GRAS Notice (GRN) No. 691 http://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/NoticeInventory/default.htm GRAS Conclusion for the Use of Bacillus coagulans SANK 70258 Spores Preparation (LACRIS-S) in Select Foods SUBMITTED BY: Mitsubishi-Kagaku Foods Corporation 1-1 , Marunouchi 1-chome Chiyoda-ku, Tokyo 100-8251 JAPAN SUBMITTED TO: U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition Office ofFood Additive Safety 5001 Campus Drive College Park, MD 20740 CONTACT FOR TECHNICAL OR OTHER INFORMATION: Mary M. Murphy Senior Managing Scientist Exponent, Inc. 1150 Connecticut A venue, NW Suite 1100 Washington, DC 20036 February 3, 2017 ~~~~~Wfe(O) Page 1 of 113 FEB 6 2017 Table of Contents Table of Contents 2 List ofTables 5 List of Figures 6 List of Acronyms 7 Part 1: Signed Statements and Certification 9 Name and Address ofNotifier 9 Name ofGRAS Substance 9 Intended Use and Consumer Exposure 9 Basis for Conclusion ofGRAS Status 9 Pre-Market Approval Exclusion Claim 9 Availability of Information I 0 Exemptions from Disclosure 10 Certification Statement I 0 Part 2. Identity, Method of Manufacture, Specifications, and Physical or Technical Effect 11 Identity 11 Source ofOrganism and Use in a Spores Preparation II Description and Characterization of Organism I1 Biochemical Characterization 12 Genotypic Identification 14 16S rDNA Testing I4 DNA-DNA Hybridization Testing IS Summary of Characterization Data I 6 Method of Manufacture I 6 Specifications 19 Page 2 of 113 Shelf-Life Stability 21 Part 3. Dietary Exposure 22 Food Uses in the United States 22 Supplement Uses in the United States 23 International Uses 23 Proposed Use and Level 23 Estimated Daily Intakes 24 Part 4. Self-Limiting Levels of Use 25 Part 5. Experience Based on Common Use in Food before 1958 26 Part 6. Narrative 27 Introduction 27 Identification ofthe Microorganism 27 Antibiotic Resistance and Potential Production ofVirulence Factors 28 Antibiotic Susceptibility 28 Virulence Activity 29 Summary 30 Pre-Clinical and Clinical Evidence of Safety 30 Published Pre-Clinical Data on B. coagulans 30 Genotoxicity Data 30 Acute Oral Toxicity 31 Sh011-term Oral Toxicity 31 Subchronic Oral Toxicity 32 Chronic Oral Toxicity 33 Acute Skin Irritation 34 Acute Eye Irritation 34 Reproductive Toxicity 34 Unpublished Pre-Clinical Studies on the B. coagulans strain SANK 70258 Spores Preparation 39 Subchronic Oral Toxicity 39 Oral Feeding Studies 40 Pre-Clinical Safety Summary 40 Clinical Data 41 Fate ofB. coagulans in the Human Gastrointestinal Tract 41 B. coagulans SANK 70258 41 Other Strains 44 Page 3 of 113 Summary ofClinical Data 44 Conclusion Regarding Safety and General Recognition of Safety 50 Discussion ofInformation Inconsistent with GRAS Determination 51 Part 7. List of Supporting Data and Information in GRAS Notice 52 Appendices 59 Appendix A. 16S rONA Sequence Analysis ofBacillus coagulans SANK70258 Reports and DNA-DNA Hybridization Test Rep01t 60 Appendix B. Compositional Analyses ofBacillus coagulans and the LACRIS-S Preparation 76 Appendix C. Antibiotic Susceptibility Testing ofB. coagulans SANK 70258 in a Spores Preparation 86 Appendix D. Duopath® Cereus Enterotoxin Detection Test Report 102 Exhibit: Report of the Expert Panel 108 Page 4 of 113 List ofTables Table 1. Taxonomic characteristics of B. coagulans SANK 70258 in LACRIS-S and other GRAS strains ofB. coagulans 13 Table 2. Analytical results for Lot Analyses ofBacillus coagulans SANK 70258 18 Table 3. Product specifications and Lot Analyses ofLACRIS-S 20 Table 4. Shelf-Life Stability Test of B. coagulans in LACRIS-S 21 Table 5. Genotoxicity Studies with Bacillus coagulans 36 Table 6. Pre-Clinical Studies with Bacillus coagulans 37 Table 7. Clinical Trials with Bacillus coagulans SANK 70258 43 Table 8. Clinical Trials with Bacillus coagulans Strains Not Identified as B. coagulans SANK 70258 45 Page 5 of 113 List of Figures Figure 1. Phylogenetic tree ofthe B. coagulans strain SANK 70258 in LACRIS-S (SIID17786) and Related Species 15 Figure 2. Manufacturing flow chart ofLACRIS-S 17 Page 6 of 113 List ofAcronyms ·c degrees Celsius f.lg microgram llm micrometer ADI acceptable daily intake bp base pairs BSL-1 biosafety level 1 bw body weight CAC Codex Alimentarius Commission cART combination antiretroviral treatment CFR Code ofFederal Regulations CFU colony forming unit cGMP current Good Manufacturing Practice cm2 centimeter squared d day DNA deoxyribonucleic acid EDI Estimated Daily Intake EFSA European Food Safety Authority F female FDA U.S. Food and Drug Administration FOS fructo-oIigosaccharides g gram GI gastrointestinal GRAS Generally Recognized As Safe GRN GRAS Notice h hour HlV human immunodeficiency virus kg kilogram M male mg milligram MKF Mitsubishi-Kagaku Foods Corporation mL milliliter n number NA not applicable NBRC National Institute ofTechnology and Evaluation Biological Resource Center NIH National Institutes ofHealth NLT not less than NMT not more than Page 7 of 11 3 NOAEL no observed adverse effect level NOEL no observed effect level OECD Organization for Economic Co-operation and Development ppm parts per million QPS qualified presumption ofsafety RBC red blood cell RCT randomized controlled trial rONA ribosomal deoxyribonucleic acid RNA ribonucleic acid rRNA ribosomal ribonucleic acid RT randomized trial so Sprague Dawley URI upper respiratory infection u.s. United States USDA U.S. Department ofAgriculture WBC white blood cell wk week y year Page 8 of 113 Part 1: Signed Statements and Certification Mitsubishi-Kagaku Foods Corporation submits to the U.S. Food and Drug Administration (FDA) this generally recognized as safe (GRAS) notice in accordance with the 21 CFR part 170, subpart E. Name and Address of Notifier Mitsubishi-Kagaku Foods Corporation 1-1 , Marunouch i 1-chome Chiyoda-ku, Tokyo 100-8251 , Japan Name of GRAS Substance The substance that is the subject of this GRAS notice is a preparation of Bacillus coagulans strain SANK 70258, a spore-forming lactic acid bacteria, di luted in lactose. The spores preparation is marketed under the trade name LACRJS-S. Intended Use and Consumer Exposure The proposed use of B. coagulans SANK 70258 in a spores preparation (up to 2 x 109 CFU/serving) includes use as an ingredient in : baked goods and baking mixes; beverages and beverage bases; breakfast cereals; chewing gum ; coffee and tea; condiments and relishes; confections and frostings; dairy product analogs; fruit juices; frozen dairy desserts and mixes; fruit and water ices; gelatins, puddings and fillings; grain products and pastas; hard candy; herbs, seeds, spices, seasonings, blends, extracts, and flavorings; j ams and jellies; milk; milk products; nuts and nut products; plant protein products; processed fruits; processed vegetables and vegetable juices; snack foods; soft candy; soups and soup mixes; sugar; and sweet sauces, toppings and syrups. The proposed use of B. coagulans SANK 70258 in a spores preparation is identical to the use offour B. coagulans strains previously determined to be GRAS (GRNs 399, 526, 597, and 601) and therefore, would prov ide an alternate source ofthe microorganism in the spores preparation added to these foods. The estimated intake of B. coagulans strains from the proposed use was previously calculated at 36.4 x 109 CFU per day (GRN 399). Basis for Conclusion of GRAS Status Mitsubishi-Kagaku Foods Corporation's conclusion of GRAS status for the intended use ofB. coagulans SANK 7025~ in a spores preparation is based on scientific procedures in accord with 21 CFR §170.30(a) and. (b). Pre-Market Approval Exclusion Claim Use ofthe preparation of Bacillus coagulans strain SANK 70258 is not subject to the pre-market approval requirements ofthe Federal Food, Drug, and Cosmetic Act because Mitsubishi-Kagaku Page 9 of 113 Foods Corporation has concluded that such use is generally recognized as safe (GRAS) through scientific procedures. Availability of Information The data and information that serve as the basis for this GRAS conclusion, as well as the information that has become available since the GRAS conclusion, will be sent to the FDA upon request, or are available for the FDA' s review and copying during customary business hours at the office ofMary Murphy at Exponent, Inc., 1150 Connecticut Ave, NW, Suite 1100, Washington, DC 20036. ·Exemptions from Disclosure It is our view that none ofthe data and information in Parts 2 through 7 ofthe GRAS notice are exempt from disclosure under the Freedom oflnformation Act (FOIA). We redacted the following information from certain ofthe appendices for the reasons explained below: • We redacted the identity ofthe "Client" in Appendix A-1 because it constitutes confidential commercial information and has no bearing on MKF's conclusion ofGRAS status. • We redacted the seals of the "Scientist Responsible" in Appendices A-1, A-2, A-3 and D, as well as the signature ofthe individual referenced in Appendix B, for reasons of personal privacy. That information has no bearing on MKF's conclusion ofGRAS status. • We redacted the name ofan individual who is not employed by MKF in Appendix A-1 for reasons ofpersonal privacy. That information has no bearing on MKF's conclusion ofGRAS status. Certification Statement On behalfof Mitsubishi-Kagaku Foods Corporation, I hereby certify that, to the best ofmy knowledge, this GRAS notice is a complete, representative, and balanced submission that includes unfavorable, as well as favorable information, known to me and pertinent to the evaluation ofthe safety and GRAS status ofthe use ofthe substance. (b) (6) I Name: Yukino Nagai Date Title: Group Manager Regulatory Affairs Group Quality Assurance Department Company: Mitsubishi-Kagaku Foods Corporation Page 10 of 113 Part 2. Identity, Method of Manufacture, Specifications, and Physical or Technical Effect Identity The substance that is the subject ofthis GRAS conclusion is a preparation of Bacillus coagulans strain SANK 70258, a spore-forming lactic acid bacteria, diluted in lactose. The preparation is marketed under the trade name LACRJS-S. Throughout this document, Bacillus coagulans strain SANK 70258 is to as B.