.50 < 2.50 EN 15510:2007 Arsenic (mg/kg) < 2 < 2 < 2 < 2 < 2 EN 15510:2007 Mercury (mg/kg) < 0.02 < 0.02 < 0.02 < 0.02 < 0.02 UNEEN 13806:2003 Microbial Aerobic mesophilic plate count (APC) (CFU/g) < 40 < 40 < 40 < 40 < 40 ISO 48331: 2014 Yeasts (CFU/g) < 1 × 102< 1 × 102< 1 × 102< 1 × 102< 1 × 102ISO 215272: 2008 Moulds (CFU/g) < 1 × 102< 1 × 102< 1 × 102< 1 × 102< 1 × 102ISO 215272: 2008 Salmonella spp. (in 25 g) n.d. n.d. n.d. n.d. n.d. ISO 65791:2017/A1 Total coliforms (CFU/g) < 10 < 10 < 10 < 10 < 10 ISO 4832: 2006 E. coli (CFU/g) < 10 < 10 < 10 < 10 < 10 ISO 166492: 2001 Abbreviations: AOAC, Association of Official Analytical Collaboration; APC, aerobic mesophilic plate count; CFU, colony forming units; EC, European Commission; EN, European Standard; n.d., not detected; HPLC, highperformance liquid chromatography; ICPOES, Inductively Coupled Plasma Optical Emission Spectroscopy; rRNA, ribosomal ribonucleic acid; UNE, Spanish standardisation body; WGS, whole genome sequence. aThe Panel notes that ‘dietary fibre’ refers to nondigestible polysaccharides. 18314732, 2025, 5, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9371 by Readcube (Labtiva Inc.), Wiley Online Library on [05/08/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 7 of 15 SAFETY OF C. BUTYRICUM TOA AS A NF Silicon (Si) analysis was performed in order to detect any siliconbased residues from the antifoaming agent (i.e. dimethyl polysiloxane (E 900)) used during the manufacturing process, and levels of 18–19 mg/kg of Si were detected. These amounts correspond to about 50 mg of the antifoaming agent (i.e. dimethyl polysiloxane (E 900)). Considering the maximum proposed intakes of the NF, which could reach up to 1 g NF/day (see Table3), the daily intake of dimethyl polysiloxane would be 50 μg (equivalent to 0.7 μg/kg bw per day for adults). Taking into account the ADI for dimethyl polysiloxane of 17 mg/kg bw per day established in 2020 (EFSA FAF Panel,2020), the Panel considers that this does not raise a safety concern. Information was provided on the accreditation of the laboratories that conducted the analyses presented in the application. 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. The tests were carried out at normal storage conditions at 25°C in a dry place (< 60% relative humidity (RH)) for a period of 25 months. The batches were analysed for microbial contaminants and physicochemical characteristics. All batches after 25 months of storage appeared to be solid in the form of fine white homogeneous powder. The CFU content in the NF decreased from an average of 3.8 × 109 CFU/g at the beginning of the storage period to an average of 1.7 × 109 CFU/g after 25 months. Moisture and volatile matter (%) increased from 1% to about 2.4%. The analysed microbial parameters (yeasts, moulds, total aerobic microbial count (TAMC), Salmonella spp., total coliforms) remained within the limits as set in the specifications. Based on these results, the applicant proposed a shelflife of 25 months for the NF. The Panel considers that the data provide sufficient information with respect to the stability of the NF. 3.5 | Specifications The specifications of the NF, as proposed by the applicant, are indicated in Table2. The Panel considers that the information provided on the specifications of the NF is sufficient and does not raise safety concerns. 3.6 | History of use of the NF and/or of its source 3.6.1 | History of use of the source C. butyricum was reported to be widely used in Asia, particularly in Japan, Korea and China (Vos etal.,2009). C. butyricum is found in many environments, including vegetables, soured milk and cheeses (Cassir etal.,2016). 3.6.2 | History of use of the NF C. butyricum TOA has been marketed for humans as part of a combination of three bacteria (i.e. BIOTHREE®) in China, Taiwan and Japan for many decades. TABLE 2 Specifications of the NF. Description: The NF is produced by anaerobic fermentation of Clostridium butyricum TOA, which is harvested by centrifugation, dried and mixed with potato starch Appearance: Odourless white/greyish powder Parameter Specification C. butyricum TOA 1–7 × 109 CFU/g Moisture < 5% Coliforms < 10 CFU/g TAMC ≤ 1 × 103 CFU/g TYMC ≤ 1 × 102 CFU/g Salmonella spp. Not detected in 25 g Abbreviations: CFU, colony forming units; TAMC, total aerobic microbial count; TYMC, total yeast and mould count. 18314732, 2025, 5, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9371 by Readcube (Labtiva Inc.), Wiley Online Library on [05/08/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 8 of 15 | SAFETY OF C. BUTYRICUM TOA AS A NF BIOTHREE® (including C. butyricum TOA) was assessed as a feed additive by the EFSA FEEDAP Panel in 2022. The FEEDAP Panel concluded that BIOTHREE® is safe for the target species (chickens, turkeys, all avian species) under the proposed conditions of use. The Panel also concluded that the additive is safe for the consumers of products derived from animals receiving the additive (EFSA FEEDAP Panel,2022). Following that assessment, BIOTHREE®, which includes C. butyricum TOA, was authorised as a feed additive by Commission Implementing Regulation (EU) 2024/2185.10 3.7 | Proposed uses and use levels 3.7.1 | Target population The target population proposed by the applicant is children from 3 months onwards, adolescents and adults. 3.7.2 | Proposed uses and use levels The applicant intends to market the NF as a food supplement. The maximum proposed doses per day, which range from 1 × 108 CFU/day for infants to 1 × 109 CFU/day for adults, as well as the corresponding maximum amounts of the NF in mg per day, are indicated in Table3. 3.8 | Absorption, distribution, metabolism and excretion (ADME) No ADME data were provided for the NF. The applicant stated that the NF, consisting of live microorganisms, can be expected to pass through the gastrointestinal tract unabsorbed and that ADME studies with the NF were therefore not considered necessary. The applicant submitted a study performed with another C. butyricum strain, i.e. CBM588 (Sato & Tanaka,1997). Following a single administration to rats of 107 spores of C. butyricum CBM588, the number of C. butyricum CBM588 cells in the intestinal contents was counted. The results of this study suggest that the administered C. butyricum spores germinated in the upper small intestine, grew mainly from the distal small intestine to the colon and were excreted from the rat intestine within 3 days (Sato & Tanaka,1997). 3.9 | Nutritional information The applicant provided a nutritional analysis of the NF as intended to be marketed (i.e. including the potato starch). According to this analysis, the NF is mostly composed of carbohydrates (95%–96%) with minor amounts of protein (about 0.6%) and fat (below 0.2%). The Panel considers that, taking into account the composition of the NF and the proposed conditions of use, the consumption of the NF is not nutritionally disadvantageous. 3.10 | Toxicological information The applicant provided nine toxicological studies, which were performed with lysed C. butyricum TOA (without potato starch), culture supernatant of the NF (i.e. derived from the manufacturing process of the NF) and BIOTHREE®, respectively. 10Commission Implementing Regulation (EU) 2024/2185 of 3 September 2024 concerning the authorisation of a preparation of Bacillus subtilis FERM BP07462, Enterococcus lactis FERM BP10867 and Clostridium butyricum FERM BP10866 as a feed additive for all poultry species for fattening, all poultry species reared for laying or breeding and ornamental birds (holder of authorisation: Toa Biopharma Co., Ltd.). C/2024/5963. OJ L, 2024/2185. TABLE 3 Proposed use of the NF as a food supplement in various population groups. Population group Age Max. use level (CFU/day) Max. amount of NF (mg/day) Infants excluding those below 3 months 3 to < 12 months 1 × 108 CFU/day 100 mg/day Young children 1 to < 3 years 1 × 108 CFU/day 100 mg/day Other children 3 to < 10 years 3 × 108 CFU/day 300 mg/day Adolescents 10 to < 18 years 6 × 108 CFU/day 600 mg/day Adults ≥ 18 years 1 × 109 CFU/day 1000 mg/day Abbreviation: CFU, colony forming units. 18314732, 2025, 5, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9371 by Readcube (Labtiva Inc.), Wiley Online Library on [05/08/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 9 of 15 SAFETY OF C. BUTYRICUM TOA AS A NF With the exception of the subacute study (Sakamaki etal.,1989), the studies were conducted in compliance with principles of good laboratory practice (GLP) (OECD, 1998) and followed respective test guidelines (OECD, 1997, 2010, 2016a, 2016b) and Ministry of Health and Welfare (Japan), respectively). The studies, which were claimed proprietary by the applicant, are listed in Table4. 3.10.1 | Genotoxicity Originally the applicant undertook genotoxicity testing by using as test item the food supplement BIOTHREE®, which includes the NF, i.e. C. butyricum TOA (at 1 × 108 CFU/g), but also contains B. subtilis TOA (at 1 × 108 CFU/g) and E. faecium T110 (at 1 × 109 CFU/g). The bacterial reverse mutation test (Unpublished,2018a) was carried out with five Salmonella typhimurium strains (TA1535, TA1537, TA98, TA100 and TA102), both with and without metabolic activation, in two independent assays (the second one according to the preincubation protocol). The test item (BIOTHREE®) was used in the form of a dimethylsulfoxide (DMSO) extract at 2 g/mL (i.e. 2.4 × 109 CFU/mL). The top dose volumes were 100 and 50 μL/plate of DMSO extract in the conditions without and with preincubation, respectively. No increases in the number of revertants were noted with any strain under any condition. In the invitro mammalian cell micronucleus test (Unpublished,2018b), the genotoxic activity of BIOTHREE® was assessed in TK6 lymphoblastoid human cells using a DMSO extract at 2 g/mL (i.e. 2.4 × 109 CFU/mL). The top concentration tested was 1% of DMSO extract. Two lower concentrations (0.5 and 0.25%) were also analysed. Following a preliminary cytotoxicity assay, the genotoxicity assay was carried out using a shortterm 3 h treatment with and without metabolic activation, followed by a recovery period and a 27 h continuous treatment without metabolic activation and no recovery period. BIOTHREE® induced no statistically or biologically significant increases in the number of micronucleated cells under any condition. Following an EFSA request for additional genotoxicity testing of the NF, for both the lysate of the NF and the culture supernatant (in separate tests), the applicant submitted the studies as described below. For the lysis of the NF (after removing TABLE 4 List of provided toxicological studies. Reference Type of study Test item Test system Dose Study No. FSRIPL 180806 (Unpublished,2018a) Bacterial reverse mutation test (GLP, OECD TG 471) BIOTHREE® (DMSOextract) S. typhimurium TA98, TA100, TA102, TA1535 and TA1537 Up to 100 μL/plate (absence and presence of S9 mix) Study No. FSRIPL 180807 (Unpublished,2018b) In vitro mammalian cell micronucleus test (GLP, OECD TG 487) BIOTHREE® (DMSOextract) TK6 lymphoblastoid human cells Up to 1% of the DMSO extract Study No. FSRIPL 221204 (Unpublished,2024a) Bacterial reverse mutation test (GLP, OECD TG 471) NF – lysate S. typhimurium TA98, TA100, TA102, TA1535 and TA1537 Up to 100 μL/plate Study No. FSRIPL 221206 (Unpublished,2024b) In vitro mammalian cell micronucleus test (GLP, OECD TG 487) NF – lysate TK6 lymphoblastoid human cells Up to 10% Study No. FSRIPL 221203 (Unpublished,2023a) Bacterial reverse mutation test (GLP, OECD TG 471) NF – culture supernatant S. typhimurium TA98, TA100, TA102, TA1535 and TA1537 Up to 500 μL/plate Study No. FSRIPL 221205 (Unpublished,2023b) In vitro mammalian cell micronucleus test (GLP, OECD TG 487) NF – culture supernatant TK6 lymphoblastoid human cells Up to 10% Study No. FSRIPL 230701 (Unpublished,2024c) In vivo mammalian alkaline comet assay combined with an invivo mammalian erythrocytes micronucleus test (OECD TG 474 and TG 489) NF – culture supernatant Sprague Dawley rats See description of the study Sakamaki etal.(1989) 28day oral toxicity study NF Sprague Dawley rats Up to 3000 mg/kg bw per day (1010 CFU/g) Study No. AZ13349 (Unpublished,2014) 90day repeated dose oral toxicity study (GLP; Guidelines for Repeated Dose Toxicity Studies (Notification No. 655, 1999, Japan)) NF (and also BIOTHREE® in two study groups) Sprague Dawley rats Up to 3000 mg/kg bw per day (NF: 3 × 108 CFU/g) Abbreviations: bw, body weight; DMSO, dimethylsulfoxide; GLP, Good Laboratory Practice; OECD, Organization for Economic Cooperation and Development; TG, Test Guideline. 18314732, 2025, 5, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9371 by Readcube (Labtiva Inc.), Wiley Online Library on [05/08/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License