A Review of Probiotic Ingredient Safety Supporting Monograph Development Conducted by the United States Pharmacopeia (USP)

Abstract

The United States Pharmacopeia (USP) is an independent, nonprofit science-based organization whose mission is to improve global health through public standards and related products for medicines, food and dietary supplements. Probiotic-based dietary supplements are increasingly popular in the marketplace and USP has developed fourteen monographs specific to probiotic ingredients, including representatives from the Genera Lactobacillus, Bacillus, Streptococcus, and Bifidobacterium. These monographs include the definition of the article, tests for identification, quantification assays (enumeration in the case of probiotics), limits for contaminants, and other quality parameters when appropriate. In addition to quality, the USP also considers the safety of probiotics for monograph development. This report includes an overview of the USP admission evaluation process for probiotics as well as a tabular summary of the probiotic monographs currently available. Pharmacopeia monographs can guide manufacturers and brand owners and protect consumers through establishment of quality standards.

Keywords:

• Dietary supplement
• monograph
• probiotics
• quality
• safety
• United States Pharmacopeia (USP)

Introduction

The United States Pharmacopeia (USP) is an independent, nonprofit science-based organization whose mission is to improve global health through public standards and related products for medicines, food and dietary supplements (Oketch-Rabah et al. 2018). In so doing, the USP serves to improve global health through this standard setting process. Within dietary supplements, the USP has developed numerous monographs for probiotic ingredients and products over the past several years. In fact, due to the increasing number of probiotic strains proposed for monograph development, the USP established a Probiotic Expert Panel (EP) to advise on quality requirements and safety considerations specific for probiotics. To date, the Probiotic EP has published several scientific peer-reviewed manuscripts to help guide manufacturers of probiotic dietary supplements. In addition, there are USP general chapters that should also be used by anyone developing probiotic dietary supplements. A summary of these references and general chapters are provided in Table 1.

Table 1. A summary of USP peer-reviewed scientific references and general chapters related to quality requirements and safety considerations specific to probiotics.

Title	Summary of Content	Reference
Improving end-user trust in the quality of commercial probiotic products	Comments and recommendations by the USP Probiotic EP related to third party certification, the process of setting standards for identity, purity, and quantification of probiotics; emerging methodologies useful for quality assessment; and technical challenges unique to managing quality of live microbial products.	Jackson et al. 2019
Considerations for determining safety of probiotics: A USP perspective.	Provides expert guidance specific to safety considerations for probiotics and a summary guide to global regulatory guidelines.	Roe et al. 2022
Improving and comparing probiotic plate count methods by analytical procedure lifecycle management	Provides tools to reduce measurement uncertainty and strengthen the reliability of probiotic enumerations by using analytical procedure lifecycle management (APLM).	Weitzel et al. 2021
USP <64> Probiotic Tests	Testing procedures for identification, enumeration, contamination, and other requirements for probiotics.	USP-NF

The USP process of developing standards for dietary ingredients including probiotics begins with an ‘admission evaluation’ process conducted by the Dietary Supplement Admission Evaluation and Labeling Expert Committee (DSAEL EC). Included in the evaluation process is information on the identity and quality of material under evaluation, toxicity data, human clinical safety studies, relevant in vitro or mechanistic studies, in silico data and adverse event (AE) data (Oketch-Rabah et al. 2018). The probiotic monographs, once developed, include the definition of the article, tests for identification at the strain level, enumeration assays (quantification), limits for contaminants, and other quality parameters when appropriate.

The scope of this report includes an overview of the USP admission evaluation process for probiotics as well as a tabular summary of the various probiotic monographs developed to date. As a note to the reader of this article, by probiotic we are referring to the formal definition as established by the Joint FAO/WHO Expert Consultation which is as follows: Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (Hill et al. 2014, FAO/WHO 2001). Additionally, data from unpublished self-affirmed Generally Recognized as Safe (GRAS) documents for which FDA did not have any questions about the GRAS self-determination based on scientific procedures for use as an ingredient in the specified conventional foods and dietary supplements were used in some cases to support conclusions on safety endpoints.

Comprehensive quality and safety assessment of USP probiotic monographs

As of the publishing of this report there are a total of 14 USP Probiotic Monographs (containing 19 strains for which admission evaluation was done) within the Dietary Supplements Compendium (DSC) which are summarized in Table 2. A brief description of the quality and safety elements included in USP’s review of probiotics under consideration for monograph development is provided below.

Table 2. A summary table of current USP probiotic monographs.

Probiotic	Bifidobacterium longum subspecies longum
Probiotic strain	Bl-05
Definition	Bifidobacterium longum subspecies longum is a Gram-positive, non-spore forming anaerobic, lactic acid producing, pleomorphic bacilli-shaped facultative hexose heterofermenters.
Comprehension or abbreviated revision	Comprehensive review conducted in December 2019.
Other Pharmacopeial Monographs & Regulatory standards (CFSAN, ODI, NNHPD, EFSA QPS, ARTG)	Health Canada listed Bifidobacterium longum subspecies longum species as a Schedule 1 substance and item 1 bacterium on the Natural Health Products regulations. EFSA granted a qualified presumption of safety status in 2007 to Bifidobacterium longum species due to its ‘long history of safe use’. The species is also listed in the Inventory of Microorganisms with Documented History of Use in Human Food published by IDF/EFFCA. Australian Register of Therapeutic Goods has information more than 100 approved single medication products that contain the species Bifidobacterium longum, however, ARTG does not specifically list B. longum subsp. longum Bl-05 as an active ingredient in the products.
Chemistry/Identity/Characteristics
Taxonomy change	The three subspecies, longum, infantis, and sui, were originally categorized as separate species, however, analysis of their DNA sequencing showed a great degree of homology, and so, the three bacteria were re-classified under one species: B. longum.
History of use	This strain has been used in many commercial dairy products available on the Brazilian market and is an ingredient in some dietary supplements.
Complete genome sequence unambiguously identifying the strain at the species level	Complete genome sequencing was performed for this strain; however, the genome was not available in the literature.
Antibiotic resistance profile (phenotypic and genotypic)	Did not show acquired resistance to antibiotics in a microdilution method following EFSA 2012 guidelines.
Presence of transmissible elements (e.g. transposase)	None
Presence of virulence factors/toxin genes (e.g. hemolysis)	No known suspect genes, virulence factors, or toxin proteins were identified. The genome of this strain was reported to have no known genes related to enterotoxigenicity.
Animal toxicity studies	In a study of acute oral toxicity in rats, no toxicities or adverse signs were observed, and this strain was not considered to be acutely toxic at a dose of 5000 mg/kg (unpublished data).
Allergenicity	None listed.
Intended use indications	Digestive health support and immunity in some dietary supplements in the US and in one product for pregnant and nursing women for digestive health and lactation
Typical intake level	At least 5.0x10^10 CFU/serving in dietary supplements, and for conventional foods at least 1.0x10^10 CFU/serving. Estimated daily intakes are up to 5.0x10^12 CFU/serving from dietary supplements or conventional foods (yogurt and other dairy products, soy products, beverages, chewing gum, confectionary snacks, and other foods). Recommended daily intake levels, per manufacturers, are 1.0x10^8 CFU to 5x10^9 CFU, and probiotic blends have recommended daily intake levels of 5x10^9 CFU to 5x10^10 CFU.
Clinical studies and adverse events reporting
Human clinical studies tracking safety endpoints	Clinical studies that evaluate the safety of this strain in healthy subjects are not available in public domain. There is one study evaluating healthy volunteers trying various probiotic yogurts with B. longum subsp. longum Bl-05 (1x10^7 CFU/g) and Lactobacillus acidophilus La14 in one of the products. Adverse events monitoring or safety parameters were not described. No side effects mentioned in the study. A self-affirmed GRAS report for this strain referenced clinical studies conducted for other strains of B. longum subsp. longum which were well tolerated with no significant adverse events reported. Intake levels ranged from 5x10^6 CFU/day to 6x10^11 CFU/day and averaged 1x10^9 CFU/day to 1x10^10 CFU/day with study duration of up to 12 months.
Adverse events reporting	Generally regarded as safe, this strain has had some adverse events reported, however, these were possibly due to underlying medical conditions. CAERS data returned no adverse event reports for the timeframe of January 2004–March 2019. The Canada Vigilance Program database did not report any adverse event reports when searched for this specific strain, however using the search term B. longum subsp. longum resulted in 15 adverse event reports with majority cases involving products containing multiple ingredients or concomitant consumption of other products leading to uncertain causality assignment. The Australian Therapeutic Goods Administration database did not find any adverse event reports, however, 23 adverse event reports were reported using the term B. longum between January 1971 – July 2019. The strain was not specified in this search and causality assignment was uncertain due to involving products containing multiple ingredients or concomitant consumption of other products. The Medicines and Healthcare Products Regulatory Agency – Yellow Card database, EudraVigilance European database, and New Zealand MedSafe reported no adverse event reports related to this strain.
Quality assessment
Nucleic acid-based identification assay	PCR sample preparation with primer set produces an amplification of around 164 base pairs.
Enumeration assay	NLT 100% of the labeled viable cell count, in CFU/g
Contaminants	None reported in the literature. The USP monograph has specifications for total combined molds and years count that does not exceed 10^2 CFU/g. Total non-lactic acid bacterial count is less than 5x10^3 CFU/g. It meets the requirements for absence of E. coli and Salmonella species in 40 g. Meets requirements of test for absence of Listeria in 25 g.
Packaging & storage	Preserve in high barrier foil laminate bags and store at or below 4 °C
Labeling	Ingredient should be labeled with genus, species, and strain names with the formulated enumeration in CFU/g.
Probiotic	Bacillus coagulans
Probiotic strain	Unique IS-2
Definition	Bacillus coagulans is a Gram-positive, spore-forming, rod-shaped, aerobic to microaerophilic bacterium.
Comprehension or abbreviated revision	Abbreviated review conducted in July 2021.
Other Pharmacopeial Monographs & Regulatory standards (CFSAN, ODI, NNHPD, EFSA QPS, ARTG)	Bacillus coagulans Unique IS-2 is the subject of FDA GRAS notice GRN 526 for use as an ingredient in a wide variety of food types. Health Canada listed this species as a Schedule 1 substance and item 1 bacterium under the Natural Health Products regulations. EFSA granted QPS status to the species based on its long history of safe use. The species is also listed in the Inventory of Microorganisms with Documented History of Use in Human Food published by IDF/EFFCA.
Chemistry/Identity/Characteristics
Taxonomy change	In 1935, it was described as Lactobacillus sporogenes in the fifth edition of the Bergey’s Manual of Systematic Bacteriology, then reclassified in 1957 under the Bacillus genus based on its biochemical properties, then reclassified in 2020 as Weizmannia coagulans based on phylogenetic analysis (Gupta 2020).
History of use	B. coagulans was first described in 1915 in association with the coagulation of evaporated milk at the Iowa Agricultural Experiment. This specific strain of probiotic is widely available in foods and dietary supplements in the US.
Complete genome sequence unambiguously identifying the strain at the species level	The genome for this strain has been published (Upadrasta et al. 2016).
Antibiotic resistance profile (phenotypic and genotypic)	Did not show acquired resistance to antibiotics in a microdilution method following EFSA 2018 guidelines.
Presence of transmissible elements (e.g. transposase)	None
Presence of virulence factors / toxin genes (e.g. hemolysis)	The published information shows this strain to be a nonpathogenic and nontoxigenic bacterium.
Animal toxicity studies	Rats were administered 3250 or 6500 mg/kg of this strain for 14 days, and no treatment-related changes in clinical signs, body weight, feed intake, and gross pathology or mortality was produced (FDA 2015).
Allergenicity	None listed.
Intended use indications	Dietary supplement products and food
Typical intake level	This strain in variety of food categories at maximum use level of 2x10^9 CFU/serving which can result in maximum daily intake of 3.64x10^10 CFU/day.
Clinical studies and adverse events reporting
Human clinical studies tracking safety endpoints	In a prospective, open-label, phase II clinical study evaluating the efficacy and safety of this strain in participants with acute diarrhea for 10 days, safety parameters included the monitoring of the incidence and type of adverse events, physical examinations, vital signs, and clinical lab tests. The researchers concluded that this strain was safe in these subjects with acute diarrhea (Sudha, 2012). In two other studies evaluating this strain, no adverse events or toxicity were mentioned who were given either 2x10^10 CFU or 4x10^10 CFU daily for 60 days, however, safety parameters or monitoring of adverse events were not described in these two studies (Sudha, 2012). A double blind, randomized, placebo-controlled, parallel group multicentric study concluded that this strain is safe for use in constipation treatment (Madempudi et al. 2020). GRN 526 references at least 15 clinical studies conducted for other strains of this species, and no adverse effects were noted. One of the studies, children were given 1.5 × 10^8 CFU/day of this species preparation for 1 year and it resulted in no adverse effects. Lastly, one study reported 24 episodes of Bacillus bacteremia in 18 febrile cancer patients between January 1978 and June 1986 where one was identified to be related to B. coagulans which indicates that this species may only be opportunistic in a highly immunocompromised population. There is no direct evidence that this species causes infection following oral ingestion (Banerjee et al. 1988).
Adverse events reporting	No adverse case reports were found for this strain in the literature and non in GRN 526. CFSAN had one AER listed for this species where a myocardial infarction was reported in a 52-year-old male, however the strain was not specified, and the consumer was taking multiple supplements. The Canada Vigilance Adverse Reaction Online Database had 4 AERs listed for the species, however the strains were not specified and the AERs involved the intake of multiple products, therefore, causality could not be determined. No AERs for this strain were listed in the WHO Uppsala Monitoring Center VigiAccess, but there were 22 AERs for this species. The most commonly reported AERs were skin and subcutaneous tissue disorders (8 cases) and gastrointestinal disorders (6 cases). The strains were not specific in any of these AERs, so a causality assignment is not possible since no other information was provided in terms of the product that was used. No AERs were listed for this strain in the Australian TGA, but there was one AER for the species in a 3-year-old female who experienced vomiting, however a causality assignment is not possible since she also had taken a multivitamin gummy intended for adults at the time and limited information was provided in the AER that involved multi-ingredient products. Finally, no AERs related to this strain were found in the Medicines and Healthcare Products Regulatory agency – Yellow Card database, EudraVigilance European database, or New Zealand’s MedSafe.
Quality assessment
Nucleic acid-based identification assay	PCR sample preparation with primer set 1 produces an amplification product of 480–504 base pairs. The PCR sample preparation with primer set 2 does not produce an amplification product of 280–297 base pairs. PCR sample preparation with Primer set 3 produces an amplification product of 440–452 base pairs.
Enumeration assay	NLT 100% of the labeled viable CFU/g
Contaminants	None reported in the literature. The USP monograph for B. coagulans has specifications for yeast count that does not exceed 100 CFU/g. It meets the requirements for absence of E. coli and Salmonella species in 10 g. The probiotic ingredient should be tested for the absence of Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus cereus depending on the risk associated with the contamination of the above organisms based on formal risk assessment programs such as HACCP.
Packaging and storage	Preserve in tight containers in cool, dry place.
Labeling	Label with genus and species names, or genus, species, and strain names with formulated enumeration in CFU/g (or similar units)
Probiotic	Lacticaseibacillus paracasei (formerly Lactobacillus paracasei)
Probiotic strain	Lpc-37 (ATCC-SD5275)
Definition	Lacticaseibacillus paracasei is a lactic acid producing, gram-positive, rod-shaped, non-motile, non-spore-forming homofermentative bacterium. LPC-37 is a non-D-lactate producing strain.
Comprehension or abbreviated revision	Comprehensive review in October 2016.
Other Pharmacopeial Monographs & Regulatory standards (CFSAN, ODI, NNHPD, EFSA QPS, ARTG)	L. paracasei Lpc-37 is the subject of FDA GRAS notices (GRN) 736 for use as an ingredient in a wide variety of food types. FCC has published a monograph for this strain. L. paracasei is included in the EFSA Qualified Presumption of Safety list. The species is also listed in the Inventory of Microorganisms with Documented History of Use in Human Food published by IDF/EFFCA. Health Canada listed Lactobacillus paracasei species as a Schedule 1 substance and item 1 bacterium under on the Natural Health Products regulations. The ARTG lists 52 products that contain L. paracasei but none that specify this strain specifically.
Chemistry/Identity/Characteristics
Taxonomy change	Interchangeable use of L. casei and L. paracasei due to recent debate over taxonomy with several strains of L. paracasei and L. casei showing 99% identity to the 16S rRNA sequence of L. casei ATCC334 which is the current type strain for L. paracasei. Such debate ended with the reclassification in 2020 as Lacticaseibacillus paracasei (Zheng et al. 2020).
History of use	This strain has been sold commercially for more than 15 years. Major uses include probiotics, cheese fermentation, and probiotic cheese.
Complete genome sequence unambiguously identifying the strain at the species level	The L. paracasei Lpc-37 genomic sequence is deposited at the National Center for Biotechnology Information. L. paracasei Lpc-37 was isolated from a dairy source and has been characterized and classified through independent laboratories with modern genotypic methods including 16S rRNA gene sequencing, and electrophoretic whole-organism protein analysis.
Antibiotic resistance profile (phenotypic and genotypic)	Resistance to vancomycin in certain Lactobacillus including L. paracasei can be attributed to intrinsic factors relating to the cell wall composition and not due to transmissible elements. L. paracasei Lpc-37 has been confirmed to be free of enterococcus-like vancomycin resistant genes. There were no acquired antibiotic resistance detected in L. paracasei Lpc-37 during screening by the EU-funded PROSAFE project.
Presence of transmissible elements (e.g. transposase)	No transmissible elements identified and reported for lactic acid bacteria in foods or feed
Presence of virulence factors / toxin genes (e.g. hemolysis)	None
Animal toxicity studies	An unpublished study showed this strains ability to exert moderate but significant protection from intestinal inflammation with no mention of toxicity studies in this trial. Animal studies on L. paracasei Lpc-37 with single or repeated doses, short-term or long-term studies or studies on reproductive or developmental toxicity were not available from the literature.
Allergenicity	None listed.
Intended use indications	Gastrointestinal support, immune system function, detoxification process, supports urinary tract health
Typical intake level	The DSLD has 27 products with L. paracasei Lpc-37 as an ingredient, but the products were proprietary blends, therefore, the amount of this strain per dose was not found on the labels.
Clinical studies and adverse events reporting
Human clinical studies tracking safety endpoints	Multiple clinical trials were conducted with strain Lpc-37, although none were designed to evaluate the strain’s safety. No adverse effects linked to L paracasei Lpc-37 were reported in the clinical trials. The probiotic concentration ranged between 7.8 × 10^8 to 7.8 × 10^10 CFU per day, over a period ranging from 4 wk to 9 months in a population between 2 and 95 years.
Adverse events reporting	No adverse events were reported after researching multiple databases including PubMed, FDA MedWatch, Australian TGA Database of Adverse Events Notification, Canada Vigilance Adverse Reaction database, Toxicology data network, and more.
Quality assessment
Nucleic acid-based identification assay	The PCR sample preparation with Primer set produces an amplification product of 273 base pairs.
Enumeration assay	NLT 100% of labeled viable cell count in CFU/g
Contaminants	None were reported in the literature. The official USP monograph for L. paracasei LPC-37 has specifications for total combined molds and yeast count not to exceed 10^2 CFU/g. Total non-lactic acid bacterial count is less than 5x10^3 CFU/g. It meets the requirements for absence of E. coli in 10 g and Salmonella species in 40 g. Meets requirements of test for absence of Listeria in 25 g. The total enterococci count is less than 10^2 cfu/g. The total coliforms count is less than 10 cfu/g.
Packaging and storage	Preserve in high barrier foil laminate bags and store at or below 4 °C
Labeling	Label with genus, species, and strain names and with formulated enumeration in CFU/g.
Probiotic	Limosilactobacillus reuteri (formerly Lactobacillus reuteri)
Probiotic strain	1E1
Definition	Limosilactobacillus reuteri is a lactic acid producing, gram-positive, rod-shaped, non-spore-forming heterofermentative bacterium. The 1E1 strain produces 41% d-lactate and 59% l-lactate.
Comprehension or abbreviated revision	Comprehensive review conducted in December 2019.
Other Pharmacopeial Monographs & Regulatory standards (CFSAN, ODI, NNHPD, EFSA QPS, ARTG)	Health Canada has listed the L. reuteri species as a Schedule 1 substance and item 1 bacterium under NHP regulations, however, the 1E1 strain was not listed. The L. reuteri species is included in the Health Canada monograph for probiotics. EFSA granted QPS status to the L. reuteri species based on its long history of safe use. The L. reuteri species is also on Australia’s Therapeutic Goods Administration’s approved list of medical ingredients. ARTG has 75 approved single medicine products with the L. reuteri species, but no specific mention of the strain 1E1 is made.
Chemistry/Identity/Characteristics
Taxonomy change	Originally classified as Lactobacillus fermentum biotype II but in 1980 re-classified as new species called L. reuteri based on phenotypic and genetic profile differences. Then reclassified as Limosilactobacillus reuteri in 2020 based on phylogenetic analysis (Zheng et al. 2020).
History of use	L. reuteri strains have been frequently used as probiotic cultures in commercial dairy products worldwide due to its ability to produce the antimicrobial substance reuterin, and this substance is regarded as an effective food preservative.
Complete genome sequence unambiguously identifying the strain at the species level	Complete genome sequencing was done for strain L. reuteri 1E1.
Antibiotic resistance profile (phenotypic and genotypic)	This strain did not show acquired resistance to antibiotics in a microdilution method following EFSA guidelines (EFSA 2012).
Presence of transmissible elements (e.g. transposase)	None
Presence of virulence factors / toxin genes (e.g. hemolysis)	Unlikely to produce biogenic amines: histamine and tyramine. The genome of strain 1E1 showed no genes associated with enterotoxigenicity. No other data for bacteriocins and for hemolytic/mucolytic potential were available for this strain.
Animal toxicity studies	No mortality or adverse effects reported in female rats administered at 5000 mg/kg of strain L. reuteri 1E1 during a GLP-compliant 14-day acute oral toxicity study. No adverse effects on clinical signs and body weights were noted and no macroscopic lesions were observed at necropsy. No other toxicology studies were identified for this strain. Studies evaluating early administration effects of this train to neonatal piglets and jejunal gene expression in weanling pigs showed no toxicity signs (Gebert et al. 2011).
Allergenicity	None listed.
Intended us indications	Maintenance of gastrointestinal health.
Typical intake level	Intended for use in dietary supplements at intake levels of at least 5.0x10^10 CFU/serving. Estimated daily intakes are up to 5.0x10^10 CFU/day of this strain from conventional foods or dietary supplements which is more conservative when considering that an average person consumes approximately 20 servings/day of all food groups combined.
Clinical studies and adverse events reporting
Human clinical studies tracking safety endpoints	No clinical studies that evaluated the safety of L. reuteri 1E1 were identified in the literature. Other strains of this have been studied. No adverse effects related to L. reuteri treatment were reported in any of the clinical studies.
Adverse events reporting	No AERs were found for this strain or other strains of L. reutri in the literature. Following a search of CAERS data files between January 2004 – March 2019 showed no adverse events reported for this train. There were three AERs for products that contained this species, however, the strain was not specified. The Canada Vigilance Program data based showed no AERs for L. reutri between January 1965 – July 2019, but seven AERs were found for this species, however, the strain was not specified. Causality assignment was uncertain since these cases made up products that consisted of multiple ingredients or probiotics and/or involved the concomitant intake of other products. No safety data available for this strain in pregnant or lactating women. There was one study that mentioned no adverse events in infants consuming breast milk from mothers who were given a strain of L. reuteri, but the intake level was not reported (FDA GRN No. 254, 2008).
Quality assessment
Nucleic acid-based identification assay	PCR sample preparation with primer set produces an amplification of around 170 base pairs.
Enumeration assay	NLT 100% of labeled viable cell count in CFU/g
Contaminants	No contaminants reported in the literature. The USP monograph for Limosilactobacillus reuteri has specifications for total combined molds and yeast count that does not exceed 10^2 CFU/g. Total non-lactic acid bacterial count is less than 5x10^3 CFU/g. It meets the requirements for absence of E. coli in 10 g, Salmonella species in 40 g, and Listeria in 25 g.
Packaging and storage	Preserve in high barrier foil laminate bags and store at or below 4 °C.
Labeling	Label with genus, species, and strain names and with formulated enumeration in CFU/g.
Probiotic	Lactobacillus acidophilus
Probiotic strain	La-14 (ATCC-SD5212)
Definition	Lactobacillus acidophilus is a lactic acid-producing, Gram-positive, non-motile, non-spore-forming homofermentative bacterium presenting as rods, sometimes in short chains. Strain La-14 produces 60% l-lactate and 40% d-lactate.
Comprehension or abbreviated revision	Abbreviated revision conducted in August 2016.
Other Pharmacopeial Monographs & Regulatory standards (CFSAN, ODI, NNHPD, EFSA QPS, ARTG)	L. acidophilus La-14 is listed in the official Food Chemicals Codex as a microbial food culture. It was the subject of FDA GRAS notice (GRN) 502, which was filed without question for use as an ingredient in a wide variety of food types. GRN 502 also notes that in 2008, EFSA evaluated the safety of L. acidophilus species, noting no safety concerns for species of this genus. Health Canada has listed the L. acidophilus species as a Schedule 1 substance and item 1 bacterium under NHP regulations. The L. acidophilus species is included in the Health Canada monograph for probiotics. EFSA granted QPS status to the L. acidophilus species based on its long history of safe use. The species is also listed in the Inventory of Microorganisms with Documented History of Use in Human Food published by IDF/EFFCA.
Chemistry/Identity/Characteristics
Taxonomy change	None
History of use	Lactobacillus acidophilus has been added to human food since at least 1950 and is very common in dairy products worldwide, including the US where the species is the most common Lactobacillus in yogurt products. Lactobacillus acidophilus La-14 is a strain utilized in conventional foods (e.g. milk, yogurt) and in dietary supplements. It has been commercially available in the United States for more than a decade with no adverse incidents reported.
Complete genome sequence unambiguously identifying the strain at the species level	Genome sequence of this strain is deposited with the National Center for Biotechnology Information under accession number CP_005926. 16S ribosomal sequencing along with phenotypic and genotypic characterization identified and confirmed the strain as L. acidophilus La-14.
Antibiotic resistance profile (phenotypic and genotypic)	None provided
Presence of transmissible elements (e.g. transposase)	None provided
Presence of virulence factors / toxin genes (e.g. hemolysis)	None provided
Animal toxicity studies	GRN 502 discusses several targeted animal studies investigating the effects of the La-14 or NCFM strains of L. acidophilus on the microflora, intestine, and IgM and IgG, among others. Species tested included mice; healthy, immunocompromised, and colitis-induced strains. No adverse events were observed in any of the studies
Allergenicity	None listed.
Intended us indications	Used as an ingredient in a wide variety of food types.
Typical intake level	Manufacturer recommended intake ranges from 2x10^9 to 12x10^9 CFU/day.
Clinical studies and adverse events reporting
Human clinical studies tracking safety endpoints	GRN 502 lists 14 studies in which L. acidophilus NCFM and/or La-14 have been administered to healthy and diseased patients with no reported adverse events related to the treatment protocol.
Adverse events reporting	No adverse events were observed or reported for concentration between 1 × 10^9 and 2 × 10^10 CFU per serving (GRN 502).
Quality assessment
Nucleic acid-based identification assay	PCR samples preparation with primer set provides an amplification of around 184 base pairs. There should be no amplification product of 600 base pairs.
Enumeration assay	NLT 100% of the labeled viable cell count in CFU/g
Contaminants	None were reported in the literature. The official USP monograph for Lactobacillus acidophilus La-14 has specifications for total combined molds and yeast count that does not exceed 10^2 CFU/g. Total non-lactic acid bacterial count is less than 5x10^3 CFU/g. It meets the requirements for absence of E. coli in 10 g and Salmonella species in 40 g. Meets requirements of test for absence of Listeria in 25 g.
Packaging and storage	Preserve in high barrier foil laminate bags and store at or below 4 °C.
Labeling	Label with genus, species, and strain names and with formulated enumeration in CFU/g.
Probiotic	Lactobacillus acidophilus
Probiotic strain	NCFM (ATCC-SD5221)
Definition	Lactobacillus acidophilus is a lactic acid-producing, Gram-positive, non-motile, non-spore-forming homofermentative bacterium presenting as rods, sometimes in short chains. Strain NCFM produces 66% l-lactate and 34% d-lactate.
Comprehension or abbreviated revision	Abbreviated
Other Pharmacopeial Monographs & Regulatory standards (CFSAN, ODI, NNHPD, EFSA QPS, ARTG)	GRN 357 and GRN 865, which were filed without question by the FDA for use as an ingredient in a wide variety of food types. In 2008, EFSA evaluated the safety of the L. acidophilus species and noted no safety concerns. Health Canada has listed the L. acidophilus species as a Schedule 1 substance and item 1 bacterium under NHP regulations. The L. acidophilus species is included in the Health Canada monograph for probiotics. EFSA granted QPS status to the L. acidophilus species based on its long history of safe use. The species is also listed in the Inventory of Microorganisms with Documented History of Use in Human Food published by IDF/EFFCA. Production process is consistent with current Good Manufacturing Practice and meets the specifications in the GRAS Dossier.
Chemistry/Identity/Characteristics
Taxonomy change	None
History of use	Lactobacillus acidophilus has been added to human food since at least 1950 and is very common in dairy products worldwide, including the US where the species is the most common Lactobacillus in yogurt products. It is involved in a range of food and feed fermentations and applied as probiotics for humans and animals
Complete genome sequence unambiguously identifying the strain at the species level	The entire genome has been sequenced and is available from GenBank under accession number CP000033 for public reference.
Antibiotic resistance profile (phenotypic and genotypic)	Genomic sequencing did not detect any known antibiotic resistant genes.
Presence of transmissible elements (e.g. transposase)	Genome analysis revealed no transposable elements known to be harmful to humans.
Presence of virulence factors / toxin genes (e.g. hemolysis)	Genome screening revealed no elements known to be harmful to humans.
Animal toxicity studies	GRN No. 357 discusses several targeted animal studies investigating the effects of L. acidophilus NCFM on growth, microflora, intestine, and IgM and IgG, among other effects. These studies involved the use of healthy, immunocompromised and colitis-induced strains of mice. No adverse events were observed. GRN 865 reported no toxicity in female rat on administration of single oral dose of 5000 mg/kg or 1.72 × 10^12 CFU/kg of L. acidophilus NCFM
Allergenicity	None listed.
Intended us indications	Dietary supplement and food
Typical intake level	Food use is targeted to contain 1x10^9 CFU/serving typically of this strain. Ingesting daily intake of 3.8x10^11 CFU/day for 6 days did not result in any adverse signs or symptoms (FDA GRAS No. 357, 2011).
Clinical studies and adverse events reporting
Human clinical studies tracking safety endpoints	At the time of the GRAS filing, sixty-one publications reporting the results of 55 clinical trials using L. acidophilus NCFM were identified in the literature search. The vast majority of studies were randomized, blinded, placebo-control trials (32), the remaining studies were either open-label (either controlled or uncontrolled). A total of 2,476 subjects were treated in these studies and the total number of treatment days was 5,340,732. In the longest of these studies, the treatment duration was 182 days. Doses ranged from 10^6 − 4 × 10^11 cfu/day but the dose in most studies clustered around 1 − 2 × 10^10 cfu/day. Stratified by age, studies on elderly, adults, children, and infants were the subject of 4, 47, 8, and 1, respectively (some studies did not report the age of the subjects). In one study, breastfeeding women were treated and the effects on their children were monitored.
Adverse events reporting	The clinical studies conducted either reported no treatment-related adverse events, described the NCFM treatment as well tolerated, or did not report any safety-related endpoints. When adverse events were noted, they were generally confined to gastrointestinal issues, were equally distributed between treatment and control groups, were generally considered mild and reversible, and were not considered related to NCFM treatment.
Quality assessment
Nucleic acid-based identification assay	PCR sample preparation with Primer set 1 produces an amplification product of 365 base pairs with two SNPs. PCR sample preparation with Primer set 2 produces an amplification product of 433 base pairs with two SNPs. PCR sample preparation with Primer set 3 produces an amplification of around 600 base pairs. There should be no amplification product of 184 base pairs.
Enumeration assay	NLT 100% of the labeled viable cell count in CFU/g.
Contaminants	None were reported in the literature. Total combined molds and yeast count does not exceed 10^2 CFU/g. Total non-lactic acid bacterial count is less than 5x10^3 CFU/g. It meets the requirements for absence of E. coli in 10 g and Salmonella species in 40 g. Meets requirements of test for absence of Listeria in 25 g.
Packaging and storage	Preserve in high barrier foil laminate bags and store at or below 4 °C.
Labeling	Ingredient should be labeled with genus, species, and strain names and with the formulated enumeration in CFU/g.
Probiotic	Lacticaseibacillus rhamnosus (formerly Lactobacillus rhamnosus)
Probiotic strain	GG
Definition	Lacticaseibacillus rhamnosus is a Gram-positive, rod-shaped, heterofermentative, non-spore-forming bacterium. Plasmid-free strain of Lacticaseibacillus rhamnosus presents as small uniform rods in chains. The GG strain is a non-D-Lactate producer.
Comprehension or abbreviated revision	Abbreviated review conducted in December 2019.
Other Pharmacopeial Monographs & Regulatory standards (CFSAN, ODI, NNHPD, EFSA QPS, ARTG)	A GRAS notice (GRN 231) for Lactobacillus casei subsp. rhamnosus strain GG now referred to as L. rhamnosus GG was submitted to the FDA for intended use in infant formulas, with the intake levels not to exceed 10^8 CFU/g of powdered formula. The FDA filed GRN 231 without question. Health Canada has this species on the Natural Health Products Ingredients Database as a Schedule 1 substance and item 1 bacterium under NHP regulations. The L. rhamnosus species and the strain GG are included in the Health Canada monograph for probiotics. Total of 82 active licensed products were found that included this strain in the Licensed Natural Health Products Database EFSA granted QPS status to the L. rhamnosus species based on its long history of safe use. The species is also listed in the Inventory of Microorganisms with Documented History of Use in Human Food published by IDF/EFFCA. In Australia, this species is on the Therapeutic Goods Administrations approved list of medical ingredients, however, ARTG did not have this specific strain listed as one of the active ingredients in the products. EFSA granted qualified presumption of safety (QPS) status to L. rhamnosus species in 2007 based on evidence supporting ‘the long history of safe use’ for this species along with other Lactobacillus species. The L. rhamnosus species was still on the QPS list that was updated in 2020.
Chemistry/Identity/Characteristics
Taxonomy change	Its former identity was Lactobacillus casei subsp. rhamnosus. L. rhamnosus was originally considered a subspecies of L. casei, but L. rhamnosus was later reclassified as a separate species following genetic analysis. Then reclassified as Lacticaseibacillus rhamnosus in 2020 based on phylogenetic analysis (Zheng et al. 2020).
History of use	This strain was originally isolated in 1982 from the intestinal tract of a healthy human and has been used in dietary supplements and food products worldwide for over 20 years.
Complete genome sequence unambiguously identifying the strain at the species level	The genome sequence of this strain has been published.
Antibiotic resistance profile (phenotypic and genotypic)	No acquired resistance to antibiotics in microdilution method was seen (unpublished data).
Presence of transmissible elements (e.g. transposase)	None
Presence of virulence factors / toxin genes (e.g. hemolysis)	No known toxins and virulence factors and no genetic elements known to be harmful to humans were identified in the strain.
Animal toxicity studies	No mortality or adverse effects reported in Crl:CD(SD) female rats who were given 3.11x10^12 CFU/kg (equivalent to 5000 mg/kg) of this strain in an oral acute toxicity study. It was concluded that this probiotic is not toxic and calculated the oral median lethal dose to be at more than 9.0x10^11 CFU/kg (FDA GRN No. 231, 2008). GRN 231 describes the absence of toxicity or bacteremia in highly compromised, germ-free, beige-athymic (bg/bh-nu/nu) neonatal mice and in immunocompetent neonatal rodents treated with L. rhamnosus GG, indicating that the strain is generally safe in healthy and immunocompromised animals
Allergenicity	None listed.
Intended use indications	Maintenance of gastrointestinal health in adults, children, and infants
Typical intake level	Intended to be added to infant formula to up to 1x10^8 CFU/gram of infant formula. Estimated daily intake in infants is 1x10^8 CFU/day to 1x10^10 CFU/day. Estimated daily intakes in adults are up to 1x10^11 CFU/day of this strain from conventional foods or dietary supplements. Typical manufacturer recommended levels range from 1x10^9 CFU/day to 2x10^10 CFU per day of this strain. In the self-determined GRAS report, the estimated daily intakes are up to 1.0 × 10^11 CFU/day of L. rhamnosus GG from conventional foods or dietary supplements.
Clinical studies and adverse events reporting
Human clinical studies tracking safety endpoints	No adverse events were reported in 32 of the clinical studies that were found for this strain, but most of the studies did not describe any safety-related endpoints. Studies including compromised neonates, infants, and children with immune deficiencies or impaired intestinal barrier function showed that this strain was well-tolerated at intake levels of up to 1x10^11 CFU/day (FDA GRAS No. 231, 2008). Reported adverse events in clinical studies included mostly of gastrointestinal effects that were usually reversible, mild, and occurred equally in the treatment and placebo arms of the study. One study with a prospective, randomized, and double-blind design evaluated the effects of strain in children with acute gastroenteritis. Safety outcomes included extra-intestinal infection by this strain (ex. Bacteremia). No participants were noted with extra-intestinal side effects, and the rates of adverse events or side effects were not different between the treatment and placebo groups. However, 5 subjects were noted to have experienced wheezing in the treatment group with none reported in the placebo group (Schnadower et al. 2018). Another study (randomized, double-blind, placebo-controlled) was looking at this strain to reduce influenza and other respiratory virus infections in residents of long-term and chronic care facilities. The researchers noted no significant difference in the adverse events seen in the treatment and placebo groups (Wang et al. 2018). Another randomized and double-blinded controlled study in high-risk infants evaluating cumulative incidence of eczema, asthma, and rhinitis reported no major adverse events, however, no monitoring of adverse events was described (Cabana et al. 2017). In another study, no significant difference between the two groups were noted for adverse events that involved cardiovascular, gastrointestinal, genitourinary, metabolic/endocrine, musculoskeletal, neurologic or sensory, respiratory, or other body systems, and it was reported that the treatment with probiotics were well-tolerated, and no study withdrawals were related with the treatment (Dickerson et al. 2018). A retrospective study in Finland looked at increased consumption of this strain over a 9-year period after its introduction to food products in Finland 1990, and this showed no increase in the number of cases with Lactobacillus bacteremia (Salminen et al. 2002).
Adverse events reporting	No increase in adverse events were seen and the consumption of this strain was concluded as safe in an analysis of pooled adverse event data from 6 trials (Tapiovaara et al. 2016). When the FDA CAERS data filed were searched for this strain between the period of January 2004 – March 2019, four adverse event reports were generated. One of the patients in these reports tested positive for Lactobacillus, however, no details were given for the test, and the patient also had pneumonia, leukocytosis, hypotension, acute respiratory failure, and acute myocardial infarction which showed poor health to begin with. No other details were provided regarding the patient’s medical history. Causality assignment was uncertain for the other three reported adverse effects. No bacteremia or infection was reported in these 3 cases. Canada Vigilance Program database returned one AER for this strain which occurred in a 22-year-old woman who experienced vomiting (intake level was not available). In the Australian TGA, no AERs were found. Lastly, no AERs related to this strain were found in the Medicines and Healthcare Products Regulatory agency – Yellow Card database, EudraVigilance European database, or New Zealand’s MedSafe.
Quality assessment
Nucleic acid-based identification assay	PCR sample preparation with Primer set 1 produces an amplification product of 552 base pairs. PCR sample preparation with Primer set 2 produces an amplification product of 388 base pairs with one SNPs. PCR sample preparation with Primer set 3 produces an amplification of around 293 base pairs with two SNPs. PCR sample preparation with Primer set 4 produces an amplification product of 490 base pairs. There should be no amplification product of 367 base pairs.
Enumeration assay	NLT 100% of the labeled viable cell count in CFU/g.
Contaminants	None were reported in the literature. The official USP monograph for Lacticaseibacillus rhamnosus has specifications for total combined molds and yeast count that does not exceed 10^2 CFU/g. Total non-lactic acid bacterial count is less than 5x10^3 CFU/g. It meets the requirements for absence of E. coli in 10 g and Salmonella species in 40 g. Meets requirements of test for absence of Listeria in 25 g.
Packaging and storage	Preserve in high barrier foil laminate bags and store below 4 °C.
Labeling	Ingredient should be labeled with genus, species, and strain names and with the formulated enumeration in CFU/g.
Probiotic	Lacticaseibacillus rhamnosus (formerly Lactobacillus rhamnosus)
Probiotic strain	HN001
Definition	Gram-positive, rod-shaped, heterofermentative non-spore-forming bacterium. This strain of Lacticaseibacillus rhamnosus contains plasmids and presents as rods of varying length, occurring in short chains. The HN001 strain is a non-D-Lactate producer strain.
Comprehension or abbreviated revision	Abbreviated review conducted in September 2016.
Other Pharmacopeial Monographs & Regulatory standards (CFSAN, ODI, NNHPD, EFSA QPS, ARTG)	L. rhamnosus HN001 is listed in the official Food Chemicals Codex as a microbial food culture and was the subject of FDA GRAS notice (GRN) 288, which was filed without question for use as an ingredient in various food types. Health Canada has this species on the Natural Health Products Ingredients Database as a Schedule 1 substance and item 1 bacterium under NHP regulations. The L. rhamnosus species is included in the Health Canada monograph for probiotics. Total of 82 active licensed products were found that included this strain in the Licensed Natural Health Products Database. EFSA granted QPS status to the L. rhamnosus species based on its long history of safe use. The species is also listed in the Inventory of Microorganisms with Documented History of Use in Human Food published by IDF/EFFCA.
Chemistry/Identity/Characteristics
Taxonomy change	L. rhamnosus was originally considered a subspecies of L. casei, but L. rhamnosus was later reclassified as a separate species following genetic analysis. Then reclassified as Lacticaseibacillus rhamnosus in 2020 based on phylogenetic analysis (Zheng et al. 2020).
History of use	This strain has been isolated from a cheddar cheese from New Zealand that has been consumed here for more than 20 years. This probiotic strain is widely available in foods and dietary supplements in the US.
Complete genome sequence unambiguously identifying the strain at the species level	Genome of strain HN001 was sequenced and published under the Refseq accession number NZ_ABWJ00000000.
Antibiotic resistance profile (phenotypic and genotypic)	No genes encoding for antibiotic resistance were identified in the strain’s genome.
Presence of transmissible elements (e.g. transposase)	None
Presence of virulence factors / toxin genes (e.g. hemolysis)	No known toxins and virulence factors known to be harmful to humans.
Animal toxicity studies	GRN 288 reported that the published literature included 19 experimental studies in a variety of animal species, including both normal and immunodeficient mice, hamsters, rats, and rabbits. Adverse effects were seen in only 2 animal studies, which both involved animal models of questionable relevance to humans, and neither study used the HN001 strain of L. rhamnosus. The GRN 288 notice concluded that the body of evidence confirms the safety of L. rhamnosus strain HN001 for use as an ingredient in food at the intended use levels up to 10^11 CFU/day.
Allergenicity	None listed.
Intended use indications	For shelf-life of the food, including but not limited to dairy products (fluid milk and milk drinks, milk-based desserts and meal replacements, dry and powdered milk, yogurt, and cheese), ready to eat cereals, fruit juices, nectars, drinks, confections, chewing gum, and functional nutritional products.
Typical intake level	1.5x10^9 to 1.5x10^10 CFU/day
Clinical studies and adverse events reporting
Human clinical studies tracking safety endpoints	GRN 288 references 83 separate studies in humans, including 35 studies in adults, 25 studies in children, and 23 studies in infants at doses up to and exceeding 10^11 CFU/day for up to 2 years. These studies included nearly 1,700 adults, 3,400 children, and 4,100 infants. No serious adverse events were seen in any human studies.
Adverse events reporting	There were no adverse effects reported during the clinical studies.
Quality assessment
Nucleic acid-based identification assay	PCR sample preparation with Primer set produces an amplification product of 340 base pairs.
Enumeration assay	NLT 100% of the labeled viable cell count in CFU/g.
Contaminants	None were reported in the literature. The official USP monograph for Lacticaseibacillus rhamnosus has specifications for total combined molds and yeast count that does not exceed 10^2 CFU/g. Total non-lactic acid bacterial count is less than 5x10^3 CFU/g. It meets the requirements for absence of E. coli in 10 g and Salmonella species in 40 g. Meets requirements of test for absence of Listeria in 25 g.
Packaging and storage	Preserve in high barrier foil laminate bags and store at or below 4 °C.
Labeling	Ingredient should be labeled with genus, species, and strain names and with the formulated enumeration in CFU/g.
Probiotic	Bifidobacterium bifidum
Probiotic strain	Bb-06
Definition	Bifidobacterium bifidum is a Gram-positive, non-spore forming, anaerobic, lactic acid-producing, pleomorphic bacilli-shaped facultative hexose heterofermenters. They have various shapes, including short, curved rods; club-shaped rods; and bifurcated Y-shaped rods.
Comprehension or abbreviated revision	Comprehensive review conducted in December 2019.
Other Pharmacopeial Monographs & Regulatory standards (CFSAN, ODI, NNHPD, EFSA QPS, ARTG)	An expert panel evaluated the safety information in the self-affirmed GRAS report and concluded that B. bifidum Bb-06 was GRAS based on scientific procedures. This strain is not listed in the US CFR for any food uses in the United States. The species, B. bifidum, is listed on the United Natural Products Association list of old dietary ingredients, however strain Bb-06 is not found on the list. Health Canada lists this species on the Natural Health Products Ingredients Database as a Schedule 1 substance and item 1 bacterium under the NHP regulations. The species is included in the Health Canada monograph for probiotics. EFSA has given a qualified presumption of safety status to this species due to its long history of safe use. The species is also listed in the Inventory of Microorganisms with Documented History of Use in Human Food published by IDF/EFFCA. ARTG has information about 68 approved single medication products containing this species, however, this specific strain was not listed as an active ingredient in any of the products.
Chemistry/Identity/Characteristics
Taxonomy change	None
History of use	Bifidobacterium species have been utilized globally in fermented food products and commercially produced food supplements. B. bifidum has been added to human food since at least 1970 and is very common in fermented milk worldwide. B. bifidum Bb-06 is reported to be in commercial use since 2012 and has been sold worldwide that include North America, Europe, and Asia and Pacific countries.
Complete genome sequence unambiguously identifying the strain at the species level	This strain has gone through complete genomic sequencing; however, the genome is not available in the literature (unpublished data).
Antibiotic resistance profile (phenotypic and genotypic)	This strain did not show any acquired resistance to antibiotics in a microdilution method (unpublished data).
Presence of transmissible elements (e.g. transposase)	None
Presence of virulence factors / toxin genes (e.g. hemolysis)	No genes reported to be associated with enterotoxigenicity (unpublished data). This strain did show alpha-hemolytic activity. There were no known bacteriocins or toxin genes identified during the genome screening.
Animal toxicity studies	Female rats were given 5000 mg/kg of this strain by oral gavage in an acute toxicity study, and it showed no toxicity or adverse signs. This strain was not considered to be acutely toxic at a dose of 5000 mg/kg (unpublished data).
Allergenicity	None listed.
Intended us indications	Support of gastrointestinal health, immune function, and overall health.
Typical intake level	Not available.
Clinical studies and adverse events reporting
Human clinical studies tracking safety endpoints	Clinical studies that evaluate the safety of this strain as a single ingredient in healthy participants are not available. One efficacy study was found that looked at this strain in a multi-probiotic mixture given to obese subjects. The researchers noted no adverse effects with no significant differences in the blood tests for uric acid, gamma-glutamyl transferase, alanine aminotransferase, aspartate aminotransferase, urea, and creatinine between the placebo and treatment arms of the study (Gomes et al. 2017). Clinical studies conducted for other strains of B. bifidum have shown no serious adverse effects and that they are well-tolerated.
Adverse events reporting	No adverse events or case reports were identified in the literature for this strain. B. bifidum bacterium is generally regarded as safe (Quigley et al. 2017). Additionally, no adverse events were seen in studies for different strains of this bacterium when given to women during their last month of pregnancy and their infants when aged 0–6 months old (Allen et al. 2010). Canada Vigilance Program database did not result in any AERs for this strain, however, 98 AERs were noted when the search term Bifidobacterium bifidum was used between January 1965 and June 2019. Specific strains of the bacterium were not specified, and causality assignment was uncertain. In TGA, no AERs were found for this specific strain, however, three AERs were identified when using the search term Bifidobacterium bifidum between January 1971 and July 2019. Specific strains of the bacterium were not specified, and causality assignment was uncertain. Two of the AERs consisted of an anaphylactic reaction, pain, rash, nausea, and vomiting in adults and the other AER consisted of necrotizing colitis in a female infant following a product containing Bifidobacterium bifidum and Lactobacillus acidophilus. No other details were outlines in the AERs. Finally, no AERs related to this strain were found in the Medicines and Healthcare Products Regulatory agency – Yellow Card database, EudraVigilance European database, or New Zealand’s MedSafe.
Quality assessment
Nucleic acid-based identification assay	PCR sample preparation with Primer set 1 produces an amplification product of 196 base pairs. PCR sample preparation with Primer set 2 produces an amplification product of 186 base pairs.
Enumeration assay	NLT 100% of the labeled viable cell count, in CFU/g
Contaminants	None were reported in the literature. The official USP monograph for Bifidobacterium bifidum Bb-06 has specifications for total combined molds and yeast count that does not exceed 10^2 CFU/g. Total non-lactic acid bacterial count is less than 5x10^3 CFU/g. It meets the requirements for absence of E. coli in 10 g and salmonella species in 40 g. Meets requirements of test for absence of Listeria in 25 g.
Packaging and storage	Preserve in high barrier foil laminate bags and store at or below 4 °C.
Labeling	Ingredient should be labeled with genus, species, and strain names and with the formulated enumeration in CFU/g.
Probiotic	Bifidobacterium animalis subsp. lactis
Probiotic strains	Bi-07 (ATCC- SD5220), Bl-04 (ATCC- SD5219), B420 (ATCC- SD6685) and HN019 (ATCC- SD5674)
Definition	Bifidobacterium animalis subsp. lactis is a lactic-acid producing, Gram-positive, rod-shaped, non-spore-forming, anaerobic bacterium that is pleomorphic. Various rod shapes may be observed, but they are typically curved or clubbed and often branched. Strains Bl-04 and Bi-07 are of human origin while HN019 and B420 are isolated from fermented milk products.
Comprehension or abbreviated revision	Abbreviated review conducted in September 2016.
Other Pharmacopeial Monographs & Regulatory standards (CFSAN, ODI, NNHPD, EFSA QPS, ARTG)	Bifidobacterium animalis subsp. lactis strains Bi-07, Bl-04, HN019, and B420 were the subject of FDA GRN 445 for use as ingredients in a wide variety of food types. Food Chemicals Codex monographs for B. animalis subsp. lactis strains Bi-07, Bl-04, and HN019 have been effective since June 2016. Health Canada has this species on the Natural Health Products Ingredients Database as a Schedule 1 substance and item 1 bacterium under NHP regulations. The species is included in the Health Canada monograph for probiotics. EFSA granted QPS status to the species based on its long history of safe use. The species is also listed in the Inventory of Microorganisms with Documented History of Use in Human Food published by IDF/EFFCA.
Chemistry/Identity/Characteristics
Taxonomy change	None listed
History of use	The species has been added to human food since at least 1980 and is very common in dairy products worldwide including the US where the organism is the most common Bifidobacterium in yogurt products. In particular, HN019 has been safely added to foods globally in dairy products and dietary supplements for many years as well as Bi-07, BI-04 B420 all without a report of adverse effect on consumers.
Complete genome sequence unambiguously identifying the strain at the species level	The four strains have gone through complete genomic sequencing.
Antibiotic resistance profile (phenotypic and genotypic)	The four strains are sensitive to clinically relevant antibiotics. The presence of the gene tetW is intrinsic to the species Bifidobacterium animalis subsp. lactis.
Presence of transmissible elements (e.g. transposase)	Based on the GRAS document, there is no evidence that the tetW gene that is co-transcribed in tandem with this transposase has any ability to transfer resistance, and therefore poses no known risk of transfer.
Presence of virulence factors / toxin genes (e.g. hemolysis)	None listed
Animal toxicity studies	GRN 445 noted that the panel of experts found that ‘species of the genus Bifidobacterium are considered to be non-pathogenic, non-toxigenic and are considered safe for use in foods’ according to the EFSA guidance document criteria.
Allergenicity	Components of the fermentation media are not derived from major food allergens.
Intended us indications	Dietary supplements and conventional foods.
Typical intake level	The daily recommended intake of the dietary supplement is in the range of 1–2 × 10^10 CFU/day.
Clinical studies and adverse events reporting
Human clinical studies tracking safety endpoints	A total of 53 human studies conducted in adults and children using various strains of Bifdobacteria were presented and evaluated in GRN 445. It was concluded from these studies that infants, children, adults, and elderly adults can safely tolerate Bifidobacteria species at daily doses up to 6 x10^11 CFU/day for up to 2 years.
Adverse events reporting	HN019: no adverse events were reported in studies involving children, adult, elderly, healthy, mildly-ill, to critically-ill subjects consuming doses ranging from 1.9x10^7 to 3x10^11 CFU per day for 7 days to two years. Bi-07: There were no withdrawals related to adverse events during the studies. Bl-04: No adverse events were recorded in the seven clinical studies.
Quality assessment
Nucleic acid-based identification assay	Bi-07: PCR sample preparation with Primer set 1 produces an amplification product of 533 base pairs, there should be no amplification product of 479 base pairs; PCR sample preparation with Primer set 2 produces an amplification product of 492 base pairs with one SNPs. Bl-04: PCR sample preparation with Primer set produces an amplification product of 479 base pairs, there should be no amplification product of 533 base pairs. HN019: PCR sample preparation with Primer set 1 produces an amplification product of 351 base pairs with one SNPs; PCR sample preparation with Primer set 2 produces an amplification product of 531 base pairs with one SNPs. B420: PCR sample preparation with Primer set 1 produces an amplification product of 231 base pairs with one SNPs; PCR sample preparation with Primer set 2 produces an amplification product of 228 base pairs.
Enumeration assay	NLT 100% of the labeled viable cell count in CFU/g
Contaminants	None were reported in the literature. The official USP monograph for Bifidobacterium animalis subsp. lactis has specifications for total combined molds and yeast count that does not exceed 10^2 CFU/g. Total non-lactic acid bacterial count is less than 5x10^3 CFU/g. It meets the requirements for absence of E. coli in 10 g and Salmonella species in 40 g. Meets requirements of test for absence of Listeria in 25 g.
Packaging and storage	Preserve in high barrier foil laminate bags and store at or below 4 °C.
Labeling	Ingredient should be labeled with genus, species, and strain names and with the formulated enumeration in CFU/g.
Probiotic	Bifidobacterium longum subsp. infantis
Probiotic strain	Bi-26 (ATCC-SD6720)
Definition	Bifidobacterium longum subsp. infantis is a Gram-positive, non-spore forming, lactic acid-producing anaerobic, facultative hexose heterofermenters. They have various shapes, including short, curved rods, club-shaped rods, and bifurcated Y-shaped rods. Bi-26 is a non-d-lactate producer strain.
Comprehension or abbreviated revision	Comprehensive review was conducted in May 2020.
Other Pharmacopeial Monographs & Regulatory standards (CFSAN, ODI, NNHPD, EFSA QPS, ARTG)	B. longum subsp. infantis Bi-26 was not the subject of a GRAS notice or an NDIN to the FDA. An expert panel evaluated the safety information in the GRAS report and concluded that B. longum subsp. infantis Bi-26 was GRAS based on scientific procedures for use as an ingredient in conventional foods and in dietary supplements (2). B. longum subsp. infantis B-26 was not listed in the US CFR for any food uses in US. B. longum species are listed on UNPA of old dietary ingredients, but Bi-26 was not specifically indicated. Health Canada lists B. longum subsp. infantis species on the Natural Products Ingredients Database as a Schedule 1 substance and item 1 bacterium under NHP. It is also included in the Health Canada monograph for probiotics. EFSA granted a QPS status of B. longum species. The species is also listed in the Inventory of Microorganisms with Documented History of Use in Human Food published by IDF/EFFCA. The Australian Register of Therapeutic Goods has information on B. longum but does not specifically list subsp. Infantis Bi-26.
Chemistry/Identity/Characteristics
Taxonomy change	None listed
History of use	Bifidobacterium infantis is a dietary ingredient found in a number of dietary supplements currently available on the US market with only small amount containing specific B. longum subsp. infantis Bi-26 strain.
Complete genome sequence unambiguously identifying the strain at the species level	Complete genome sequencing was completed on B. longum subsp infantis B1-26, but not available in the literature. Self-determined GRAS reports comparisons to available genomes for B. longum subsp. infantis confirming genetic synteny (unpublished data).
Antibiotic resistance profile (phenotypic and genotypic)	B. longum subsp. infantis Bi-26 did not exhibit acquired resistance to antibiotics in a microdilution method following EFSA 2012 guideline (EFSA 2012).
Presence of transmissible elements (e.g. transposase)	None listed
Presence of virulence factors / toxin genes (e.g. hemolysis)	Assessment for toxin genes or bacteriocins and for hemolytic was evaluated by analyzing the B. longum subsp. infantis Bi-26 genome for suspect genes and proteins. No known suspect genes, virulence factors, or toxin proteins were identified (unpublished data).
Animal toxicity studies	No mortality or adverse events were reported in SD female rats administered 5000 mg/kg of B. longum supsp., infantis Bi-26during an acute oral toxicity study. No adverse effects on clinical signs and body weights were noted during a post-dose 14-day observation period. There were no effects on body weight or body weight gain. No macroscopic lesions were seen at necropsy. B. longum subsp. infantis Bi-26 was not considered acutely toxic. No subacute, subchronic or chronic toxicity studies were identified. No genotoxicity, reproductive, or developmental toxicity studies were identified.
Allergenicity	None listed.
Intended us indications	A self-determined GRAS report for B. longum subsp. infantis Bi-26 indicated that it is intended for use in dietary supplements in the form of capsules, tablets, or powders. In addition, B. longum subsp. infantis Bi-26 also intended for use in conventional foods.
Typical intake level	The manufacturers’ recommended daily intake levels in the supplement products are 0.6 × 10^9 to 5.0 × 10^9 CFU/day of B. longum subsp. infantis Bi-26.
Clinical studies and adverse events reporting
Human clinical studies tracking safety endpoints	Clinical studies evaluating the safety of B. longum subsp. infantis Bi-26 as a single ingredient in healthy subjects are lacking in the literature. Of studies involving other strains from this species are well tolerated in infants and adults.
Adverse events reporting	No adverse events or case reports were found in literature for B. longum subsp. infantis Bi-26. A search of the FDA Center for Food Safety and Applied Nutrition (CFSAN) Adverse Event Reporting System (CAERS) data files for the period of January 2004 - March 2019 returned a total of 18 adverse event reports (AERs) using the search terms ‘Bifidobacterium infantis’ or ‘infantis’. The strain Bi-26 was not specified in the AERs. No adverse event reports were returned using the search terms Bifidobacterium longum subsp. infantis Bi-26 and Bifidobacterium longum. Nine out of 18 AERs consisted of gastrointestinal symptoms (e.g. nausea, abdominal pain, abdominal distension, vomiting, flatulence, diarrhea, discolored feces, and diverticulitis) and two AERs consisted of hypersensitivity-related symptoms (e.g. hypersensitivity, rash, pruritus). Three of the AERs involved the concomitant intake of other products. The gastrointestinal symptoms might be related to the specific product because these symptoms occurred more frequently, but a causality assignment was uncertain for the other reported symptoms because they involved different organ systems. The Canada Vigilance Program database did not yield any AERs using the search term Bifidobacterium longum subsp. infantis Bi-26 (searched on April 16, 2020). However, 16 AERs were identified for other strain of Bifidobacterium longum subsp. infantis for the period of January 1, 1965 to December 31, 2019. Ten out of 16 AERs consisted of gastrointestinal symptoms (e.g. nausea, abdominal pain, abdominal distension, vomiting, flatulence, ulcer, gastrointestinal reflux, anal incontinence) and six AERs consisted of hypersensitivity-related symptoms (e.g. hypersensitivity, rash, pruritus, pyrexia, urticaria, lip swelling, erythema,). Three of the AERs involved the concomitant intake of other products. The gastrointestinal and hypersensitivity-related symptoms might be related to a specific product because these symptoms occurred were more frequently. However, a causality assignment was uncertain for other reported symptoms because they involved different organ systems (see adverse event table at end of the report). No AERs were found for Bifidobacterium longum subsp. infantis Bi-26 in the Australian Therapeutic Goods Administration (TGA) database of adverse event notifications. However, three AERs were identified using the search term Bifidobacterium infantis for the period of January 1, 1971 to January 1, 2020. All three AERs consisted of gastrointestinal symptoms and one AER consisted of a rash. The strain of B. infantis was not specified in the AERs. Causality assignment was uncertain because of the low number of AERs that involved products containing multiple probiotic ingredients and one AER had the concomitant intake of other products. No AERs related to Bifidobacterium longum subsp. infantis Bi-26  or Bifidobacterium infantis were found in the Medicines and Healthcare Products Regulatory Agency – Yellow Card (MHRA) database, EudraVigilance European database, or New Zealand’s MedSafe.
Quality assessment
Nucleic acid-based identification assay	PCR sample preparation with Primer set 1 produces an amplification product of 105 base pairs. PCR sample preparation with Primer set 2 produces an amplification product of 181 base pairs.
Enumeration assay	NLT 100% of the labeled viable cell count in CFU/g of the specified strain of Bifidobacterium longum subsp. infantis.
Contaminants	None were reported in the literature. The official USP monograph for Bifidobacterium longum subsp. infantis has specifications for total combined molds and yeast count that does not exceed 10^2 CFU/g. Total non-lactic acid bacterial count is less than 5x10^3 CFU/g. It meets the requirements for absence of E. coli in 10 g and Salmonella species in 40 g. Meets requirements of test for absence of Listeria in 25 g.
Packaging and storage	Preserve in high barrier foil laminate bags and store at or below 4°.
Labeling	Ingredient should be labeled with the genus, species, and strain names and with the formulated enumeration in cfu/g.
Probiotic	Lacticaseibacillus casei (formerly Lactobacillus casei)
Probiotic strain	Lc-11 (ATCC-SD5213)
Definition	Lacticaseibacillus casei is a lactic acid producing, gram positive, rod shaped, non-spore forming, facultative and anerobic bacterium that is homofermentative. The cells are non-motile, straight, and found in pairs or chains with variable lengths. The Lc-11 strain produces 5% d-lactate and 95% l-lactate.
Comprehension or abbreviated revision	Comprehensive review was conducted in May 2020.
Other Pharmacopeial Monographs & Regulatory standards (CFSAN, ODI, NNHPD, EFSA QPS, ARTG)	L. casei Lc-11 was not subject of GRAS or NDIN to the FDA. Health Canada lists the species as a schedule 1 substance under item 1 bacterium of the Natural Health Product Regulations, but Lc-11 strain was not listed specifically. L. casei is included in Health Canada monograph for probiotics. EFSA granted a qualified presumption of safety status based on a long history of safe use. The species is also listed in the Inventory of Microorganisms with Documented History of Use in Human Food published by IDF/EFFCA. The Australian Register of Therapeutic Goods (ARTG) does not specifically list Lc-11 as an active ingredient.
Chemistry/Identity/Characteristics
Taxonomy change	The genus Lactobacillus was recently subdivided into 25 genera based on phylogenetic analysis. As a result, Lactobacillus casei was reclassified as Lacticaseibacillus casei (Zheng et al. 2020).
History of use	L. casei was first proposed as a novel species in 1971. L. casei Lc-11 is described as a dairy isolate typically found in gastrointestinal tract of humans and can be found naturally fermented foods including fermented cheese and milk. (unpublished data).
Complete genome sequence unambiguously identifying the strain at the species level	The ATCC number for L. casei is SD-5213. Complete genome sequencing has been performed for L. casei however it is not available in literature (unpublished data).
Antibiotic resistance profile (phenotypic and genotypic)	L. casei Lc-11 did not exhibit acquired resistance to antibiotics in a microdilution method following an EFSA guidelines (unpublished data).
Presence of transmissible elements (e.g. transposase)	Not Listed
Presence of virulence factors / toxin genes (e.g. hemolysis)	No known suspect genes, virulence factors or toxin proteins were identified in L. casei Lc-11. (unpublished data).
Animal toxicity studies	No mortality or adverse effects was reported in Crl:CD (Sprague Dawley) female rats administer 5,000mg/kg according to EPA Teat Guideline OPPTS 870.1100, OECD’s Guideline 425, and FDA Redbook 2000 Guideline IV.C.2. No adverse effects on clinical signs and body weight were noted post dose of 14 days. No macroscopic lesions were seen therefore, L. casei not considered acutely toxic. No subacute, subchronic, chronic toxicity, genotoxicity, reproductive, or developmental toxicity studies were identified for L. casei Lc-11.
Allergenicity	None listed.
Intended use indications	Intended use in dietary supplements and conventional foods (unpublished data).
Typical intake level	Dietary supplements at intake levels of at least 5.0 × 10^10 cfu/serving. In addition, L. casei Lc-11 is intended foruse in conventional foods at levels of at least 1.0 × 1010 cfu/serving. The estimated daily intakes are up to 5.0 × 1012 cfu/day of L. casei Lc-11 from conventional foods or dietary supplements (unpublished data)
Clinical studies and adverse events reporting
Human clinical studies tracking safety endpoints	Clinical studies evaluating the safety of L. casei Lc-11 as a single ingredient in health participants are lacking in public domain. Efficacy of a multi-probiotic mixture given to obese participants was evaluated in a double blind, randomized, placebo-controlled study (Gomes et al. 2017). No adverse effects occurred during the study. Clinical studies were conducted for L. casei Shirota as described in GNR 429. No adverse effects were noted in healthy adults given L. casei Shirota for up to 5 wk in healthy participants and in health compromised adults for up to 4 years. This proved true in healthy and compromised children and infants (FDA, 2012). A self-determined GRAS report available for L. casei Lc-11 references 22 articles from 2007–2019 for other strained of L. casei used in clinical studies (unpublished data). The assessed publications included randomized, double-blind, placebo-controlled studies, partially blinded/controlled trials, and open-label studies. The studies used either L. casei alone or other species of probiotics given in combination with L. casei. The reported intake levels of the L. casei strains ranged from 1.0 × 10^7 to 1.0 × 10^10 cfu/day with average intake levels of 1 × 10^9 to 1 × 10^10 cfu/day. The treatment durations were up to 6 months. The articles either report no treatment-related adverse effects or do not describe or report the monitoring of adverse events. Assessed publications described L. casei strains as either well tolerated or do not report safety outcomes. Side effects consisted mainly of gastrointestinal complaints that were mild and reversible.
Adverse events reporting	No adverse case reports for L. casei Lc-11 were found and no adverse events related to other strains. Three cases of bacteremia associated with L. casei were identified. The three cases were in elderly people with underlying conditions. CFSAN CAERS data returned no AERs from January 2004–March 2020. The Canada Vigilance Program database returned no AERs for Lactobacillus casei Lc-11 from January 1965 to May 2021. A total of 58 AERs were identified using the search terms ‘lactobacillus casei’ (strain not listed) and ‘lactobacillus casei var. rhamnosus’. A causality assignment was uncertain because many of AERs involved multiingredient products and/or the concomitant intake of other products and/or medicine. 13 AERs were identified in the Australian Therapeutic Goods Administration (TGA) using the term’ Lactobacillus casei’ from January 1971 to September 2021. The strain was not specified. The adverse events involved hypersensitivity (anaphylactic reaction, rash, urticaria, pruritus, erythema, allergic dermatitis), gastrointestinal symptoms (nausea, vomiting, abdominal pain, diarrhea). Causality assignment for L. casei was uncertain because the cases involved products with multiple ingredients or probiotics, or concomitant intake of other products European EudraVigilance database identified 16 AERs with no strain indicated. No AERs related to L. casei were found in the Medicines and Healthcare Products Regulatory Agency (MHRA) Yellow Card database or New Zealand’s MedSafe
Quality assessment
Nucleic acid-based identification assay	PCR sample preparation with Primer set produces an amplification product of 176 base pairs.
Enumeration assay	NLT 100% of the labeled viable cell count in CFU/g.
Contaminants	None were reported in the literature. The official USP monograph for Lactocaseibacillus casei Lc-11 has specifications for total combined molds and yeast count that does not exceed 10^2 CFU/g. Total non-lactic acid bacterial count is less than 5x10^3 CFU/g. It meets the requirements for absence of E. coli in 10 g and Salmonella species in 40 g. Meets requirements of test for absence of Listeria in 25 g.
Packaging and storage	Preserve in high barrier foil laminate bags and store at or below 4 °C.
Labeling	Ingredient should be labeled with the genus, species, and strain names and with the formulated enumeration in cfu/g.
Probiotic	Streptococcus thermophilus
Probiotic strain	St-21 (ATCC SD-5207)
Definition	Streptococcus thermophilus is a lactic acid producing, gram positive, cocci shaped, non-spore forming, facultatively anaerobic bacterium that is homofermentative. Cells are non-motile, commonly found in linear chains. St-21 is a non-D-lactate producer strain.
Comprehension or abbreviated revision	Comprehensive review was conducted in July 2021.
Other Pharmacopeial Monographs & Regulatory standards (CFSAN, ODI, NNHPD, EFSA QPS, ARTG)	S. thermophilus St-21 was not listed in any pharmacopeias. S. thermophilus St-21 was not the subject of a GRAS notice or a NDIN to the FDA. A self-determined GRAS report for S. thermophilus St-21 was provided from the sponsor. S. thermophilus St-21 was not listed under 21 CFR §182 for substances generally recognized as safe for food uses in the US. Streptococcus thermophilus is listed under 21 CFR §131, Subpart B, for Requirements for Specific Standardized Milk and Cream. Streptococcus thermophilus is listed along with Lactobacillus delbrueckii subsp. bulgaricus as lactic acid-producing bacteria used in bacterial cultures to produce yogurt (21 CFR §131.200). Health Canada lists Streptococcus salivarius subsp. thermophilus as a Schedule 1 substance and item 1 bacterium under the NHP regulations, however St-21 strain was not specifically listed. In addition, Streptococcus salivarius subsp. thermophilus was listed in the Health Canada monograph for probiotics. EFSA granted QPS status of S. thermophilus in 2007 noting use as dairy starter microorganism for manufacture of fermented milk and cheese. The Streptococcus thermophilus species was still on the QPS list that was updated in 2020. Australian Register of Therapeutic Goods (ARTG) has information regarding medicine prodructs containing S. thermophilus however it did not specifically list St-21 strain as the active ingredient.
Chemistry/Identity/Characteristics
Taxonomy change	S. thermophilus formerly known as Streptococcus salivarius subsp. thermophilus. Related to Lactococcus lactis but phylogenetically closer to streptococcal species of Viridans, further characterized as belonging to the salivarius group of Viridans. In mid 1980s S. thermophilus was reclassified as S. salivarius subsp thermophilus based on studies assessing DNA base composition. In 1991 a more stringent study using DNA-DNA reassociation experiments demonstrated S. thermophilus as a district species and therefore a taxonomic name of the species was shifted back to former S. thermophilus name (unpublished data; Delorme 2008; FDA 2019).
History of use	S. thermophilus is the only reported streptococcal species to originate from dairy products and is a widely used started in manufacture of dairy products including yogurt and cheese (Delorme, 1008) S. thermophilus is described as highly adapted to dairy environment and present at low levels in raw milk from animals (Harnett et al. 2011). It is thought that S. thermophilus is the only food species among commensal and opportunistic pathogens and evolved by combination of loss of function and horizontal gene transfer events from other dairy species (Delorme 2008).
Complete genome sequence unambiguously identifying the strain at the species level	Complete genome sequencing was performed for S. thermophilus St-21 (unpublished data). However, the genome of S. thermophilus St-21 is not available in the literature. Comparison of the genome has confirmed core similarity and genetic synteny to other publicly available genomes for S. thermophilus strains (unpublished data).
Antibiotic resistance profile (phenotypic and genotypic)	Antibiotics resistance of S. thermophilus was assessed using microdilution method according to EFSA 2012 guideline. S. thermophilus St-21 did not exceed the microbiological cutoff values (mg/L) defined by EFSA 2012 guideline indicating that under the conditions of the assay the strain did not exhibit acquired antibiotic resistance (unpublished data). In updated 2018 EFSA guidelines S. thermophilus St-21 was still within cut off limits and did not show phenotype resistance to tested antibiotics.
Presence of transmissible elements (e.g. transposase)	None listed
Presence of virulence factors / toxin genes (e.g. hemolysis)	No virulence or toxic genes were identified in genome of S. thermophilus St-21. S. thermophilus St-21 lacks histidine and tyrosine decarboxylase genes therefore unlikely to produce biogenic amines (unpublished data; Pradhan, 2020)
Animal toxicity studies	No mortality or adverse effects were reported in female rats administered 5000 mg/kg of S. thermophilus St-21 in oral acute toxicity study. No adverse effects on clinical signs and body weights were noted post 14-day exposure. No macroscopic lesions were seen at necropsy. S. thermophilus St-21 was not considered acutely toxic in rats when given by oral route. No short term, long-term, carcinogenicity, genotoxicity, reproductive and developmental, or potential interaction animal studies were found.
Allergenicity	None listed.
Intended us indications	Products containing S. thermophilus St-21 listed in DSLD claim to aid in health digestion, maintain intestinal lining, aid in detoxification process, and support a health gut microbiome, whereas those listed in Supplement Owl claim aid in gastrointestinal support, women’s health, daily wellness, and digestive and immune support.
Typical intake level	The manufacturer recommended daily intake values ranged from 1.5x10^8 to 5.0x10^9 cfu which is below the estimated intake levels reported in self-determined GRAS report (unpublished data).
Clinical studies and adverse events reporting
Human clinical studies tracking safety endpoints	Clinical studies evaluating safety of S. thermophilus St-21 as single ingredient are not available and efficacy studies are lacking. In a case study evaluating the effect of a symbiotic preparation in a patient with S typhimurium infection provided 4.5x10^8 cfu daily S. thermophilus St-21 for 10 days. No side effects or adverse findings were mentioned, and the patient show improvement in symptoms (Piatek et al. 2019). Clinical studies on other strains of S. thermophilus. The reported intake levels of S. thermophilus strains in the studies ranged from 1.0 × 10^9 to 5.0 × 10^11 cfu daily. The treatment durations were up to 210 days. The studies were conducted in healthy adults and adults with medical conditions and in pregnant women, infants and children. The clinical studies described S. thermophilus strains as either well tolerated or did not report on any safety outcome. Side effects consisted mainly of gastrointestinal complaints that were reversible and mild in nature. No serious adverse events were reported.
Adverse events reporting	Self-determined GRAS report for S. thermophilus St-21 reports no cases of infection linked to the intake of St-21 strain (unpublished data). No adverse event reports were found in FDA Center for Food Safety and Applied Nutrition CAERS search. The Canada Vigilance Adverse Reaction Online Database did not yield any AERs searching St-21 strain but 8 AERs were listed for Streptococcus thermophilus including genital and vaginal bacterial infection, bacteremia especially in patients with weak intestinal barrier function or immune compromised. However, the available information was insufficient to determine causality. No AERs were listed for St-21 strain in the Australian Therapeutic Goods Administration database. No AERs were found for Streptococcus thermophilus St-21 and Streptococcus thermophilus in the Medicines and Healthcare Products Regulatory Agency-Yellow Card database, EurdaVigilance European database, WHO Uppsala Monitoring Center VigiAccess and New Zealand’s MedSafe.
Quality assessment
Nucleic acid-based identification assay	PCR sample preparation with Primer set 1 produces an amplification product of 217 base pairs. PCR sample preparation with Primer set 2 produces an amplification product of 179 base pairs with one SNPs.
Enumeration assay	NLT 100% of labeled viable cell count, in cfu/g
Contaminants	None were reported in the literature. The official USP monograph for Streptococcus thermophilus St-21 has specifications for total combined molds and yeasts count does not exceed 10^2 cfu/g. The total non-lactic acid bacteria count is less than 5 × 10^3 cfu/g Meets the requirements of the test for absence of E. coli in 10 g. It meets requirements of the test for absence of Salmonella species in 40 g. Meets the requirements of the test for absence of Listeria in 25 g
Packaging and storage	Preserve in high barrier foil laminate bags and store at or below 4 °C until use
Labeling	Ingredient should be labeled with the genus, species and strain names and with the formulated enumeration in cfu/g.
Probiotic	Ligilactobacillus salivarius (formerly Lactobacillus salivarius)
Probiotic strain	Ls-33 (ATCC SD-5208)
Definition	Ligilactobacillus salivarius is a lactic acid producing, Gram positive, rod shaped, non-spore forming, facultatively anaerobic bacterium that is homofermentative. Ls-33 is a non-D-lactate producer strain.
Comprehension or abbreviated revision	Comprehensive review was conducted in July 2021.
Other Pharmacopeial Monographs & Regulatory standards (CFSAN, ODI, NNHPD, EFSA QPS, ARTG)	L. salivarius Ls-33 is not listed in any pharmacopeias and not subject of a GRAS notice or a NDIN. Health Canada lists L. salivarius as a schedule 1 substance under item 1 bacterium, however Ls-33 strain was not specifically listed. L. salivarius is included in Health Canada monograph for probiotics. EFSA has granted QPS status to L. salivarius species due to long history of safe use. The Australian Register of Therapeutic Goods has information regarding L. salivarius however, Ls-33 not specifically listed as the active ingredient in products.
Chemistry/Identity/Characteristics
Taxonomy change	The genus Lactobacillus was recently subdivided into 25 genera based on phylogenetic analysis. As a result, Lactobacillus salivarius was reclassified as Ligilactobacillus salivarius (Zheng et al. 2020).
History of use	L. salivarius is reported to be used in human food and dairy since at least 1996 (Coulin et al. 2006). Self-determined GRAS reports Ls-33 used in foods and supplements for more then 20 years (unpublished data). L. salivarius has been found in traditional fermented foods (Aljuobori et al. 2014; Bernardeau, 2006). L salivarius has also been used to prevent and treat chronic diseases such as asthma, cancer, atopic dermatitis and halitosis (Chaves, 2017).
Complete genome sequence unambiguously identifying the strain at the species level	Complete genome of L. salivarius was performed, but specific literature is not available for Ls-33 strain (unpublished data)
Antibiotic resistance profile (phenotypic and genotypic)	Antibiotic resistance of L. salivarius Ls-33 was assessed using microdilution method according to EFSA 2012 guideline. L. salivarius Ls-33 did not exceed the microbiological cut off values defined by Lactobacillus facultative heterofermentative species in EFSA 2012 guidelines.
Presence of transmissible elements (e.g. transposase)	No transposons, transposase, insertion sequences, phage or plasmids were gound in genome that are considered a health concern per PATRIC.
Presence of virulence factors / toxin genes (e.g. hemolysis)	No virulence or toxic genes were identified in genome of L. salivarius Ls-33 (unpublished data).
Animal toxicity studies	No mortality or adverse effects reported in SD female rats administered 5000 mg/kg L. salivarius Ls-33 strain in oral acute toxicity study. No adverse effects on clinical signs and body weight post 14-day observation period. No macroscopic lesions were seen at necropsy. L. salivarius Ls-33 is not considered acutely toxic (unpublished data). No subacute or subchronic toxicity studies were identified for Ls-33 strain.
Allergenicity	None listed.
Intended us indications	Products containing L. salivarius Ls-33 listed in DSLD claimed to provide benefits to colon health, gastrointestinal system, immune system, and one claimed to serve as a beauty tonic. Products in Supplement OWL claim to provide gastrointestinal support, support intestinal flora, and digestive and colon health.
Typical intake level	Numerous products containing L. salivarius are listed in both the DSLD and Supplement OWL; however, as part of proprietary blends no information on serving or daily intake were provided on specific ingredients of the blend.
Clinical studies and adverse events reporting
Human clinical studies tracking safety endpoints	Clinical studies evaluating the safety of L. salivarius Ls-33 as a single ingredient in health subject are lacking in literature. One study evaluating immunomodulatory response in health participant given 2.0 × 1010cfu/day for 3 wk, 2 studies evaluated effects on biomarkers related to inflammation and metabolic syndrome, and one evaluating multi-probiotic mixture given to colicky infants all reported not adverse effects (Paineau et al. 2008; Gobel, 2012; Larsen et al. 2013). Studies defined Ls-33 strain as well tolerated or did not report safety outcomes. Self-determined GRAS report references 27 articles published during the period from 2002 to 2019 for other strains of L. salivarius used in clinical studies. The assessed publications included randomized, double-blind, placebo-controls studies, partially blinded/controlled trials, or open label studies. The studies used either L. salivarius alone or other species of probiotics given in combination with L. salivarius. The reported intake levels of L. salivarius strains in the studies ranged from 1.0 × 10^8 to 2.0 × 10^10 cfu daily. The treatment durations were up to 2 years. The studies were conducted in healthy adults and adults with atopic dermatitis, supra/subgingival plaque, chronic spontaneous urticaria, ulcerative colitis, and in pregnant women, infants and children.  The clinical studies described L. salivarius as either well tolerated or did not describe/report monitoring of adverse events. Side effects consisted of mainly gastrointestinal complaints which were mild and reversible. No serious events were reported (unpublished data).
Adverse events reporting	Self-determined GRAS for L. salivarius Ls-33 reports no serious adverse events or cases of opportunistic infection have been linked to its consumption (unpublished data). However, 2 cases of infection were described for the species. FDA CFSAN CAERS search returned no adverse reports using the term Lactobacillus salivarius Ls-33 and Ligilactobacillus salivarius. The Canada Vigilance Adverse Reaction Online Database did not yield any AERs using the search terms ‘Lactobacillus salivarius Ls-33’ but 18 AERs were listed for either term ‘Lactobacillus salivarius’ and ‘Ligilactobacillus salivarius’. The strain was not listed. The Australian Therapeutic Goods Administrations (TGA) did not yield results for Lactobacillus salivarius Ls-33. Six AERs were listed for the term ‘Lactobacillus salivarius’ but the strain was not specified. No AERs were found for ‘Lactobacillus salivarius Ls-33’ and ‘Lactobacillus salivarius’ in the Medicines and Healthcare Products Regulatory Agency – Yellow Card (MHRA) database, EudraVigilance European database, WHO Uppsala Monitoring Center VigiAccess and New Zealand’s MedSafe. No AERs were listed for Ligilactobacillus salivarius.
Quality assessment
Nucleic acid-based identification assay	PCR sample preparation with Primer set 1 produces an amplification product of 159 base pairs. PCR sample preparation with Primer set 2 produces an amplification product of 241 base pairs with one SNPs.
Enumeration assay	NLT 100% of labeled viable cell count, in cfu/g
Contaminants	None were reported in the literature. The official USP monograph for Ligilactobacillus salivarius Ls-33 has specifications for total combined molds and yeast count that does not exceed 10^2 CFU/g. Total non-lactic acid bacterial count is less than 5x10^3 CFU/g. It meets the requirements for absence of E. coli in 10 g and Salmonella species in 40 g. Meets requirements of test for absence of Listeria in 25 g.
Packaging and storage	Preserve in high barrier foil laminate bags and store at or below 4 degrees
Labeling	Ingredient should be labeled with genus, species and strain name and with the formulated enumeration in cfu/g.
Probiotic	Lactiplantibacillus plantarum (formerly Lactobacillus plantarum)
Probiotic strain	Lp-115 (ATCC-SD5209)
Definition	L. plantarum is a lactic acid producing, gram positive, rod-shaped, non-spore forming, facultatively anaerobic bacterium that is heterofermentative. Cells are non-motile, usually straight, and commonly found in short chains. Lp-115 strain produces 55% d-lactate and 45% l-lactate.
Comprehension or abbreviated revision	Abbreviated review was conducted in July 2021.
Other Pharmacopeial Monographs & Regulatory standards (CFSAN, ODI, NNHPD, EFSA QPS, ARTG)	A GRAS notice (GRN 722) was submitted for L. plantarum Lp-115, which was filed by the FDA with no questions. The GRAS submission for FDA GRN 722 was submitted under its former name of Lactobacillus plantarum Lp-115, which was filed by the FDA with no question. NDINs 1164 and 1165 have been submitted for the L. plantarum species. The NDINs use the data for L. plantarum Lp-115 to support the safety of the other unspecified strains. Health Canada lists L. plantarum as a schedule 1 substance under item 1 bacterium under NHP regulations but does not specifically list Lp-115. L. plantarum is included in Health Canada monograph for probiotics. EFSA granted QPS status of L. plantarum. Thespecies is also listed in the Inventory of Microorganisms with Documented History of Use in Human Food published by IDF/EFFCA.
Chemistry/Identity/Characteristics
Taxonomy change	The genus Lactobacillus was recently subdivided into 25 genera based on phylogenetic analysis. As a result, Lactobacillus plantarum was reclassified to Lactiplantibacillus plantarum (Zheng et al. 2020).
History of use	This species is used commercially as a starter culture in food fermentation and as a probiotic in dietary supplements and food. At the time of the GRAS filing, L. plantarum Lp-115 has been commercially available for more than 20 years, where 78 000kg have been marketed over the past 5 years as a dietary supplement representing 5 billion servings (FDA GRAS No. 722, 2018).
Complete genome sequence unambiguously identifying the strain at the species level	Complete genome sequencing was performed for L. plantarum Lp-115 (FDA GRAS No. 722, 2018). However, the genome of L. plantarum Lp-115 is not available in the literature. GRN 722 reports that comparisons of the genome of L. plantarum Lp-115 to other publicly available genomes for L. plantarum strains has confirmed the core similarity and genetic synteny of L. plantarum Lp-115.
Antibiotic resistance profile (phenotypic and genotypic)	GRN 722 discusses studies demonstrating that L. plantarum Lp-115 is susceptible to antibiotics and it lacks acquired, functional, or transferable antibiotic resistance genes (FDA GRAS No. 722, 2018). L. plantarum Lp-115 did not exceed the microbiological cutoff values (mg/L) defined for Lactobacillus plantarum in the EFSA 2012 guideline indicating that under the conditions of the assay the strain did not exhibit acquired antibiotic resistance. Furthermore, it is still within the cutoff values from the updated version of the guidelines (FDA GRAS No. 722, 2018). The genome analysis of the Lp-115 strain showed no matches for known acquired clindamycin resistance genes. L. plantarum Lp-115 was found to have several genes that encode for prophage proteins, but no known antibiotic resistance genes were found to be associated with the prophage proteins within the same operon of genes. No statistical hits were found in a BLAST analysis of the Lp-115 genome using the DNA and protein sequences of known ermB and ermC genes from L. plantarum obtained in a search of the public database NCBI (FDA GRAS No. 722, 2018).
Presence of transmissible elements (e.g. transposase)	Transposases were also identified within L. plantarum Lp-115, but no known genes for clindamycin resistance (i.e. erythromycin genes) were found in the vicinity of the transposases (FDA GRAS No. 722, 2018).
Presence of virulence factors / toxin genes (e.g. hemolysis)	GRN 722 describes that the genome of L. plantarum Lp-115 was analyzed for bacteriocins, toxin genes, and genes associated with hemolysin however, Lp-115 was found to not have the genes that produce hemolysin (FDA GRAS No. 722, 2018). Three bacteriocins were identified but are not dangerous to host and no toxin genes were identified to be virulent to host. L. plantarum Lp-115 was found to lack histidine decarboxylase and tyrosine decarboxylase genes therefore unlikely to produce biogenic amines.
Animal toxicity studies	In acute toxicity study, SD female rats administered 5,000mg/kg L. plantarum Lp-115 by oral gavage showed no toxicity or adverse signs, therefore it was not considered acutely toxic (FDA GRAS No. 722, 2018). No genotoxicity, reproductive, or developmental toxicity studies were identified for L. plantarum Lp-115.
Allergenicity	None listed.
Intended us indications	As described in GRN 722, L. plantarum Lp-115 has been on the market for over 20 years. Products listed in DSLD containing L. plantarum Lp-115 have claims including support of a healthy gut microbiome, to purge undesirable bacteria and waste, maintain GI balance, support digestion, and support micronutrient absorption. Products listed in Supplement OWL claim to support digestive health, provide gastrointestinal support, and provide immune support.
Typical intake level	GRN 722 for L. plantarum Lp-115 indicated that it is intended for use in dietary supplements at levels of at least 5.0 × 10^10 cfu/serving. In addition, L. plantarum Lp-115 is also intended for use in conventional foods at levels of at least 1.0 × 10^9 cfu/serving. The estimated daily intakes are up to 5.0 × 10^10 cfu/day L. plantarum Lp-115 from dietary supplements and 1.0 × 10^10 cfu/day from foods, leading to a total daily intake of 6.0 × 10^10 cfu/day from both (FDA GRAS No. 722, 2018). Overall, for supplements in DSLD (CR51) and Supplement OWL, the manufacturers’ recommended daily intake values for L. plantarum Lp-115 ranged from 2.0 × 10^8 to 3.0 × 10^9 cfu.
Clinical studies and adverse events reporting
Human clinical studies tracking safety endpoints	GN722 references results of 4 clinical efficacy trials with L. plantarum Lp-115 was consumed at intake levels of 2.0 × 10^11 cfu/day in adults up to 90 days. L. plantarum Lp-115 was well tolerated although three of the four studies did not describe or report on any safety parameters (Barreto et al. 2014; Costa et al. 2014; Paineau et al. 2008; Zhang et al. 2013). One study that monitored adverse events was conducted in patients with liver transplant given a mixture of a fiber plus a probiotic ingredient including L. plantarum Lp-115 at 5x10^9 cfu, twice daily via feeding tubes or orally for seven days (Zhang et al. 2013). Adverse effects reported included diarrhea and abdominal cramps but disappeared with temporary reduction. Based on these studies the authors concluded L. plantarum Lp-115 to be well tolerated with no serious side effects (Zhang et al. 2013). In addition, GRN 722 concludes that the clinical studies presented supports the safety of L. plantarum Lp-115 at intakes up to 1.0 × 10^10 cfu/day from foods (FDA GRAS No. 722, 2018).
Adverse events reporting	Three cases of infections have been reported for L. plantarum species, but no cases identified for L. plantarum Lp-115. FDA CFSAN CAERS returned no adverse events reports for Lp-115 strain. Canada Vigilance Adverse Reaction Online Database did not yield any AERs for the term Lactobacillus plantarum Lp-115. No AERs were listed for Lactobacillus plantarum Lp-115 in the Australian TGA. No AERs were reported from the WHO Uppsala Monitoring Center VigiAccess for Lactobacillus plantarum Lp-115 strain.
Quality assessment
Nucleic acid-based identification assay	PCR sample preparation with Primer set 1 produces an amplification product of 165 base pairs. PCR sample preparation with Primer set 2 produces an amplification product of 212 base pairs with one SNPs.
Enumeration assay	NLT 100% of the labeled viable cell count in cfu/g or cfu/dose
Contaminants	None were reported in the literature. The official USP monograph for Lactiplantibacillus plantarum Lp-115 has specifications for total combined molds and yeast count that does not exceed 10^2 CFU/g. Total non-lactic acid bacterial count is less than 5 × 10^3 CFU/g. It meets the requirements for absence of E. coli in 10 g and Salmonella species in 40 g. Meets requirements of test for absence of Listeria in 25 g.
Packaging and storage	Preserve in high barrier foil laminate bags and store at or below 4 °C.
Labeling	Ingredient should be labeled with the genus, species, and strain names and with the formulated enumeration in cfu/g.
Probiotic	Bacillus clausii
Probiotic strain	UBBC07
Definition	Bacillus clausii is a Gram positive, rod shaped, spore forming, motile, aerobic, facultative alkaliphilic bacterium.
Comprehension or abbreviated revision	Comprehensive review was conducted in May 2020.
Other Pharmacopeial Monographs & Regulatory standards (CFSAN, ODI, NNHPD, EFSA QPS, ARTG)	B. clausii UBBC07 was not the subject of a GRAS notice or a NDIN to the FDA. An expert panel evaluated the safety information in the GRAS report and concluded that B. clausii UBBC07 was GRAS based on scientific procedures for use as an ingredient in conventional foods (Unique Biotech, 2016). B. clausii UBBC07 is not listed for use in foods under US Code of Federal Regulations or the United Natural Products Association (UNPA) of old dietary ingredients. Health Canada lists B. clausii species as a Schedule 1 substance and item 1 bacterium under NHP guidelines, however UBBC07 strain was not listed specifically. B. clausii species was not included in Health Canada’s monograph for probiotics. EFSA granted a QPS status to B. clausii species.
Chemistry/Identity/Characteristics
Taxonomy change	Bacillus clausii was reclassified in 2020 as Alkalihalobacillus clausii based on phylogenetic analysis (Patel 2020).
History of use	Bacillus clausii has more than 50 years of use in animal and human nutrition. Bacillus clausii species have been used for treating diarrhea and gastrointestinal effects related to antibiotic treatments. The self-affirmed GRAS report for B. clausii UBBC-07 reports that Bacillus clausii species have been detected in dairy products that includes yogurt and ice cream (Unique Biotech, 2016).
Complete genome sequence unambiguously identifying the strain at the species level	B. clausii strain UBBC07 was first isolated from soil that was contaminated with human feces and was identified by performing 16S rRNA sequencing (Sudha, 2013). The whole genome of UBBC07 was sequenced. The whole-genome shotgun project has been deposited at GenBank under accession no LATY00000000 (Upadrasta 2016).
Antibiotic resistance profile (phenotypic and genotypic)	The presence of known antibiotic resistance and toxin genes in B. clausii strain UBBC07 were evaluated (Lakshmi, 2017). B. clausii UBBC07 was found to be phenotypically resistant to three of the antibiotics: clindamycin, erythromycin and chloramphenicol. B. clausii UBBC07 did not exceed the microbiological cutoff values (mg/L) for the other antibiotics indicating that the strain did not exhibit acquired antibiotic resistance to those antibiotics (Unique Biotech, 2016; Lakshmi, 2017) A published analysis was performed using whole genome sequencing to establish that the antibiotic resistance of B. clausii strain UBBC07 is intrinsic and not transferable (Lakshmi, 2017). Additionally, the whole genome sequence of B. clausii UBBC07 was analyzed and resistance genes for the antibiotics clindamycin, erythromycin and chloramphenicol were determined to be present on chromosomal DNA, which is considered ‘intrinsic’ and not transferable (Lakshmi, 2017).
Presence of transmissible elements (e.g. transposase)	Mobile genetic elements flanking or adjacent to the antibiotic resistance genes for clindamycin, erythromycin and chloramphenicol in B. clausii UBBC07 were not mentioned in the published analysis (Lakshmi, 2017) or in the self-affirmed GRAS report for B. clausii UBBC07(Unique Biotech, 2016).
Presence of virulence factors / toxin genes (e.g. hemolysis)	Whole-genome sequencing performed for B. clausii UBBC07 showed the absence of known genes that encode for enterotoxins and hemolysins (Upadrasta et al. 2016). A search for putative virulence factors revealed that virulence putative factors found in the genome for B. clausii UBBC07 are involved in defensive mechanism, cellular activities, adhesion and sporulation, but known toxin genes were absent (Unique Biotech, 2020)
Animal toxicity studies	Acute toxicity in rodents with B. clausii UBBC07 administered 5000 mg/kg, no mortality or signs of toxicity were observed during 15-day observation period. A subacute oral toxicity study was conducted in rodents with administered dosage of 100 mg/kg, 500 mg/kg, and 1000 mg/kg daily for 28 days. No mortality or signs of toxicity were observed at up to 1000 mg/kg of B. clausii UBBC07. In addition, no significant effect on general health, body weight, food consumption, hematological or clinical chemistry profile. No chronic toxicity studies were identified for B. clausii UBBC07. No genotoxicity or reproductive or developmental toxicity studies were identified for B. clausii UBBC07.
Allergenicity	No allergenicity was identified for UBBC07 strain, however, a clinical study reported B. clausii may exert immuno-modulating effects in allergic subjects (Ciprandi, 2005).
Intended us indications	B. clausii UBBC07 is used as a probiotic in the prevention or treatment of diarrhea and for side effects that related to antibiotic treatment (Sudha, 2013; Jayanthi, 2015). One product containing B. clausii UBBC07 as the only ingredient has label claims for control of diarrhea and improvement of intestinal microbial balance during antibiotic treatment. Other claims include immunostimulatory and immunomodulatory activity, anti-bacterial activity against gram positive pathogens, and synthesis of vitamin B complex and vitamin K.
Typical intake level	Not available.
Clinical studies and adverse events reporting
Human clinical studies tracking safety endpoints	Published clinical data are available for the safety and efficacy of B. clausii strain UBBC07 supplementation in adults and children with diarrhea. No significant adverse effects were observed in the studies at intake levels of 4 × 10^9 cfu daily (Sudha, 2013; Sudha, 2019). A prospective, Phase II clinical study evaluated the efficacy and safety of B. clausii UBBC07 in patients with acute diarrhea (Sudha, 2013) who took 1 capsule twice daily for 10 days (daily concentration of 4 × 10^9 cfu) and the authors reported no significant effect on safety parameters and was effective in alleviating diarrhea symptoms. A double blind, randomized, placebo-controlled, parallel group study evaluated the effects of B. clausii UBBC07 in infants/children, between 6 months to 5 years, with acute diarrhea. The intake daily level was 4 × 10^9 cfu for 5 days. The study showed the duration of diarrhea and frequency of defecation were significantly decreased, no monitoring of adverse effects or safety parameters were not defined (Sudha, 2019).
Adverse events reporting	No significant adverse effects or toxicity were reported in available clinical studies conducted for B. clausii UBBC07 (Jayanthi, 2015). No adverse event cases were identified for B. clausii strain UBBC07 in the literature. Search in FDA CFSAN CAERS returned no adverse events for Bacillus clausii and Bacillus clausii UBBC07. Canada Vigilance Program Database did not yield any AERs as well as the Australian TGA, Medicines and Healthcare Products Regulatory Agency – Yellow Card (MHRA) database and the New Zealand’s MedSafe for Bacillus clausii and Bacillus clausii UBBC07. The EudraVigilance European database yielded 196 AERs for Bacillus clausii. The strain was not specified and the available information was insufficient to determine causality (e.g. product information, intake levels, concomitant use of other products or medications, and/or medical history of the patient).
Quality assessment
Nucleic acid-based identification assay	PCR sample preparation with Primer set UBBC02 produces an amplification product of 516 base pairs. PCR sample preparation with Primer set UBBC03 produces an amplification product of 447 base pairs.
Enumeration assay	NLT 100% of the labeled viable cfu/g.
Contaminants	No adulterants were identified for B. clausii UBBC07 in the literature. A review reported that commercial Bacillus subtilis products were found to be mislabeled and contained other Bacillus species including B. clausii (Duc et al. 2004). No toxic constituents were reported in the literature for B. clausii UBBC07. The proposed USP monograph for Bacillus clausii has specifications for total combined molds and yeasts count that does not exceed 100 cfu/g. It meets the requirements of the tests for the absence of Escherichia coli and Salmonella species, both in 10 g. The total combined molds and yeasts count does not exceed 100 cfu/g. It meets the requirements of the tests for the absence of Escherichia coli and Salmonella species, both in 10 g. The probiotic ingredient should be tested for the absence of Listeria monocytogenes, Staphylococcus aureus, and Pseudomonas aeruginosa depending on the risk associated with the contamination of the above organisms based on formal risk assessment programs such as HACCP.
Packaging and storage	Preserve in tight containers in a cool, dry place
Labeling	Ingredient should be labeled with the genus, species, and strain name and with formulated enumeration in cfu/g.

The USP pathway for setting standard does not differ based on the safety evaluation pathway. Although, the magnitude of the quality and safety assessment of probiotic strains relies upon the extensiveness of the documentation provided by the company (known as the sponsor) providing data to support monograph development. The evaluation is either comprehensive or abbreviated.

A comprehensive evaluation is performed in absence of a GRAS dossier or New Dietary Ingredient (NDI) notification that has been submitted to FDA for the probiotic strain. In some cases, a GRAS dossier may become available after a monograph is already in development. This is the case for Lacticaseibacillus paracasei Lpc-37 (Table 2).

USP researches and evaluates diverse sources of safety information including historical use of probiotic species and/or strain, global pharmacopeial or regulatory authority monographs, approved lists such as the Qualified Presumption of Safety (QPS) list from the European Food Safety Authority (EFSA) (EFSA 2021), non-clinical (e.g. toxicology) studies, human clinical trials, case studies, and adverse event reporting databases.

The abbreviated admission evaluation path is often taken when a GRAS dossier or NDI notification has been submitted to the FDA for review and not objected to by the Agency. Both dossiers generally contain the required information to assess the safety of the probiotic strain. Although, a comprehensive evaluation may still be performed in the event the dossiers lack safety and quality information required for the evaluation.

Identification

The first step in evaluating a probiotic is in careful identification of the microorganism. Thus, its taxonomic status should be unambiguously established. The organism under assessment should be identified at species level based on up-to-date methodologies and current knowledge. For probiotic bacteria, whole genome sequence (WGS) analysis is required for the identification and characterization of the microorganism. This can be achieved by computational approaches for taxonomic assignments (e.g. phylogenomics or average nucleotide identity (ANI)), or by comparing the sequences commonly used for taxonomic identification (e.g. 16S rRNA gene), or other characteristic genes (e.g. housekeeping genes) to relevant databases (Roe et al. 2022; EFSA 2018; Health Canada 2023). The sponsor must provide documentation indicating the sequencing strategy and instrumentation used, the assembly method applied (bioinformatic approach, de novo or re-sequencing strategy, statistical measure of sequence quality, number of contigs and their length, and the annotation protocol used). Additionally, the documentation must demonstrate a clear comparison between the genome of the strain under investigation and the genomes of closely related type strains, unequivocally affirming the identification of the species to which the strain belongs.

In addition to the genotypic characterization, the probiotic strain must be phenotypically characterized. Phenotyping must be assessed based on characteristics routinely used to distinguish the species from others. This includes a series of testing for sufficient confirmation of observable traits of the species such as Gram classification, motility, presence of spores, bacterial morphology, cluster type (chain, pair, single), aerobiosis status, fermentation pathway and biochemical profile using Analytical Profile Index (API).

Safety evaluation

While global regulatory authorities generally concur on the fundamental safety principles for probiotics, there exists limited consensus regarding the precise types of studies and methodologies to validate their safety. When assessing the safety of a probiotic strain, USP investigates the following: in silico analysis to verify for the presence of antibiotic resistance genes, virulence and toxigenic factors, history of safe use of the species in food and dietary supplements, any available toxicology studies (in vitro and in vivo), clinical studies (if safety parameters are included), case studies, and adverse event (AE)reporting data. These parameters are discussed in more detail below.

The WGS serves not only as a tool for identification as discussed above, but also plays a crucial role in safety assessment. It is imperative to choose probiotic strains that do not contribute to the dissemination of functional antibiotic resistance in food and supplements (Ouwehand et al. 2016). The potential for antibiotic resistance in probiotic strains raises concern, particularly if this resistance can be transferred directly or through intermediary microbes to potentially harmful members of the microbiota. This could result in the emergence of antibiotic-resistant pathogens that are difficult to treat. The revised EFSA guidance from 2018 emphasizes the necessity for conducting both a phenotypic (Minimum Inhibitory Concentration, MIC) and genotypic analysis of antimicrobial resistance. It does not confine the analysis to functional antimicrobial resistance surpassing a predetermined threshold or cutoff (EFSA 2018). This approach aligns with the safety standards adopted by numerous countries around the world. In addition to genotypic analysis, the sponsor must also provide phenotypic testing for antimicrobial susceptibility.

A safety assessment must also consider the potential for a probiotic to express any virulence or toxin genes, which could lead to disease. Therefore, the sponsor must provide data showing the strain does not possess genetic elements responsible for producing virulence factors and/or toxigenic activity such as biofilm production, hemolysis, or toxins. Utilizing highly curated databases and genomic-scale analytical resources such as the Pathosystems Resource Integration Center (PATRIC), are good bioinformatic tools enabling in identifying acquired virulence and toxigenic genes (Roe et al. 2022).

Traditional toxicology studies are sometimes available for various probiotics under consideration for monograph development. When available, these studies are incorporated into the safety assessment for a probiotic. However, the utility of traditional animal toxicology studies designed for testing chemicals for relevance in assessing probiotic safety has been questioned (Roe et al. 2022).

A history of safe use for many microbial species is more commonly used to establish safety for many probiotics. Various genera and species of the family Lactobacillaceae and species of the genus Bifidobacterium for example have been historically consumed in the diet and as such are recognized as safe. The safe use of these species in foods and supplement products worldwide and the occurrence for many as normal commensals of mammalian microbiota adds further to documentation of safety. Furthermore, a well-documented history of use through consumption in large quantities, on a daily basis as fermented foods is recognized by authority bodies such as EFSA (Bernardeau et al. 2006; EFSA 2007). Thus, the intended use and known intake levels of the probiotic under review is included in the evaluation.

Clinical trials can serve as a source of safety information for a particular probiotic species or strain if safety endpoints are clearly evaluated as part of the study. However, it should be recognized that the majority of human clinical studies with probiotics reported in the literature were not designed as safety and tolerability studies. Thus, adequate reporting of adverse events from the clinical study may not be reported. In addition, there is a lack of consistency in terms of what safety endpoints should be assessed in clinical trials with probiotics. The reporting of adverse events is a critical component of any human clinical trial. A clear definition of what and how adverse events will be collected must be clearly reported in the study report. In order for a clinical trial to be helpful in a review of a probiotic, the adverse events should be fully reported in terms of grade (mild, moderate, severe, life threatening) and a determination on its causal relationship to the treatment is critical (Ioannidis et al. 2004).

In addition to adverse events reported from clinical trials a literature search for any case reports of adverse events is also conducted for a given probiotic species/strain. Various international databases for capturing AE reports are also searched including the US FDA Center for Food Safety and Applied Nutrition Adverse Event Reporting (CAERS), Canada Vigilance Program, Australia’s Therapeutic Goods Administration (TGA), World Health Organization (WHO) Uppsala Monitoring Center VigiAccess, Medicines and Healthcare products Regulatory Agency Yellow Card Database, Europe’s EudraVigilance system, and New Zealand’s Medicines and Medical Devices Safety Authority (NZ MedSafe).

Quality parameters of a probiotic monograph review

In addition to review of the safety aspects of the probiotic strain under consideration for monograph development, the quality of the various assays provided by the sponsor are also reviewed.

The nucleic acid-based assay for strain-level identification is the centerpiece of the probiotic monograph. The sponsor is required to provide a comprehensive PCR validation report including details such as the assay design, PCR conditions (including the reaction formula breakdown and PCR amplification cycles), as well as the primer sets along with their corresponding acceptance criteria. The validation report must demonstrate specificity of their primers while using non-targeted samples, strains from other species, and targeted samples, closely related strains from the same species including the strains already published in the species monograph.

In addition to the section for species identification, the species monograph also includes a chapter dedicated to enumeration. Typically, lactobacilli enumeration assays follow the method outlined in Probiotic Tests ˂64>. The same holds true for bifidobacteria, with the additional inclusion of cysteine chloride in the specified media as outlined in the species monograph. For other genera, or if a sponsor necessitates the use of its own enumeration method, the company must provide a scientifically validated enumeration method for its probiotic strain. This should entail details on the peptone diluent, sample preparation, and incubation conditions. The acceptance criteria for this assay are a minimum of 100% of the labeled viable cell count in CFU/g.

By default, the contaminants assays refer to the methods listed under Probiotic Tests ˂64>. The main contaminants are total combined yeasts and molds, non-lactic acid bacteria, Escherichia coli and Salmonella species. The methods of analysis indicated are currently accepted methods commonly used in industry. Although, users may substitute such methods by other validated test methods.

Furthermore, the absence of Listeria monocytogenes, Staphylococcus aureus, or Pseudomonas aeruginosa in addition to the absence of the specified microorganisms mentioned above should be tested and confirmed if a probiotic ingredient or a finished product containing a probiotic ingredient poses a risk associated with the contamination of the above microorganisms based on formal risk assessment programs such as Hazard Analysis and Critical Control Points (HACCP). The absence of Clostridium perfringens and Cronobacter sakazakii in addition to the absence of the listed specified microorganisms should be tested and confirmed if a probiotic ingredient or finished product is intended for infant use. The test methods for Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium perfringens, and Cronobacter sakazakii must be officially accepted methods or appropriately validated methods.

Discussion

Probiotic dietary supplement products are increasingly popular among consumers. Marketplace projections have estimated that globally, probiotic sales could reach over 90 billion USD by 2026 (MarketsandMarkets 2021). With the increase in popularity of this type of dietary supplement it is important that pharmacopeia monographs are available to guide manufacturers and brand owners and to protect consumers through establishment of quality standards. The USP has developed 20 probiotic monographs through an ‘admission evaluation’ conducted by scientific expert volunteers who are members of the DSAEL EC. A summary of those probiotic monographs is presented herein.

The quality and safety parameters reviewed by the DSAEL EC and/or incorporated in to monographs for probiotics are summarized in Table 3. The EFSA QPS approach follows this same basic premise for safety assessment of probiotics (EFSA 2020 a and b). More recently, the International Scientific Association for Probiotics and Prebiotics convened a meeting of experts to review similar quality and safety parameters of probiotics. The recommendations of these experts are similarly aligned with the approaches presented herein (Merenstein et al. 2023).

Table 3. Essential parameters of a comprehensive quality and safety review for a USP probiotic monograph.

Basic Safety Information: Genomic Analysis – whole genome sequencing Strain identification Identification of any virulence factors, hemolytic activity, toxin production Genotypic assessment of antimicrobial resistance genes Antibiotic resistance testing – Minimum Inhibitory Concentration (MIC) determination and assessment of potential transferable antimicrobial resistance genes Other considerations: Biogenic amine, D(-)-lactate production, Mucin degradation History of safe use (Genus, species, and strain levels) Intended use Intake levels

Animal toxicity studies Repeated dose studies Human clinical studies tracking safety endpoints and adverse events Adverse Event Databases searches Quality Assessment: Nucleic acid-based identification assay Enumeration assay Contaminants Packaging and Storage Labeling

A final consideration by the DSAEL EC is whether to recommend cautionary labeling specific to the probiotic strain under review to mitigate known risk. Any specific cautions by regulatory authorities cited for a particular species or strain would be considered. Other cautionary statements that have been proposed in the literature or by regulatory authorities are related to the use of probiotics by immunocompromised individuals. For example, Sanders et al. (2010) recommends; …’in patients with weakened intestinal barrier function, immune compromised state or central venous catheter use, probiotic consumption should in any case be carefully considered’. Health Canada monograph for probiotics has the following caution statements for the intake of probiotics; ‘Consult a health care practitioner/health care provider/health care professional/doctor/physician prior to use if you have fever, vomiting, bloody diarrhea or severe abdominal pain’ and to ‘Stop use and consult a health care practitioner/health care provider/health care professional/doctor/physician if symptoms of digestive upset (e.g. diarrhea) occur, worsen and/or persists beyond 3 days’. In addition, the use of probiotics is contraindicated in immune-compromised individuals with the statement: ‘Do not use this product if you have an immune-compromised condition (e.g. AIDS, lymphoma, patients undergoing long-term corticosteroid treatment)’. To date, the DSAEL EC has not required cautionary labeling statements such as these in any of the probiotic monographs discussed herein. The DSAEL EC would typically base a decision on whether or not to add cautionary labeling statements to monographs based on careful review of adverse event reports.

The final step in the review process conducted by the DSAEL EC is to determine whether a dietary ingredient (e.g. probiotic strain) as a component of a dietary supplement qualifies for admission into the United States Pharmacopeia and National Formulary (USP-NF). The Admission Criteria and Safety Classification for Dietary Supplements Guideline establishes a Class A and Class B classification system that categorizes dietary ingredients according to the level of safety concern (USP 2021). Specifically, the safety classification and admission criteria are presented in Table 4 below.

Table 4. The admission criteria and safety classification for dietary supplements guideline class A and Class B classification system.

Class A: Admitted into the Compendia: Articles for which the available evidence does not indicate a serious risk to health* or other public health concern that precludes inclusion of a quality monograph into the compendia.

Class B: Not admitted into the Compendia: Articles for which the available evidence indicates a serious risk to health* or other public health concern that precludes inclusion of a quality monograph into the compendia. *Serious risk to health means that the use of the article could: (A) result in: (i) death; (ii) a life–threatening experience; (iii) inpatient hospitalization; (iv) a persistent or significant disability or incapacity; or (v) a congenital anomaly or birth defect; or (B) require, based on reasonable medical judgment, a medical or surgical intervention to prevent an outcome described under subparagraph (A).

Even when a dietary ingredient is classified as Class A and admitted for monograph development, USP continues to monitor for safety information related to that dietary ingredient. This monitoring is ongoing and may serve as a basis for possible safety reevaluation. It should be noted that USP’s evaluation of a dietary supplement ingredient is performed solely for determining admission into the compendia and should not be interpreted as any statement of intrinsic safety or effectiveness of the dietary ingredient under review.

Safety assessments of probiotic bacterial species can be challenging. There is a lack of standardized methodologies for assessing the safety of probiotics. As a result, not all strains have the same level of characterization. USP is working toward addressing the lack of standardized methodologies as evidenced by publications listed in Table 1. Additional efforts are ongoing by the USP Probiotics EP and will be published as available.

In summary, this report can serve as an overview of the USP admission evaluation process for probiotics including the key quality and safety parameters that are important for admission to the compendia. The tabular summary of the various probiotic monographs can serve as quick reference of those developed to date.

Authors’ contributions

MEB, NA, BK, and ALR collected and analyzed the data presented in this manuscript. All authors participated in writing aspects of this manuscript and have responsibility for final content.

Acknowledgments

The authors wish to thank Nidhi Kanchan (The Ohio State University College of Pharmacy) and Sarah Smiley (Lake Erie College of Osteopathic Medicine) for their help in developing the tabular summaries of probiotic monographs.

Disclosure statement

The authors are either employed by USP (NA, BK) or currently work (ALR) or have worked (MEB) for companies who manufacture and/or distribute dietary supplements including probiotic products.

Data availability statement

Data described in the manuscript are available through access to the USP-NF at https://www.uspnf.com

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.

Notes on contributors

Marie-Eve Boyte

Marie-Eve Boyte is a microbiologist graduated from Université Laval. She is currently the vice-chair of the USP Probiotic Expert Panel and the co-chair of the International Probiotics Association (IPA) Scientific Committee. She has more than 15 years of experience in the dietary supplement industry with strong expertise in the probiotic field.

Nadeem Akhtar

Nadeem Akhtar is a toxicologist holding an MS (Pharm.) in Pharmacology & Toxicology from the National Institute of Pharmaceutical Education and Research (NIPER) in S.A.S. Nagar, Punjab, India, currently working as Senior Scientist II (Toxicology) in Dietary Supplements and Herbal Medicines (DSHM) team at USP. He has more than 9 years of experience in the drug, cosmetics, and dietary supplement areas.

Binu Koshy

Binu Koshy is a microbiologist with a Ph.D. in Microbiology and a Post doctoral research experience from Indian Institute of Science (IISc) Bangalore, India. He is currently working as Senior Scientist II in Dietary Supplements and Herbal Medicines (DSHM) team at USP with more than 15 years’ experience in dietary supplement areas with expertise in probiotics and proteins.

Amy L. Roe

Amy L. Roe is a board-certified (ABT) toxicologist with a PhD in toxicology from the University of Kentucky. She is currently Chair of the USP Dietary Supplements Admission Evaluation and Labeling Expert Committee. She has over 24 years of experience as a practicing toxicologist in industry across both the drug and dietary supplement spaces.