Table 1.

General regulatory structure and reference of microbial strains appropriate for use in foods or food supplements in example countries. Where specifically addressed in guidance or regulation, details of safety requirements including identification (ID), whole genome sequencing (WGS), determination of virulence factors, toxin production, infectivity and toxicity in addition to considerations for assessment of antimicrobial/antibiotic resistance (AbR) are listed.

General Regulatory Structure		Requirements for Establishing Safety
Country	Microbial List and Regulatory Reference	ID	WGS	Virulence Factors	Toxin Production	Infectivity/ Toxicity	AbR Potential
Australia	Therapeutic Goods Permissible Ingredients https://www.legislation.gov.au/Details/F2021L01449	Not specified for those species in the substances for use in listed medicines
Brazil	Food Supplements: http://antigo.anvisa.gov.br/documents/10181/5809185/IN_76_2020_COMP.pdf/28f071f8-8079-4671-a1ec-b47c8dd30917	ID – Genotypic and Phenotypic ID required WGS – required Virulence Factors – Adverse metabolic activity must be investigated Toxins and hemolysins must be investigated Adherence and translocation must be reported Resistance to antimicrobials must be investigated
Canada	NNHPD: http://webprod.hc-sc.gc.ca/nhpid-bdipsn/atReq.do?atid=probio Food: https://www.canada.ca/en/health-canada/services/food-nutrition/food-labelling/health-claims/accepted-claims-about-nature-probiotic-microorganisms-food.html	ID – Genotypic and Phenotypic ID required WGS (complete) and homology to type strains Demonstration of the absence of genetic elements responsible for the production of virulence factors Demonstration of lack of toxigenic activity (i.e. production of toxins) Toxicity not applicable if included in probiotics monograph Full phenotypic and genotypic investigation/explanation of resistance; Demonstration of lack of horizontal antimicrobial resistance transfer ability
China	For Food Products: http://www.nhc.gov.cn/sps/pztq/201612/712553a5f7554e0e9ec1dfdbcc91e99a.shtml Novel Food Products: Regulations on the safety review of new food ingredients 国家卫生计生委关于印发⟪新食品原料申报与受理规定⟫和⟪新食品原料 安全性审查规程⟫的通知 (nhc.gov.cn) For Health Food: http://www.nhc.gov.cn/sps/s3585/200804/8eafce69b178442eace396b7bc7bf8ef.shtml Technical guidelines for safety inspection and evaluation of strains used as health food raw materials or a health food finished product (2020 edition)	Not specified for those species in the list of microbial species Taxonomic ID of the strain is required Special properties must be identified If microorganisms do not have history of usual consumption the following is needed: Acute Toxicity/Pathogenicity, three Genotoxicity, 90 Days feeding toxicity, Teratogenicity and Reproduction Toxicity required Antimicrobial resistance should be reported Not specified for those species in the list of microbial species Phenotypic and genotypic strain level and taxonomy defined Complete WGS and homology to type strains required WGS search to demonstrate the absence of genetic elements responsible for the production of virulence factors required WGS search for genetic elements responsible for toxin metabolism required and some microbes may require in-vitro testing Toxicity testing includes intraperitoneal injection and oral gavage
Denmark	Food Supplements: https://www.foedevarestyrelsen.dk/english/Food/Food-supplements/Pages/default.aspx	Microbial species identification is required Demonstration of absence of potentially pathogenic properties in humans or animals required Demonstration of lack of formation of toxins in harmful quantities during the particular application Toxicity testing may be necessary It must be shown that microbe does not possess transferable antibiotic resistance
EU	QPS - https://zenodo.org/record/4428353#.YD-upBrsY2w Novel Foods: https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2016.4594	ID - Genotypic and Phenotypic ID required Detection of genes associated with virulence should be provided via complete strain characterization by fully assembled and validated WGS Toxin potential - Note qualification on QPS list for some species; Toxin potential must be considered for novel foods in regard to potentially adverse metabolic characteristics Animal toxicity testing may be required for novel foods Antimicrobial resistance risk assessment is required for all; determination of intrinsic or acquired resistance and potential transmissible antimicrobial resistance genes is required
India	Foods with added probiotic ingredients (Schedule VII): https://fssai.gov.in/upload/uploadfiles/files/Compendium_Nutra_29_09_2021.pdf	ID - both Genotypic and Phenotypic identification is required WGS is optional and ID may be carried out using any latest molecular techniques Assessment of the acute, subacute and chronic toxicity of ingestion of extremely large amounts of probiotics must be demonstrated If the strain under evaluation belongs to a species that is a known mammalian toxin producer or of hemolytic potential, it must be tested for each, respectively. Assessment of undesirable side effects must be investigated The risk of transferable antimicrobial resistance must be investigated
Italy	Guidelines on Prebiotics and Probiotics: http://www.salute.gov.it/imgs/C_17_pubblicazioni_1016_ulterioriallegati_ulterioreallegato_0_alleg.pdf	Taxonomy confirmed as a species with a history of use WGS - Species and strain ID using genetic methods; whole genome sequencing is an option Probiotics shall not be carriers of acquired and/or transmissible antibiotic resistance
Malaysia	Reg. 26A. Probiotic Culture: https://members.wto.org/crnattachments/2014/sps/MYS/14_4284_00_x.pdf	ID at the strain level is required Demonstration of lack of transmissible antimicrobial resistance genes
United States	For Food Products: https://www.federalregister.gov/documents/2016/08/17/2016-19164/substances-generally-recognized-as-safe For Dietary Supplements: https://www.federalregister.gov/documents/2016/10/04/2016-23931/dietary-supplements-new-dietary-ingredient-notifications-and-related-issues-revised-draft-guidance	Taxonomic ID of the strain is required If well-characterized microorganisms within the same species have a history of use, provide percentage homology to demonstrate similarities and differences For strains within species that do not have history of use in foods, 90-day subchronic toxicity testing may be required Antimicrobial resistance should be reported Biogenic amine production should be reported Phenotypic and genotypic strain level and taxonomy defined Identify any human pathogens phylogenetically related to the probiotic Identify any toxins produced by the species or those phylogenetically related to the probiotic Complete WGS and homology to well characterized strains within the species Report resistance to clinically relevant antibiotics and genetic transfer potential WGS search to demonstrate the absence of genetic elements responsible for the production of virulence factors required If no history of use, appropriate human or animal studies that report persistence in body and ability to translocate outside of the GI tract