GRAS Notice (GRN) No. 759 https://www.fda.gov/food/generally-recognized-safe-gras/gras-notice-inventory DSM SUMMARY OF DATA FOR A GRAS CONCLUSION Purified Steviol Glycosides (Rebaudioside M) produced by Yarrowia lipolytica January 16, 2018 Prepared by: Katherine Vega Senior Manager Regulatory Affairs DSM Nutritional Products North America 45 Waterview Boulevard Parsippany, New Jersey 07054 Jeanine van de Wiel DSM Food Specialties Alexander Fleminglaan 1 2613 AX Delft Netherlands DSM-ID4045-001 1 DSM UJGNf SC1£tfCI. lltlc.MTU LMNG Contents Part 1: Signed Statements and Certification ...................................................................................................... 5 1.1 Submission of GRAS Notice ........................................................................................................... 5 1.2 Name and Address of Notifier ....................................................................................................... 5 1.3 Name of the Substance.................................................................................................................. 5 1.4 Intended Conditions of Use ........................................................................................................... 5 1.5 Statutory Basis for the GRAS Conclusion ....................................................................................... 5 1.6 Exclusion from Premarket Approval Requirements ...................................................................... 5 1.7 Availability of Information ............................................................................................................. 6 1.8 Freedom of Information Act (FOIA) Exemptions ........................................................................... 6 1.9 Certification ................................................................................................................................... 6 Part 2: Identity, Method of Manufacture, Specifications, and Physical or Technical Effect of the Notified Substance ........................................................................................................................................................... 7 2.1 Identity ........................................................................................................................................... 7 2.2 Specifications ................................................................................................................................. 8 2.3 Description of the production organism ....................................................................................... 9 2.3.1 Classification of the organism: Yarrowia lipolytica ................................................................ 9 2.3.2 Modifications to the production microorganism ................................................................... 9 2.3.3 Parental strains ..................................................................................................................... 10 2.3.4 Genetic engineering of the production strain .................................................................. 10 2.3.5 Antibiotic resistance ......................................................................................................... 11 2.3.6 History of safe use of Yarrowia lipolytica ............................................................................. 11 2.4 Manufacturing process for DSM steviol glycosides (rebaudioside M) ........................................ 14 2.4.1 Overview ............................................................................................................................... 14 2.4.2 Raw Materials ....................................................................................................................... 15 2.4.3 Fermentation Process ........................................................................................................... 15 2.4.4 Recovery Process .................................................................................................................. 15 2.4.5 Methods used to control the product specifications ........................................................... 15 2.4.6 Method to ensure stability of the production organism ...................................................... 16 2.4.7 Global capabilities ................................................................................................................. 16 2.5 Batch Analyses ............................................................................................................................. 16 2.6 Stability ........................................................................................................................................ 19 2.6.1 Stability of Rebaudioside A ................................................................................................... 19 2.6.2 Stability of Rebaudioside M .................................................................................................. 20 Part 3: Dietary Exposure ................................................................................................................................... 22 3.1 Intended Food Uses ..................................................................................................................... 22 3.2 Estimated Daily Intake ................................................................................................................. 22 3.3 Estimated exposure based on caloric sweetener consumption .................................................. 23 Part 4: Self-limiting levels of use ...................................................................................................................... 25 Part 5: Common use in food prior to 1958 ....................................................................................................... 25 Part 6: Narrative of the basis for the GRAS conclusion .................................................................................... 25 6.1 Overview ...................................................................................................................................... 25 6.2 Absorption, distribution, metabolism, excretion (ADME) ........................................................... 28 6.3 Safety data of steviol glycosides .................................................................................................. 30 2 DSM UJGNf SC1£tfCI. lltlc.MTU LMNG 6.3.1 Overview ............................................................................................................................... 30 6.3.2 Acute toxicity ........................................................................................................................ 30 6.3.3 Short-term and Subchronic Toxicity ..................................................................................... 31 6.3.4 Genotoxicity .......................................................................................................................... 32 6.3.4.1 In vitro genotoxicity of steviol and steviol metabolites.............................................. 32 6.3.5 Chronic toxicity .................................................................................................................... 32 6.3.6 Developmental and Reproductive Toxicity........................................................................... 33 6.3.7 Human Studies ...................................................................................................................... 33 6.4 Other safety considerations ........................................................................................................ 47 6.4.1 Intermediates and byproducts of steviol glycoside biosynthesis ......................................... 47 6.4.2 Biogenic amines .................................................................................................................... 48 Part 7: Supporting Data and Information ......................................................................................................... 49 7.1 Expert Panel Statement ............................................................................................................... 49 7.2 References ................................................................................................................................... 53 7.3 Annexes ....................................................................................................................................... 62 List of Tables and Figures Figure 2-1 Molecular structure of the steviol glycoside rebaudioside M ....................................................... 7 Figure 2-2 Overview of the steviol glycoside biosynthetic pathway .............................................................. 9 Table 2-1 List of patent and patent applications for the production organism .......................................... 10 Figure 2-3 Overview of the manufacturing process for DSM steviol glycosides (Reb M) produced by Yarrowia lipolytica ....................................................................................................................... 14 Table 2-2 Batch analysis data for DSM purified steviol glycosides (rebaudioside M) produced by Yarrowia lipolytica ....................................................................................................................................... 17 Table 2-3 Stability data for DSM purified steviol glycosides (Reb M) produced by Yarrowia