GRAS Notice for Pichia kudriavzevii ASCUSDY21 for Use as a Direct Fed Microbial in Dairy Cattle Prepared for: Division of Animal Feeds, (HFV-220) Center for Veterinary Medicine 7519 Standish Place Rockville, Maryland 20855 Submitted by: ASCUS Biosciences, Inc. 6450 Lusk Blvd Suite 209 San Diego, California 92121 GRAS Notice for Pichia kudriavzevii ASCUSDY21 for Use as a Direct Fed Microbial in Dairy Cattle TABLE OF CONTENTS PART 1 – SIGNED STATEMENTS AND CERTIFICATION ................................................................................... 9 1.1 Name and Address of Organization .............................................................................................. 9 1.2 Name of the Notified Substance ................................................................................................... 9 1.3 Intended Conditions of Use .......................................................................................................... 9 1.4 Statutory Basis for the Conclusion of GRAS Status ....................................................................... 9 1.5 Premarket Exception Status .......................................................................................................... 9 1.6 Availability of Information .......................................................................................................... 10 1.7 Freedom of Information Act, 5 U.S.C. 552 .................................................................................. 10 1.8 Certification ................................................................................................................................. 10 PART 2 – IDENTITY, METHOD OF MANUFACTURE, SPECIFICATIONS AND PHYSICAL OR TECHNICAL EFFECT ............................................................................................................. 11 2.1 Identity ........................................................................................................................................ 11 2.1.1 Taxonomic Classification ..................................................................................................... 11 2.1.2 Source of the Microorganism ............................................................................................. 11 2.1.3 Description of the Microorganism ...................................................................................... 11 2.1.4 Identification of the Microorganism ................................................................................... 13 2.1.4.1 Ribosomal DNA Marker Analysis ..................................................................................... 13 2.1.4.2 Whole Genome Sequence Analysis and Annotation ...................................................... 14 2.1.4.3 Whole Genome Sequence Comparison .......................................................................... 14 2.1.4.4 Section Summary ............................................................................................................ 15 2.1.5 Plasmid Analysis .................................................................................................................. 15 2.1.6 In-vitro and In-silico Analysis of Antimicrobial Susceptibility ............................................. 15 2.1.6.1 Section Summary ............................................................................................................ 16 2.1.7 Antimicrobial Production .................................................................................................... 17 2.1.8 Toxigenicity and Pathogenicity ........................................................................................... 17 2.1.8.1 Section Summary ............................................................................................................ 22 2.1.9 Genetic Stability .................................................................................................................. 23 2.1.10 Summary of Organism Safety Based on Genomics ............................................................. 23 2 2.2 Method of Manufacture ............................................................................................................. 23 2.2.1 Raw Materials and Processing Aids .................................................................................... 23 2.2.2 Manufacturing Process ....................................................................................................... 23 2.2.3 Production Controls ............................................................................................................ 24 2.3 Product Specifications and Batch Analyses ................................................................................ 25 2.3.1 Product Specifications for Freeze Dried P. kudriavzevii ASCUSDY21 (Notified Substance) ........................................................................................................... 25 2.3.2 Batch Analyses for Freeze Dried P. kudriavzevii ASCUSDY21 (Notified Substance) .......................................................................................................................... 25 2.3.3 Product Specifications for the Fat Encapsulated Product ................................................... 25 2.3.4 Batch Analyses for Fat Encapsulated Product..................................................................... 26 2.3.5 Additional Analytical Data ................................................................................................... 26 2.4 Stability ....................................................................................................................................... 26 2.4.1 Stability Data ....................................................................................................................... 26 2.4.1.1 Stability Study at 5°C ....................................................................................................... 27 2.1.4.2 Stability Study at 25°C ..................................................................................................... 27 2.1.4.3 Stability Study at 40°C ..................................................................................................... 28 2.4.2 In-Feed Stability .................................................................................................................. 28 2.4.3 Homogeneity Data .............................................................................................................. 28 2.4.4 Manufacturing Summary .................................................................................................... 29 2.5 Effect of the Notified Substance ................................................................................................. 29 2.5.1 Rumen Microbiome ............................................................................................................ 30 2.5.2 Impact of Failure of the Notified Substance ....................................................................... 31 2.5.3 Summary ............................................................................................................................. 34 PART 3 – TARGET ANIMAL AND HUMAN EXPOSURE .................................................................................. 35 3.1 Target Animal Exposure .............................................................................................................. 35 3.1.1 Exposure to the Direct Fed Microbial Strain ....................................................................... 35 3.1.2 Exposure to the Other Components of the Fat Encapsulated Product .............................. 35 3.1.3 Background Exposure to the Microorganism ..................................................................... 36 3.2 Human Exposure ......................................................................................................................... 36 PART 4 – SELF-LIMITING LEVELS OF USE ..................................................................................................... 37 PART 5 – EVIDENCE BASED ON COMMON USE BEFORE 1958 .................................................................... 38 3 PART 6 – NARRATIVE ................................................................................................................................... 39 6.1 Functionality ............................................................................................................................... 39 6.2 Identity ........................................................................................................................................ 41 6.3 Literature Search ......................................................................................................................... 41 6.4 Natural Occurrence ..................................................................................................................... 41 6.4.1 Prevalence in Animals ......................................................................................................... 41 6.4.2 Microbiome Safety .............................................................................................................. 42 6.4.3 Environmental Occurrence ................................................................................................. 43 6.4.4 Section Summary ................................................................................................................ 43 6.5 History of Use in Manufacture of Food and Feed Ingredients