GRAS Notice (GRN) No. 697 https://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/NoticeInventory/default.htm GRAS Notice for Dried Euglena gracilis (ATCC PTA-123017) Prepared for: Office of Food Additive Safety (FHS-200) Center for Food Safety and Applied Nutrition Food and Drug Administration 5100 Campus Drive College Park, MD 20740 March 16, 2017 GRAS Notice for Dried Euglena gracilis (ATCC PTA-123017) Table of Contents Page Part 1. §170.225 Signed Statements and Certification .... ..... ... ... ......................................... ..... .. 3 1.1 Name and Address of Notifier .................................................. .............. .... ...... ... 3 1.2 Common Name of Notified Substance ..... ........................................................... 3 1.3 Conditions of Use .......................................... .... ...... .. ......................... .... .. ... ........ 4 1.4 Basis for GRAS ................................................................................................... 5 1.5 Availability of Information ..................................................................................... 5 1.6 Freedom of Information Act, 5 U.S.C. 552 ........................................................... 5 Part 2. §170.230 Identity, Method of Manufacture, Specifications, and Physical or Technical Effect ................................................................................................. .. ........... 5 2.1 Description ...................................................................................................... ... 5 2.1.1 Dried Algae (Euglena gracilis ATCC PTA-123017) .................................. 6 2.2 Source Organism ................................................................................................. 8 2.2.1 Phenotypic Identity .............. .......... .............. ............................................ 8 2.2.2 Genotypic Identity ................................................................... .. ... .. ........ 10 2.3 Manufacturing ................................................................................... ... .. ............ 12 2.4 Product Specifications and Batch Analyses ....................................................... 14 2.4.1 Proposed Product Specifications ................. .. ....................... ..................14 2.4.2 Batch Analyses ......................................................................... .............. 14 2.4.3 Additional Analyticallnformation ............................................................. 15 2.5 Stability ...................................................... .............. ......... .. ......... .. .................... 19 · Part 3. §170.235 Dietary Exposure ........................................................................................... 21 3.1 History of Use in Food ....................................................................................... 21 3.3 Estimated Consumption of Dried Algae and Paramylon Isolate from All Intended Conditions of Use in Food ............................................................... ... .24 3.3.1 Dietary Intake Survey .......................................................................... ... 24 3.3.2 Estimated Consumption of Dried Algae from Proposed Food-Uses ........25 Part 4. § 170.240 Self-Limiting Levels of Use ...... .. .................................................................. .. 27 Part 5. §170.245 Experience Based on Common Use in Food Before 1958 ............................. 27 Part 6. §170.250 Narrative and Safety Information ......... .. ............... ....... ..... .............................27 6.1 Introduction ....................................................................................... .. ............. .. 27 6.2 Literature Search ................................_............................................................ .. 28 6.3 Absorption, Distribution, Metabolism, and Excretion .......................................... 28 6.3.1 In Vitro Fermentation Studies- Dried Euglena gracilis and Paramylon ..............................................................................................29 6.4 Toxicology Studies .............................................................................................31 6.4.1 Acute Toxicity ................................. ....................... ... ......... ....... .. ......... .. .31 6.4.2 Repeat-Dose Toxicity .......................................................... ................. .. 32 6.4.3 Genotoxicity Studies ......................... .. ......... .... ........... ....... .. ... ..... ....... ... .36 Algal Scientific 1 March 16 h, 2017 6.4.4 Anti-Carcinogenicity Studies .................................... ........ .. ... ... ..... ..... .... .37 6.5 Other Published Studies ......................................................... .. .. ... .. ... ..... ......... .38 6.5.1 Poultry ..................................................................... .... ...... ..... .... ... .... ... .. 40 6.6 Toxicity Studies and Safety of Related beta-1 ,3-Giucans..... .. .......................... ..42 6.7 Safety of Source Organism ............................................................................... .42 6.7.1 Pathogenicity and Toxicogenicity ....................... ................................... .42 6.8 Expert Panel Evaluation .................................................................................... .44 6.9 Conclusions ......................................................................... ........ ... ... .. .... ..... ... ..44 Part 7. §170.255 List of Supporting Data and Information ........................ .......... .. ...... ..... .... ..... .45 List of Figures and Tables Figure 2.1.2-1 Structural Representations of Paramylon ...................................................... 7 Figure 2.2.1-1 Structural Characteristics of Euglena gracilis Klebs ........................... , ..... ...... 8 Figure 2.2.1-2 Biosynthesis of Paramylon and Wax Esters by Euglena gracilis is Influenced by Culture Conditions ............................................................. .... .. 9 Figure 2.2.2.1-1 Species Identity and Taxonomic Classification .......................................... .. .11 Figure 2.3-1 Schematic of the Production Process of Dried Euglena gracilis .............. ... ... 13 Figure 3.2-1 History of Safe Food Use - Examples of Dietary Supplement Products and Food Products Containing Euglena gracilis ...................................... .. .. .22 Figure 6.3.1-1 Bacterial Cultures Grown in Modified M9 Medium (pH 6.5) for Growth of Bifidobacterium Spp............................................................................... .. 30 Figure 6.3.1-2 Bacterial Cultures Grown in Modified M9 Medium (pH 6.5) for Growth of Lactobacillus spp......................................................................................31 Table 1.3-1 Summary of the Individual Proposed Food Uses and Use Levels for Dried Algae and Paramylon Isolate in the U.S ............................................... 4 Table 2.4.1-1 Product Specifications- Dried Algae (Euglena gracilis) ........................... .... 14 Table 2.4.2-1 Results of 3 Batch Analyses of Dried Algae (Euglena gracilis) .............. ....... 15 Table 2.4.3.1-1 Method of Analysis for beta-Giucan ...................................................... .. ...... 16 Table 2.4.3.1-2 Results of beta-Giucan Analyses of Dried Algae (Euglena gracilis) ...... .. ..... .16 Table 2.4.3.2-1 Results of Per-0-methylation and Linkage Analysis of Dried Algae ...... .... .... 17 Table 2.4.3.3-1 Results of Glycosyl Composition Analysis ..... .............................................. .. 18 Table 2.4.3.4-1 Mineral Analysis of 3 Lots of Dried Algae (Euglena gracilis) .................. ... .. .. 18 Table 2.4.3.4-2 Vitamin and Carotenoid Analysis of 3 Lots of Dried Algae (Euglena gracilis) ........................................................................................................ 19 Table 2.5-1 Results of an Accelerated Shelf-life Study on Dried Algae (Euglena gracilis) (Lot No. 020916-AM-1) and Paramylon (Lot No. 021016­ BG-1) ................................................................................................... ........ 19 Table 2.5-2 Shelf-life Data Results of 2 Batches of Dried Algae (Euglena gracilis) .... ...... 20 Table 3.3.2-1 Summary of the Estimated Daily Intake of Dried Algae from Proposed Food-Uses in the U.S. by Population Group (2011-2012 NHANES Data) ....................................................................................................... ..... 26 Table 3.3.2-2 Summary of the Estimated Daily Per Kilogram Body Weight Intake of Dried Algae from Proposed Food-Uses in the U.S. by Population Group (2011-2012 NHANES Data) ...............................................................26 Table 6.4.2.1-1 Treatment Allocation and Mean Intake of Test Articles ................................. 33 Table 6.5.1-1 Studies Conducted Using Euglena gracilis Pertinent to the Safety and Utility for Poultry .......................................................................................... .41 Algal Scientific 2 March 16'h, 2017 (b) (6) 1.3 Conditions of Use Algal Scientific intends to market dried Euglena gracilis algae as an ingredient in the U.S. for use in conventional food and beverage products across a number of food categories at use levels up to 500 mg/serving. The individual proposed food-uses and use-levels for dried