GRAS Notice (GRN) No ORIGINAL SUBMISSION

Transcription

1 GRAS Notice (GRN) No ORIGINAL SUBMISSION

2 ASSCICIA-rES, LLC Jacklight Lane Bend, OR November 5, 2014 Food and Drug Administration Center for Food Safety & Applied Nutrition Office of Food Additive Safety (HFS-255) 5100 Paint Branch Parkway College Park, MD [FR~~~~'W~[Q) NOV OFFICE OF FOOD ADDITIVE SAFETY Attention: Dr. Paulette Gaynor Re: GRAS Notification- High Purity Steviol Glycosides (~ 95%) Dear Dr. Gaynor: On behalf of Productora Alysa SpA from Quilue, Chile, we are submitting for FDA review Form 3667 and the enclosed CD containing a GRAS notification for High Purity Steviol Glycosides (~ 95%) Primarily Consisting of Rebaudioside A. The attached documentation contains the specific; information that addresses the safe human food uses for the subject notified substance as discussed in the GRAS guidance document. We also wish to advise you that the CD provided for agency review is free of viruses. If additional information or clarification is needed as you and your colleagues proceed with the review, please feel free to contact GRAS Associates via telephone or . We look forward to your feedback. Sincerely, (b) (6) RobertS. McQuate, Ph.D. CEO & Co-Founder GRAS Associates, LLC Jacklight Lane Bend, OR mcquate@gras-associates.com Enclosure: GRAS Notification for Productora Alysa SpA- High Purity Steviol Glylcosides (~ 95%) 1 of 1

3 fri re (G ~ ~'&~ [Q) Form Approved: OMB No ; Expiration Date: 02/29/2016 (See last page NOV for OMB Statement) FDA USE ONLY OFFICE OF GRNNUMBER DATE OF RECEIPT FOOD ADDITIVE SAFETY ooo 55 5 DEPA JIL;.I'f VI '"""'L 1 n 1-\NU nur. AN SERVICES Food and Drug Administration GENERALLY RECOGNIZED AS SAFE (GRAS) NOTICE ESTIMATED DAILY INTAKE NAME FOR INTERNET KEY\IVORDS INTENDED USE FOR INTERNET Transmit completed form and attachments electronically via the Electronic Submission Gateway (see Instructions); OR Transmit completed form and attachments in paper format or on physical media to: Office of Food Additive Safety (HFS-200), Center for Food Safety and Applied Nutrition, Food and Drug Administration, 5100 Paint Branch Pkwy., College Park, MD PART I-INTRODUCTORY INFORMATION ABOUT THE SUBMISSION 1. Type of Submission (Check one) C8J New D Amendment to GRN No. D Supplement to GRN No. 2. C8J All electronic files induded in this submission have been checked and found to be virus free. (Check box to verify) 3a. For New Submissions Only: Most recent pre submission meeting (if any) with FDA on the subject substance (wwlmmldd): 3b For Arnendn1ents or St;pplernents is CJrnerlcinlent or sllpp!err1t:jrt subm1ttecl to;:-: frorn D D NA PART II-INFORMATION ABOUT THE NOTIFIER Name of Contact Person Javier Sainz Position General Manager 1a. Notifier Company (if applicable) Productora Alysa SpA Mailing Address (number and street) Calle Tres No. 600 City State or Province Zip Code/Postal Code Quilpue Fifth Region Country Chile Telephone Number Fax Number Address call first jsainz@prodalysa.cl 1b. Agent or Attorney (if applicable) Name of Contact Person Richard Kraska Company (if applicable) GRAS Associates, LLC Mailing Address (number and st.reet) Riverview Center Blvd, Suite 212 Position Chief Scientific Officer I City State or Province Zip Code/Postal Code Country Bonita Springs!Florida I United States of America Telephone Number Fax Number Address kraska@gras-associates.com FORM FDA 3667 (2113) Page 1 of 4

4 PART Ill -GENERAL ADMINISTRATIVE INFORMATION 1. Name of Substance High Purity Steviol Glycosides (purity no less than 95%) primarily consisting of Rebaudioside A 2. Submission Format: (Check appropriate box(es)) 3 D Electronic Submission Gateway [8J Electronic files on physical media D Paper with paper signature page If applicable give number and type of physical media 4. Does this submission incorporate any information in FDA's files by reference? (Check one) DYes (Proceed to Item 5) [8J No (ProceE!d to Item 6) D GRAS Notice No CC;RN D b) (Jf-ZAS Aff!rrnat:on D f:(!od D eli Food Master F No FMF No F/\P 6. Statutory basis for determination of GRAS status (Check one) [gj Scientific Procedures (21 CFR (b)) D Experience based on common use in food (21 CFR (c)) 7. Does the submission (including information that you are incorporating by reference) contain information that you view as trade secret or as confidential commercial or financial infom1ation? D Yes (Proceed to Item 8) [gj No (Proceed to Part IV) 8 Have you clesig'lated H'~fot rnat!o( :n c~li tnat :1pp1v) c Yes see CJUach8::.1 Des1gnat1on inforn12t1c)n ts des:~1natecj at No PART IV- INTENDED USE 1. Describe the intended use of the notified substance including the foods in which the substance will be used, the levels of use in such foods, the purpose for which the substance will b~! used, and any special population that will consume the substance (e.g., when a substance would be an ingredient in infant formula, identify infants as a special population). Intend to use as table top sweetener and general purpose non-nutritive sweetener for incorporation into foods other than infant formulas and meat and poultry products. 2. Does the intended use of the notified substance include any use in meat, meat food product, poultry product, or egg product? (Check one) DYes [gj No FORM FDA 3667 (2113) Page 2 of4

5 PART V- IDENTITY 1. lnfonnation about the Identity of the Substance Name of Substance 1 Registry Used Registry No. 2 (CAS, EC) Biological Source (if applicable) Substance Category (FOR FDA USE ONLY) High purity steviol glycosides primarily consisting of Rebaudioside A 2 Rebaudioside A CAS Stevia rebaudiana Bertoni 3 1 1nclude chemical name or common name. Put synonyms (whether chemical name, other scientific name, or common name) for each respective item (1-3) in Item 3 of Part V (synonyms) 2 Registry used e.g., CAS (Chemical Abstracts Servio~) and EC (Refers to Enzyme Commission of the International Union of Biochemistry (IUB), now carried out by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB)) 2. Description Provide additional information to identify the notified substance(sj, which may include chemical formula(sj, empirical formula(s), structural formula(s), quantitative composition, characteristic properties (such as molecular weight(s)), and general composition of the substance. For substances from biological sources, you should include scientific information sufficient to identify the source (e.g., genus, species, variety, strain, part of a plant source (such as roots or leave s), and organ or tissue of an animal source), and include any known toxicants that could be in the source. High purity steviol glycosides primarily consistinq of rebaudioside A extracted from the leaves ofstevia rebaudiana Bertoni and subsequently purified to meet the detailed specifications provided on pages 19 and 20 and within Table 3 of volume 1. Chemical structure provided on pages 14 and in Figures 1 in volume 1. Molelcular weight: Rebaudioside A daltons. See Table 2 on page 13 of volume 1. Chemical formula: Rebaudioside A- C44H70023 as found in Table 2 on page 13 of volume 1. or relevant: Rebaudioside A-> 13-[(2-0-(3-D-glucopyranosyi-3-Q-(3-D-glucopyranosyi-(3-D- glucopyranosyl) oxy] kaur-16-en-18-oic acid, (3-Dnosyl ester - from page 13 oft able 2 in volume Add Continuation Page FORM FDA 3667 (2113) Page 3 of4

6 PART VI-- OTHER ELEMENTS IN YOUR GRAS NOTICE (CileCk list to /1e/p 8/I:OU/e your SUIJIIIISSI0/1 IS CO!IIj)iete- Ci7eck c1// tilcll cipp/y) IZJ Any additional information about identity not cc vered in Part V of this form IZJ Method of Manufacture IZJ Specifications for food-grade material IZJ Information about dietary exposure IZJ Information about any self-limiting levels of use (which may include a statement that the intended use of the notified substance is not-self-limiting) D Use in food before 1958 (which may include a statement that there is no infonnation about use of the notified substance in food prior to 1958) IZJ Comprehensive discussion of the basis for the determination of GRAS status IZJ Bibliography Other lnfonnation Did you include any other information that you want FDA to consider in evaluating your GRAS notice? IZJ Yes D No Did you include this other information in the list of attachments? IZJ Yes 0No PART VII- SIGNATURE 1. The undersigned is informing FDA that Productora Alysa SpA ~--~~~ (name of notifier) has concluded that the intended use(s) of High Purity Steviol Glycosides (purity no less than 95%) primarily consisting of Rebaudioside.. (name of notified substance) described on this form, as discussed in the attached notice, is (are) exempt from the premari<et approval requirements of section 409 of the Federal Food, Drug, and Cosmetic Act because the intended use(s) is (are) generally recognized as safe. 2. 1ZJ Product ora Alysa SpA agrees to make the data and information that are the basis for the (name of notifiet1 determination of GRAS status available to FDA if FDA asks to see them. Product ora Alysa SpA agrees to allow FDA to review and copy these data and information during :, customary business hours at the following location if FDA asks to do so. (name of notifierj Calle Tres No. 600 Belloto Norte, Quilpue, CHILE (address of notifier or other location) _P_ro_d_u_ct o_ra_a--=-iy_s_a-,-s-=-p_a,--.,--:----- agrees to send these data and information to FDA if FDA asks to do so. (name of notifierj OR 0 The complete record that supports the determination of GRAS status is available to FDA in the submitted notice and in GRP No. (GRAS Affirmation Petition No.) (b) (6) Printed Name and Title Richard Kraska, Chief Scientific Officer Date (mm/dd/yyyy) 11/05/2014 FORM FDA 3667 (2113) Page 4 of4

7 PART VIII- LIST OF ATTACHMENTS List your attached files or documents containing your submission, forms, amendments or supplements, and other pertinent information. Clearly identify the attachment with appropriate de!scriptive file names (or titles for paper documents), preferably as suggested in the guidance associated with this form. Number your attachments consecutively. When submitting paper documents, enter the inclusive page numbers of each portion of the document below. Attachment Number Attachment Name Folder Location (select from menu) (Page Number(s) for paper Copy Only) GRAS Assessment of High Purity Steviol Glycosides vol. l.pdf GRAS Assessment of High Purity Steviol Glycosides vol. 2-- Appendices A through M.pdf; contains supporting information I I I I I I I I I I I I I I I I I I OMB Statement: Public reporting burden for this.:;ollection of information is estimated to average 150 hours per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to: Department of Health and Human Services, Food and Drug Administration, Office of Chief Information Officer, 1350 Piccard Drive, Room 400, Rockville, MD (Please do NOT return the form to this address.). An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB control number. FORM FDA 3667 (2/13) Page 5 of4

8 GRAS ASSESSMENT of HIGH PURITY STEVIOL GLYCOSIDES ( 95%) Food Usage Conditions for General Recognition of Safety for Productora Alysa SpA Calle Tres No. 600 Quilpué, Fifth Region Chile VOLUME 1 OF 2 Evaluation by GRAS Expert Panel Richard C. Kraska, Ph.D., DABT Robert S. McQuate, Ph.D. Robert W. Kapp, Jr., Ph.D., Fellow ATS, ERT (UK) November 5, 2014

9 TABLE OF CONTENTS I. GRAS EXEMPTION CLAIM. 5 A. Claim of Exemption from the Requirement for Premarket Approval Pursuant to Proposed 21 CFR (c)(1). 5 B. Name & Address of Notifier 5 C. Common Name & Identity of Notified Substance... 6 D. Conditions of Intended Use in Food. 6 E. Basis for GRAS Determination.. 6 F. Availability of Information 6 II. INTRODUCTION... 6 A. Objective... 6 B. Foreword... 7 C. Summary of Regulatory History of Stevia & Stevia-Derived Sweeteners 7 D. FDA Regulatory Framework III. CHEMISTRY & MANUFACTURE OF STEVIA PURE. 12 A. Common or Usual Name B. Description. 12 C. Chemistry of Steviol Glycosides D. Accepted Identity Specifications for Food Grade Steviol Glycosides E. Manufacturing Processes Scientific & Patent Literature PAS Manufacturing Process for Purified Stevia Pure F. Product Specifications & Supporting Methods JECFA Specifications for Steviol Glycosides Specifications for Stevia Pure Preparations & Supporting Methods. 20 G. Stability Data Stability Data on Steviol Glycosides Stability Data for Stevia Pure Preparations H. Sweetness Equivalence of Stevia Pure 22 IV. INTENDED FOOD USES & ESTIMATED DIETARY INTAKE A. Intended Uses B. Estimated Daily Intake of Rebaudioside A Preparations 23 C. Other Information on Human Exposure to Stevia: Use as Food Ingredient & Other Uses.. 24 V. SAFETY INVESTIGATIONS FOR STEVIOL GLYCOSIDES A. Safety Considerations for Steviol Glycosides: Recent Reports & Reviews by Expert Bodies & Other Scientists.. 24 B. Safety Information for Rebaudioside A VI. GRAS CRITERIA & PANEL SAFETY FINDINGS A. GRAS Criteria B. Safety Studies of High Purity Steviol Glycosides C. Common Knowledge Elements of GRAS Determinations GRAS ASSOCIATES, LLC Page 2 of 45

10 TABLE OF CONTENTS continued VII. CONCLUSIONS VIII. REFERENCES TABLES Table 1. FDA s GRAS Notice Inventory on Rebaudioside & Steviol Glycosides Preparations 8 Table 2. Chemical Identity of Rebaudioside A & Stevioside.. 13 Table 3. Specifications for Stevia Pure Preparations.. 19 Table 4. PAS Stevia Pure Storage Stability Data of Steviol Glycosides 22 Table 5. Daily Intake of Sweeteners (In Sucrose Equivalents) & Estimated Daily Intakes of High Purity Stevia Pure Preparations.. 23 FIGURES Figure 1. Chemical Structure of Rebaudioside A.. 14 Figure 2. Chemical Structure of Stevioside 14 Figure 3. Chemical Structures of Various Steviol Glycosides. 15 Figure 4. PAS Production Process for High Purity Steviol Glycosides (Stevia Pure). 17 VOLUME 2 -- APPENDICES APPENDIX A Certificates of cgmp Compliance 3 APPENDIX A-1 Bureau Veritas Certificate of Compliance.. 4 APPENDIX A-2 Bureau Veritas ISO 22000:2005 Certificate... 5 APPENDIX A-3 Bureau Veritas HACCP Certificate.. 6 APPENDIX B Specifications & Certificates of Analysis for Production Processing Aids. 7 APPENDIX B-1 Dow Chemical Documentation for Food Additive Status for Cation Exchange Resins 8 APPENDIX B-2 Dow Chemical Documentation for Food Additive Status for Anion APPENDIX B-3 Exchange Resins 9 Dow Chemical Documentation for Food Additive Status for Anion Exchange Resins & Adsorbent. 10 APPENDIX B-4 Supplier Letter of Regulatory Compliance for Spiral Wound Element APPENDIX C Analytical Method for Steviol Glycosides Quantitation. 12 APPENDIX D HPLC Analysis Report for Five Lots of Stevia Pure.. 27 APPENDIX E Certificates of Analysis for Multiple Production Batches of Stevia Pure 43 APPENDIX E-1 Certificate of Analysis for Stevia Pure 289A.. 44 APPENDIX E-2 Certificate of Analysis for Stevia Pure 292A.. 46 APPENDIX E-3 Certificate of Analysis for Stevia Pure 297B.. 48 APPENDIX E-4 Certificate of Analysis for Stevia Pure 299B.. 50 APPENDIX E-5 Certificate of Analysis for Stevia Pure 310A.. 52 APPENDIX F Pesticide Analytical Reports for Stevia Pure from Analab APPENDIX F-1 Test Report for Pesticides for Stevia Pure GRAS ASSOCIATES, LLC Page 3 of 45

11 TABLE OF CONTENTS continued APPENDIX F-2 Test Report for Pesticides for Stevia Pure APPENDIX G Shelf Stability Testing Report for Stevia Pure 60 APPENDIX H Sweetness Intensity Test Report for Stevia Pure.. 64 APPENDIX I Estimated Daily Intake Levels of Steviol Glycosides. 68 APPENDIX J Summary of Published Safety Reviews.. 73 APPENDIX K Studies on Principal Metabolite: Steviol. 78 APPENDIX L Studies on Steviol Glycosides Preparations that are Primarily Mixtures of Stevioside & Rebaudioside A 81 APPENDIX M Studies on Steviol Glycosides Preparations that are Primarily Rebaudioside A.. 93 GRAS ASSOCIATES, LLC Page 4 of 45

12 I. GRAS EXEMPTION CLAIM A. Claim of Exemption From the Requirement for Premarket Approval Pursuant to Proposed 21 CFR (c)(1) 1 Productora Alysa SpA ( PAS ) has determined that its high purity steviol glycosides ( 95%) preparations, with rebaudioside A as the principal component---referred to as Stevia Pure---which meet the specifications described below, is Generally Recognized As Safe (GRAS) in accordance with Section 201(s) of the Federal Food, Drug, and Cosmetic Act. This determination was made in concert with an appropriately convened panel of experts who are qualified by scientific training and experience. The GRAS determination is based on scientific procedures as described in the following sections. The evaluation accurately reflects the intended conditions of food use for the designated stevia-derived sweetener. Signed: (b) (6) Robert S. McQuate, Ph.D. Date: November 5, 2014 GRAS Associates, LLC Riverview Center Blvd Suite 212 Bonita Springs, FL B. Name & Address of Notifier Productora Alysa SpA Calle Tres No. 600 Quilpué, Fifth Region, Chile As the notifier, Productora Alysa SpA accepts responsibility for the GRAS determination that has been made for its high purity steviol glycosides preparations, primarily containing rebaudioside A, as described in the subject notification; consequently, this high purity steviol glycosides preparations, having a purity of no less than 95% total steviol glycosides, which meet the conditions described herein, are exempt from premarket approval requirements for food ingredients. 1 See 62 FR 18938, 17 April Accessible at Accessed on June 30, GRAS ASSOCIATES, LLC Page 5 of 45

13 C. Common Name & Identity of the Notified Substance High purity steviol glycosides, primarily containing rebaudioside A ---abbreviated as Reb A or reb A ---is the common name for the notified substances; also see Section III.A. D. Conditions of Intended Use in Food The high purity steviol glycosides preparations, primarily containing rebaudioside A, are intended to be used as a table top sweetener and as a general purpose non-nutritive sweetener for incorporation into foods in general, other than infant formulas and meat and poultry products, at per serving levels reflecting good manufacturing practices principles in that the quantity added to foods should not exceed the amount reasonably required to accomplish its intended technical effect. E. Basis for GRAS Determination Pursuant to 21 CFR , PAS s high purity steviol glycosides ( 95%) preparations, with rebaudioside A as the principal component, extracted from the leaves of Stevia rebaudiana Bertoni have been determined to be GRAS on the basis of scientific procedures as discussed in the detailed description provided below. F. Availability of Information The data and information that serve as the basis for this GRAS notification will be sent to the US Food and Drug Administration (FDA) upon request or will be available for review and copying at reasonable times at the offices of GRAS Associates, LLC, located at Riverview Center Blvd, Suite 212, Bonita Springs, FL II. INTRODUCTION A. Objective At the request of PAS, GRAS Associates, LLC ( GA ) has undertaken an independent safety evaluation of PAS s steviol glycosides preparations, Stevia Pure. Stevia Pure preparations are extracted from the leaves of Stevia rebaudiana Bertoni and purified to yield a minimum of 60% rebaudioside A, with a total steviol glycosides content 95%. The purpose of the evaluation is to ascertain whether the intended food uses of steviol glycosides as a general purpose non-nutritive sweetener as described in Section IV.A are generally recognized as safe, i.e., GRAS, under the intended conditions of use. GRAS ASSOCIATES, LLC Page 6 of 45

14 B. Foreword Productora Alysa SpA provided GA with substantial background information needed to enable the GRAS assessment to be undertaken. In particular, the information provided addressed the safety/ toxicity of steviol glycosides; the history of use of stevia in food; and compositional details, specifications, and method of preparation of the notified substance. PAS was asked to provide adverse reports, as well as those that supported conclusions of safety. Safety/toxicity studies performed with animals were noted to have value, along with available human testing. PAS was also asked to supply past and present human food use information. Knowing how much steviol glycosides ---including Reb A--- has been safely consumed, i.e., the use levels, is critical in extrapolating to safe exposures for highly purified steviol glycosides when consumed as a food ingredient. The composite safety/toxicity studies, in concert with exposure information, ultimately provide the specific scientific foundation for the GRAS determination. In addition to the product specifications, chemical properties, manufacturing, and safety related information, PAS also provided some consumption/exposure information, along with other related documentation. This was augmented with an independent search of the scientific and regulatory literature extending through November 5, A GRAS assessment based primarily on the composite safety information, i.e., based on scientific procedures, was undertaken. Those references that were deemed pertinent to the objective at hand are listed in Section VIII. C. Summary of Regulatory History of Stevia & Stevia-Derived Sweeteners Stevia-derived sweeteners are permitted as food additives in South America and in several countries in Asia, including China, Japan, and Korea. In recent years, these sweeteners have received food usage approvals in Mexico, Australia, New Zealand, Switzerland, France, Peru, Uruguay, Colombia, Senegal, Russia, Malaysia, Turkey, Taiwan, Thailand, Israel, Canada, and Hong Kong (EFSA, 2010; NutraIngredients, 2010; Health Canada, 2012). In the US, steviol glycosides have been used as a dietary supplement since 1995 (Geuns, 2003). Based on available information from FDA s GRAS Notice Inventory 2 website as of October 13, 2014, the agency has issued 32 no questions letters on GRAS notices on rebaudioside A, rebaudioside D, rebaudioside M, or steviol glycosides, including those undergoing enzyme treatment. A summary of these filings is presented in Table 1. The Joint Expert Committee on Food Additives (JECFA) has reviewed steviol glycosides at its 51 st, 63 rd, 68 th and 73 rd meetings. In 2000, JECFA published the original review on steviol glycosides (WHO, 2000). JECFA established a temporary ADI (acceptable daily intake) of 0-2 mg/kg (on a steviol basis) at its 63 rd meeting (WHO, 2006). Additionally, JECFA finalized food grade specifications (FAO, 2007a), although they were subsequently updated in 2008 (FAO, 2008) and 2010 (FAO, 2010) (see below). At the 69 th meeting, the temporary status of the ADI was removed, and the ADI was raised to 0-4 mg/kg bw/day (on a steviol basis) as a result of the JECFA review of recently completed clinical studies with steviol glycosides (WHO, 2008). In 2009, JECFA published a final monograph addendum on steviol glycosides (WHO, 2009). 2 FDA s GRAS Inventory Website was last updated on September 1, Accessible at: (Accessed 10/13/14). GRAS ASSOCIATES, LLC Page 7 of 45

15 Table 1. FDA s GRAS Notice Inventory on Rebaudioside & Steviol Glycosides Preparations a, b COMPANY FDA GRAS IDENTIFIER 1. Merisant GRN Cargill Inc. GRN McNeil Nutritionals LLC 4. Blue California 5. Sweet Green Fields LLC 6. Wisdom Natural Brands 7. Sunwin USA LLC & WILD Flavors 8. Sunwin USA LLC & WILD Flavors GRN 275 GRN 278 GRN 282 GRN 287 GRN 303 GRN Pyure Brands, LLC GRN PureCircle USA Inc GRN GLG Life Tech Ltd c GRN NOW Foods GRN GLG Life Tech Ltd c GRN GLG Life Tech Ltd c GRN Guilin Layn Natural Ingredients, Corp. 16. BrazTek International Inc. 17. Sinochem Qingdao Co. Ltd. 18. Shanghai Freemen Americas LLC GRN 354 GRN 365 GRN 367 GRN 369 MATERIAL IDENTITY High-Purity Reb A >95% High-Purity Reb A >97% Purified Steviol Glycosides Reb A Principal Component High-Purity Reb A >97% High-Purity Reb A >97% Purified Steviol Glycosides >95% - Reb A and Stevioside Principal Component High-Purity Reb A >95%/ >98% Purified Steviol Glycosides >95% - Reb A and Stevioside Principal Component High-Purity Reb A 95%/ 98% Purified Steviol Glycosides Reb A Principal Component High-Purity Reb A >97% Enzyme Modified Steviol Glycosides Preparation (EMSGP) High-Purity Stevioside >95% High-Purity Steviol Glycosides >97% High-Purity Reb A >97% Purified Reb A High-Purity Steviol Glycosides >95% Purified Reb A INTENDED FOOD USES Variety of food categories & table top sweetener General-purpose sweetener, excluding meat & poultry products Table top sweetener General-purpose & table top sweetener General-purpose sweetener, excluding meat & poultry products General-purpose sweetener, excluding meat, poultry products & infant formulas General-purpose sweetener, excluding meat, poultry products & infant formulas General-purpose sweetener, excluding meat, poultry products & infant formulas General-purpose & table top sweetener, excluding meat, poultry products & infant formulas General-purpose & table top sweetener, excluding meat, poultry products & infant formulas General-purpose sweetener, excluding meat & poultry products General-purpose sweetener in foods, excluding meat & poultry products, at levels determined by good manufacturing practices General-purpose & table top sweetener, excluding meat, poultry products & infant formulas General-purpose & table top sweetener, excluding meat, poultry products & infant formulas General-purpose & table top sweetener, excluding meat, poultry products & infant formulas General-purpose sweetener, excluding meat & poultry products General-purpose & table top sweetener, excluding meat, poultry products & infant formulas General-purpose sweetener, excluding meat & poultry products GRAS ASSOCIATES, LLC Page 8 of 45

16 19. Toyo Sugar Refining Co., Ltd. & Nippon Paper Chemicals Co., Ltd. GRN 375 Enzyme Modified Steviol Glycosides 20. GLG Life Tech Ltd c GRN 380 Purified Reb A 21. Chengdu Wagott Pharmaceutical 22. Chengdu Wagott Pharmaceutical GRN 388 GRN 389 Purified Reb A Steviol Glycosides with Stevioside as the Principal Component 23. Daepyung Co., Ltd. GRN 393 Purified Reb A 24. Daepyung Co., Ltd. GRN MiniStar International, Inc. GRN Daepyung Co., Ltd. GRN Daepyung Co., Ltd. GRN PureCircle USA, Inc. GRN Almendra, Ltd. GRN Qufu Xiangzhou Stevia Products Co., Ltd. GRN PureCircle USA, Inc. GRN GLG Life Tech Ltd. c GRN 493 Steviol Glycosides with Reb A and Stevioside as the Principal Components Purified Reb A Enzyme Modified Steviol Glycosides Enzyme Modified Steviol Glycosides High-Purity Reb D >95% High-Purity Reb A >97% High-Purity Reb A >98% Purified Steviol Glycosides Reb M (Reb X) Principal Component High-Purity Steviol Glycosides >95% with Reb A and Stevioside as the Principal Components General-purpose sweetener in foods, excluding meat and poultry products, at levels determined by good manufacturing practices General purpose & table top sweetener, excluding meat & poultry products General purpose & table top sweetener, excluding meat & poultry products General purpose & table top sweetener, excluding meat & poultry products General purpose & table top sweetener, excluding meat & poultry products General purpose & table top sweetener, excluding meat & poultry products General-purpose sweetener, excluding meat, poultry products & infant formulas. General-purpose sweetener, excluding meat, poultry products & infant formulas. General-purpose sweetener, excluding meat, poultry products & infant formulas. General-purpose sweetener, excluding meat, poultry products & infant formulas. General-purpose sweetener, excluding meat, poultry products & infant formulas. General-purpose sweetener, excluding meat, poultry products & infant formulas. General-purpose sweetener, excluding meat, poultry products & infant formulas. General purpose & table top sweetener, excluding meat & poultry products a This table was derived, in part, from McQuate (2011). bgrn 512, addressing high purity rebaudioside M, was submitted by GLG Life Tech Corporation and filed by FDA on April 28, 2014, and is presently under review by FDA; GRN 516, addressing steviol glycosides with rebaudioside A and stevioside as principal components, was submitted by Almendra (Thailand) Limited and filed by FDA on May 9, 2014, and is presently under review by FDA; GRN 536, addressing high purity rebaudioside C, was submitted by GLG Life Tech Corporation and filed by FDA on August 18, 2014, and is presently under review by FDA. c The name of this company is now GLG Life Tech Corporation. In early 2009, a number of parties, including the government of Australia and the Calorie Control Council, submitted a request to the Codex Committee on Food Additives in which it was proposed that the JECFA specifications for steviol glycosides should be modified to allow inclusion of rebaudioside D and rebaudioside F as specifically named acceptable glycosides that would be considered as part of the minimum 95% steviol glycosides composition (CCFA, 2009). This proposed modification was endorsed by the Codex Alimentarius Committee in July 2009; it was on the agenda for discussion at the JECFA Meeting in June, 2010 (FAO/WHO, 2009), and JECFA subsequently took final action in approving the modified steviol glycosides specifications to include rebaudioside D and rebaudioside F (FAO, 2010). GRAS ASSOCIATES, LLC Page 9 of 45

17 In 2008, Switzerland s Federal Office for Public Health (2008) approved the use of stevia as a sweetener citing the favorable actions of JECFA. Subsequently, France published its approval for the food uses of rebaudioside A with a purity of 97% (AFSSA, 2009). Also in 2008, the Food Standards Australia New Zealand (FSANZ) completed its evaluation of an application for use of steviol glycosides in foods. FSANZ recommended that the Australia and New Zealand Food Regulation Ministerial Council (Ministerial Council) amend the Australia New Zealand Food Standards Code to allow the use of steviol glycosides in food (FSANZ, 2008). In December 2010, FSANZ recommended accepting the increased usage levels as requested since no public health and safety issues were identified (FSANZ, 2010). Subsequently, FSANZ approved an increase in the maximum permitted level (MPL) of steviol glycosides (expressed as steviol equivalents) in ice cream, water based beverages, brewed soft drinks, formulated beverages, and flavored soy beverages up to 200 mg/kg, and in plain soy beverages up to 100 mg/kg (FSANZ, 2011). As of May 2010, the government of Hong Kong amended its food regulations to allow the use of steviol glycosides as a permitted sweetener in foods (Hong Kong Centre for Food Safety, 2010). This action followed in the aftermath of the detailed safety evaluation and favorable findings as reported by JECFA. On September 18, 2009, based on a review of the international regulation of Stevia rebaudiana and the clinical evidence for safety and efficacy, the Natural Health Products Directorate, Health Canada (2009) adopted the following guidelines for the use of stevia and steviol glycosides in Natural Health Products (NHPs). The revised recommendation for the maximum limit for steviol glycosides in NHPs is in accordance with the full ADI of 4 mg steviol/kg bw established by JECFA (WHO, 2008). In light of JECFA s 2008 findings, and in response to a June 2008 request by the European Commission for European Food Safety Authority (EFSA) to deliver a scientific opinion on the safety of steviol glycosides as a sweetener for use in the food categories specified in the dossiers from three petitioners, EFSA reexamined the safety of steviol glycosides (EFSA, 2010). After considering all the data on stability, degradation products, metabolism and toxicology, the EFSA Panel established an ADI for steviol glycosides, expressed as steviol equivalents, of 4 mg/bw/day, which is similar to JECFA s determination. 3 In addition, on May 25, 2011, EFSA published a determination that the daily dietary intake for use of rebaudioside A as a flavoring substance in a variety of foods would be less than the ADI for steviol glycosides (EFSA, 2011a). The international community continues to exhibit much interest in the food uses of steviol glycosides, with additional advances reported in early July The Codex Alimentarius Commission has adopted proposed maximum use levels for steviol glycosides in all major food and beverage categories, and this action is expected to favorably influence authorizations of stevia 3 From a historical perspective, it is noted that the UK s Advisory Committee on Novel Foods and Processes for the Ministry of Agriculture, Fisheries and Food on September 24, 1998 rejected an application for use of steviol glycosides as a sweetener in herbal teas because the applicant had not provided all of the information necessary to enable an assessment to be made. See (Accessed on June 30, 2014). In 1999, the Scientific Committee on Food for the European Commission concluded that there are no satisfactory data to support the safe use of these stevia plants and leaves (European Commission, 1999a). In another opinion also dated June 17, 1999, the Committee also reiterated its earlier opinion that stevioside is not acceptable as a sweetener on the presently available data (European Commission, 1999b). GRAS ASSOCIATES, LLC Page 10 of 45

18 uses in India, Indonesia, Thailand, and the Philippines (FoodNavigator, 2011). An article published online by FoodNavigator (2013) states the following: with approvals now in Vietnam, the Philippines, Malaysia, Singapore and Thailand, Indonesia is the only [Southeast Asian nation] where stevia hasn t been given the rubber stamp. Furthermore, the International Alliance of Dietary/Food Supplement Associations (IADSA) reported that the Codex Alimentarius Commission agreed to adopt the use of steviol glycosides for addition to chewable food supplements as had been requested by IADSA (NewHope360, 2011). The appropriate European regulatory bodies, including the joint FAO/WHO Expert Committee on Food Additives (JECFA) and the European Food Safety Authority (EFSA), have now agreed that steviol glycosides are safe for all populations to consume and are a suitable sweetening option for diabetics. Effective December 2, 2011, the EU approved their use as food additives (EU, 2011). On September 10, 2012, the South African Department of Health issued an amendment to labeling regulations indicating: in the case of the sweetener steviol glycosides, it shall be described as Steviol Glycosides or Steviol Extract. On the same date, steviol glycosides were added to the List of Permissible Sweeteners (Republic of South Africa Department of Health, 2012a, b). The Food Safety and Standards Authority of India (FSSAI) convened on September 20, 2012, and approved the use of steviol glycosides as a non-nutritive sweetener in a variety of foods. The FSSAI specified that: the steviol glycosides must meet the specifications and purity as established by JECFA; table top sweetener tablets may contain 7 mg of steviol equivalents per 100 mg carrier/filler, as well as established maximum use levels specific to 11 distinct food categories including dairy, beverage, and chewing gum applications (FSSAI, 2012) On November 30, 2012, Health Canada published its final clearance for use of steviol glycosides as a sweetener in foods (Health Canada, 2012). In March 2014, Health Canada updated the List of Permitted Sweeteners (Lists of Permitted Food Additives) 4 to include steviol glycosides in applications as a table-top sweetener, and as an ingredient in a variety of foods, beverages, baked goods, meal replacement bars, condiments, and confectionary and gums (Health Canada, 2014). Since December 10, 2012, multiple food registrations have been granted by FDA Philippines to stand-alone steviol glycosides sweeteners or foods containing steviol glycosides as ingredients: FR , Steviten Light Brand Steviol Glycosides 95% Sweetener Powder; FR , Del Monte Pineapple Chunks in Extra Light Syrup Reduced Calorie with Steviol Glycosides from Stevia; FR , Diebetamil Zero Calorie Sweetener with Stevia (stick pack); and FR , Sawayaka Stevia Sweetener (1 g sticks) (Republic of the Philippines, Food and Drug Administration, 2013). Finally, steviol glycosides are listed under INS number 960 in the Food Additives Permitted Under the Singapore Food Regulations document prepared by the Agri-Food & Veterinary Authority (AVA) of Singapore, and this information can be accessed on their website as of September 24, 2013 (AVA, 2013). 4 Available at: (Accessed on 9/16/14). GRAS ASSOCIATES, LLC Page 11 of 45

19 D. FDA Regulatory Framework In order to be incorporated into conventional foods, food ingredients must undergo premarket approval by FDA as food additives or, alternatively, the ingredients must be determined to be generally recognized as safe (GRAS). The authority to make GRAS determinations is not restricted to FDA. In fact, GRAS determinations may be provided by experts who are qualified by scientific training and experience to evaluate the safety of food and food ingredients under the intended conditions of use. 5 In 1997, FDA altered the GRAS determination process by eliminating the formal GRAS petitioning process. At that time, the petitioning process was replaced with a notification procedure. 6 While outlining the necessary content to be considered in making a GRAS determination, FDA encouraged that such determinations should be provided to FDA in the form of a notification. However, notifying FDA of such determinations is strictly voluntary. III. CHEMISTRY & MANUFACTURE OF STEVIA PURE A. Common or Usual Name High purity steviol glycosides is the common or usual name of the non-nutritive sweetener derived from Stevia rebaudiana Bertoni that is the subject of the GRAS evaluation. The compositional features of the subject high purity steviol glycosides ( 95%), primarily containing rebaudioside A, are described in more detail in this section. Stevia Pure is the general term used by PAS in referring to the notified substance. In the scientific literature, steviol glycosides have been referred to as stevia, stevioside, steviol glycosides, and stevia glycoside. JECFA adopted the term, steviol glycosides, for the family of steviol derivatives with sweetness properties that are derived from the stevia plant. Presently, the term, stevia, is used more narrowly to describe the plant or crude extracts of the plant, while Reb A like stevioside is the common name for another one of the specific glycosides that is extracted from stevia leaves. B. Description In 2010, Food Chemicals Codex (FCC) prepared a monograph with a description and specifications for rebaudioside A. In this monograph, rebaudioside A is described as a white to offwhite, hygroscopic fine crystal, granule, or powder having a sweet taste (FCC, 2010). It is freely soluble in ethanol:water 50/50 (v/v), and is sparingly soluble in water and in ethanol. Rebaudioside A is obtained from the leaves of the Stevia rebaudiana Bertoni plant in a multistep separation and purification process. The principal steps of manufacturing include extraction of steviol glycosides from the leaves using an aqueous or aqueous alcoholic (ethanol or methanol) solvent, and purification of rebaudioside A from the resulting mixture of steviol glycosides by resin absorption followed by recrystallization from an aqueous or aqueous alcoholic (ethanol or methanol) solvent. 5 See 21 CFR 170.3(i)(3). 6 See 62 FR 18938, 17 April At Accessed on June 30, GRAS ASSOCIATES, LLC Page 12 of 45

20 It is primarily composed of rebaudioside A, a glycoside of the ent-kaurenoid diterpenoid aglycone known as steviol (FCC, 2010). C. Chemistry of Steviol Glycosides At its 51 st meeting, JECFA reviewed the safety related information on steviol glycosides, including the identity and chemistry of these compounds. The following chemistry related description of steviol glycosides is taken from the original JECFA monograph (WHO, 2000). Stevioside is a glycoside of the diterpene derivative steviol (ent-13-hydroxykaur-16-en-19-oic acid). Steviol glycosides are natural constituents of the plant Stevia rebaudiana Bertoni, belonging to the Compositae family. The leaves of S. rebaudiana Bertoni contain eight different steviol glycosides, the major constituent being stevioside (triglucosylated steviol), constituting about 5-10% in dry leaves. Other main constituents are rebaudioside A (tetraglucosylated steviol), rebaudioside C, and dulcoside A. S. rebaudiana is native to South America and has been used to sweeten beverages and food for several centuries. The plant has also been distributed to Southeast Asia. Stevioside has a sweetening potency times that of sucrose and is stable to heat. In a 62-year-old sample from a herbarium, the intense sweetness of S. rebaudiana was conserved, indicating the stability of stevioside to drying, preservation, and storage(soejarto et al., 1982; Hanson & De Oliveira, 1993). Of the nine different steviol glycosides, the two principal sweetener components of stevia extracts have been identified as rebaudioside A and stevioside. The chemical identities and key chemical identifiers for the two major components are presented in Table 2. Table 2. Chemical Identity of Rebaudioside A & Stevioside REBAUDIOSIDE A Common Name Chemical name Chemical formula Formula weight Rebaudioside A 13-[(2-O-β-D-glucopyranosyl-3-O-β-Dglucopyranosyl-β-D- glucopyranosyl) oxy] kaur-16-en-18-oic acid, β-d- glucopyranosyl ester C 44 H 70 O daltons CAS Number Common name Chemical name Chemical formula Formula weight STEVIOSIDE Stevioside 13-[2-O-β-D-glucopyranosyl-β-Dglucopyranosyl)oxy] kaur-16-en-18-oic acid, β-d-glucopyranosyl ester C 38 H 60 O daltons CAS Number GRAS ASSOCIATES, LLC Page 13 of 45

21 The chemical structure of rebaudioside A is presented in Figure 1, and the chemical structure of stevioside is presented in Figure 2. In the Chemical and Technical Assessment (FAO, 2007b), JECFA identified the sweetener components. They updated the list of common glycosides and their chemical structures, which are slightly different from compounds depicted in older publications (Nanayakkara et al., 1987; Suttajit et al., 1993). They are shown in Figure 3. Figure 1. Chemical Structure of Rebaudioside A Figure 2. Chemical Structure of Stevioside GRAS ASSOCIATES, LLC Page 14 of 45

22 Figure 3. Chemical Structures of Various Steviol Glycosides a, b a From FAO, 2007b. b The indicated C.A.S. No. for Rubusoside as reported in the cited reference is incorrect and should be In a number of reviews by different authors (Kinghorn and Soejarto, 1989; Kinghorn, 2002; Kennelly, 2002; Geuns, 2003), the structures of the components of steviol glycosides have been described. Through a series of chemical reactions and analyses, the structures, stereochemistry, and absolute configurations of steviol and isosteviol were established over a 20-year period after the seminal work of Bridel and Lavielle (1931) in France. The work by Ogawa et al. (1980, cited in Brandle, et al., 1998) on synthetic transformation of steviol into stevioside supported the proposed structures. Two other sweet glycosides, Reb A and Reb B, were obtained from methanol extracts of stevia leaves, along with the major sweet principle constituent, stevioside, and a minor constituent steviolbioside, which was first prepared from stevioside by alkaline hydrolysis by Wood et al. (1955, cited in Brandle et al., 1998). Subsequently, it was suggested that Reb B was an artifact formed from Reb A during isolation (Brandle et al., 1998; Kennelly, 2002). In addition, stevioside can be converted both chemically and enzymatically to Reb A. Further fractionation led to the isolation and identification of three other sweet glycosides, respectively named Reb C, Reb D, and Reb E. It was reported that Reb A and Reb D could be converted to Reb B by alkaline hydrolysis showing that only the ester functionality differed (Brandle et al., 1998). Dulcosides A and B were also described by Kobayashi et al. (1977). Later, dulcoside B and Reb C were shown to be structurally identical. GRAS ASSOCIATES, LLC Page 15 of 45

23 D. Accepted Identity Specifications for Food Grade Steviol Glycosides In addition to the manufacturing process, the composition of Stevia rebaudiana Bertoni extract depends upon the composition of the harvested leaves, which, in turn, is influenced by soil, climate, etc. (FAO, 2007b). As discussed in Section III.F.1., JECFA recommended that food grade specifications for steviol glycosides consist of a minimum of 95%, on a dried weight basis, of seven specific steviol glycosides (FAO, 2007a), and this has more recently been expanded to include the original seven specific steviol glycosides plus Reb D and Reb F (FAO, 2010). The component glycosides of particular interest for their sweetening property are stevioside and Reb A. In addition to Reb D and Reb F, the other five glycosides are found at substantially lower levels in the preparations of steviol glycosides, and recognized by JECFA, are Reb C, dulcoside A, rubusoside, steviolbioside, and Reb B. E. Manufacturing Processes Manufacturing processes for stevia-derived sweeteners have been described in the published scientific and patent literature. These processes are summarized below along with PAS s manufacturing process for Stevia Pure, which is also specifically discussed in Section III.E Scientific & Patent Literature In general, steviol glycosides are typically obtained by extracting leaves of Stevia rebaudiana Bertoni with hot water or alcohols (ethanol or methanol). This extract is a dark particulate solution containing all the active principles, plus leaf pigments, soluble polysaccharides, and other impurities. Some processes remove the grease from the leaves before extraction by employing solvents such as chloroform or hexane (Kinghorn, 2002). There are several extraction patents for the isolation of steviol glycosides. Kinghorn (2002) has categorized the extraction patents into those based on solvent, solvent plus a decolorizing agent, adsorption and column chromatography, ion exchange resin, and selective precipitation of individual glycosides. In recent patents, methods such as ultrafiltration, metallic ions, supercritical fluid extraction with CO 2, and extract clarification with zeolite have been employed. At the 68 th JECFA meeting, steviol glycosides were defined as the products obtained from the leaves of Stevia rebaudiana Bertoni. As described by JECFA, the typical manufacturing process starts with extracting leaves with hot water, and the aqueous extract is then passed through an adsorption resin to trap and concentrate the component steviol glycosides. The resin is then washed with methanol to release the steviol glycosides, and the product is recrystallized with ethanol or methanol. Ion-exchange resins may be used in the purification process. The final product is commonly spray-dried. 2. PAS s Manufacturing Process for Purified Stevia Pure PAS employs a fairly typical steviol glycosides manufacturing process that is used in the industry for the production of stevia-derived sweeteners with the exception that water is the only solvent used in the process. The source of PAS s high purity steviol glycosides (>95%) preparations is the leaves of the Stevia rebaudiana (Bertoni) plant. Membrane deionized water is used to extract the steviol glycosides from the Stevia leaves, yielding an extract with approximately 35% steviol GRAS ASSOCIATES, LLC Page 16 of 45

24 glycosides, of which ~65% (w/w) is Reb A. The aqueous extract is purified to 65-70% steviol glycosides using a membrane filtration system. The steviol glycosides, including rebaudioside A and stevioside, are screened from high- and low-molecular weight compounds via mechanical separation (screening). The resulting extract is applied to a food-grade ion exchange resin, and polished water is used to elute the steviol glycosides. The extract is then concentrated from 3-5% (w/w) to 30% (w/w) by nanofiltration spiral wound membranes. The concentrated extract is then microfiltrated with spiral microfiltration membranes to reduced microbial content and remove any remaining particles, and is then pasteurized at 85 C for 55 minutes prior to spray drying. During the spray drying process, the inlet temperature is maintained at 210 C and the outlet temperature is maintained at 95 C to reduce moisture content of the finished product to less than 5%. Stevia Pure is produced under a Good Manufacturing Practices (GMP) compliant program, as certified by Bureau Veritas (certificates provided in Appendix A). The manufacturing process is summarized in a flow chart provided in Figure 4. Figure 4. PAS Production Process for High Purity Steviol Glycosides (Stevia Pure) Leaf Selection Water Extraction Membrane Purification Ion Exchange Polishing Membrane Concentration Microfiltration Pasteurization Spray Drying Product Release GRAS ASSOCIATES, LLC Page 17 of 45

25 No organic solvents are used to manufacture Stevia Pure, as certified by Bureau Veritas under the ISO CASCO 5 certification (Appendix A). The filters used in the manufacturing comply with 21 CFR , 7 and the ion exchange resins meet FDA food contact requirements. Documentation of FDA compliance for the resins and membranes is provided in Appendix B. The analytical testing method, representative HPLC chromatograms, and certificates of analysis of five representative lots of Stevia Pure are detailed in Appendices C-E, respectively. Results from pesticide analyses of representative Stevia Pure samples are provided in Appendix F. The content of steviol glycosides in the final Stevia Pure product in all cases is 95%. F. Product Specifications & Supporting Methods 1. JECFA Specifications for Steviol Glycosides The composition of extracts of Stevia rebaudiana Bertoni depends upon the composition of the harvested leaves, which are, in turn, influenced by soil, climate, and the manufacturing process itself (FAO, 2007b). In 2007, JECFA recommended that the method of assay should include a minimum requirement of 95% of the total of 7 specific steviol glycosides on a dried weight basis, and JECFA finalized food grade specifications at the 68 th JECFA meeting with publication in the FAO JECFA Monograph 4 (FAO, 2007a). Stevioside and rebaudioside A are the major component glycosides of interest because of their sweetening property. The five other associated glycosides found in preparations of steviol glycosides accepted by the JECFA specifications with the 95% requirement are rebaudioside C, dulcoside A, rubusoside, steviolbioside, and rebaudioside B. These, however, are typically found at much lower levels than stevioside or rebaudioside A. JECFA updated the specifications for steviol glycosides in 2008 (FAO, 2008), and then again in 2010, when the specifications were expanded to include the original seven specific steviol glycosides plus Reb D and Reb F (FAO, 2010). Steviol glycosides are described as a white to yellow powder, odorless to having a slight characteristic odor, and exhibiting a sweetness that is times greater than sucrose. The ingredient must consist of a minimum of 95% of nine specific steviol glycosides. The steviol glycosides are freely soluble in water and ethanol, and the 1 in 100 solutions exhibit ph values between The product should not have more than 1% ash, with no more than a 6% loss on drying at 105 o C for 2 hours. Any residual methanol levels should not exceed 200 ppm, and ethanol residues should not exceed 5,000 ppm. Arsenic levels should not exceed 1 ppm as determined by the atomic absorption hydride technique. Lead levels should not exceed 1 ppm. 7 See GRAS ASSOCIATES, LLC Page 18 of 45

26 Table 3. Specifications for Stevia Pure Preparations PHYSICAL & CHEMICAL PARA- METERS Appearance Form Appearance Color Solubility d Purity (HPLC Area) % Residual Ethanol c Residual Methanol c Loss on Drying (%) ph, 1% Solution JECFA a SPECIFICA- TIONS STEVIOL GLYCOSIDES Powder White to light Yellow Freely soluble in water FCC b SPECIFICATIONS REBAUDIOSIDE A NS > 95 NMT 5000 mg/kg NMT 200 mg/kg PAS SPECIFICATIONS STEVIA PURE RESULTS OF BATCH NUMBERS 00289A 00292A 00297B 00299B 00310A NS NS NS NS NS NS White to light yellow Pass Pass Pass Pass Pass NS NS NS NS NS NS > 95% Steviol Glycosides > 60% Rebaudioside A 96.59% 67.07% 97.34% 68.8% 97.26% 69.48% 96.35% 65.58% 95.14% 63.81% NMT 0.5% NA NA NA NA NA NA NMT 0.02% NA NA NA NA NA NA NMT 6.0% NMT 6.0% < 6.0% 2.86% 2.31% 3.36% 3.68% 3.97% Total Ash (%) NMT 1% NMT 1% <1% <1% <1% <1% <1% <1% Arsenic Lead Crystal, granule or powder White to offwhite Freely soluble in water:ethanol (50:50) Total Plate Count (cfu/g, max) Yeast & Mold (cfu/g, max) Total Coliform (mpn/g) Salmonella spp Staphylococcus aureus NMT 1 mg/kg NMT 1 mg/kg NMT 1 mg/kg < 1 mg/kg <1 mg/kg <1 mg/kg <1 mg/kg <1 mg/kg <1 mg/kg NMT 1 mg/kg < 1 mg/kg <1 mg/kg <1 mg/kg <1 mg/kg <1 mg/kg <1 mg/kg NA NA < 2,000 <10 <10 <10 <10 <10 NA NA < 100 (yeast) < 100 (mold) NA NA <3 <3 <3 <3 <3 <3 NA NA Negative in 25 g Negative Negative Negative Negative Negative NA NA <3 <3 <3 <3 <3 <3 E. coli (mpn/g) NA NA <3 <3 <3 <3 <3 <3 Pseudomonas aeruginosa NA NA Negative in 25 g Negative Negative Negative Negative Negative a Prepared at 73rd JECFA (2010). b FCC, Rebaudioside A monograph. Food Chemicals Codex (7th Ed.) c No specification is necessary for residual ethanol or methanol because neither of these solvents is used in the process. NS = not specified; NA = not applicable; NLT = not less than; NMT = not more than <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 GRAS ASSOCIATES, LLC Page 19 of 45

27 2. Specifications for Stevia Pure Preparations & Supporting Methods PAS has adopted product specifications for its purified steviol glycosides products that meet or exceed JECFA recommendations, while also complying with Food Chemicals Codex (FCC, 2010) specifications for rebaudioside A as a consumable human food substance. Certificates of analysis for five non-consecutive production batches of Stevia Pure are presented in Appendix E and are compared to the JECFA and FCC specifications in Table 3. Details of the analytical methodology employed to determine steviol glycosides is provided in Appendix C, the chromatograms for representative Stevia Pure preparations are provided in Appendix D, and certificates of analysis for five representative lots Stevia Pure preparations are provided in Appendix E. Pesticide residue screening is periodically conducted on various product lots. Test reports for analyses of pesticide residues in two representative lots of Stevia Pure are located in Appendix F. The collection of reports contained in Appendices C through F demonstrates that Stevia Pure is well characterized and meets the established purity criteria. G. Stability Data 1. Stability Data on Steviol Glycosides Steviol glycosides have been reported to be stable over the ph range 3-9 and can be heated at 100 o C for 1 hour, but, at ph levels greater than 9, it rapidly decomposes (Kinghorn, 2002). At ph 10, steviolbioside would be the major decomposition product produced from stevioside by alkaline hydrolysis (Wood et al., 1955). Chang and Cook (1983) investigated the stability of pure stevioside and Reb A in carbonated phosphoric and citric acidified beverages. Some degradation of each sweetening component after 2 months of storage at 37 o C was noted. However, no significant change at room temperature or below, following 5 months of storage of stevioside and 3 months of storage of Reb A, was noted. Exposure to one week of sunlight did not affect stevioside, but resulted in approximately 20% loss of rebaudioside A. Heating at 60 o C for 6 days resulted in 0-6% loss of rebaudioside A. Merisant (2008) conducted stability testing on rebaudioside A (1) as a powder, (2) as a pure sweetener in solution, and (3) on both cola-type and citrus carbonated beverages. In these investigations, no degradation was detected when the powder was stored at 105 C for 96 hours. It was concluded that the powder was stable when stored for 26 weeks at 40±2 C with relative humidity of 75±5%. Both published and unpublished testing results from Merisant revealed that rebaudioside A in carbonated citric acid beverages and phosphoric acid beverages did not significantly degrade during prolonged storage at refrigeration, normal ambient, or elevated ambient temperatures. Minimal loss of rebaudioside A was detected after storage at 60 C, with considerable degradation noted after 13 hours at 100 C for carbonated beverage solutions and pure sweetener solutions (Merisant, 2008). Cargill (2008) also conducted extensive stability testing on rebaudioside A as a powder under various storage conditions and under a range of phs and temperatures. Additionally, Cargill also investigated rebaudioside A stability in several representative food matrices at room temperature and elevated temperatures. Stability profiles were created for table top sweetener applications, mock beverages including cola, root beer and lemon-lime, thermally processed beverages, yogurt, GRAS ASSOCIATES, LLC Page 20 of 45

28 and white cake. The results of stability testing revealed some degradation products that had not been detected in bulk rebaudioside A. These degradation products were structurally related to the steviol glycosides that are extracted from the leaves of Stevia rebaudiana Bertoni. All the degradation products were found to share the same steviol aglycone backbone structure as found in stevioside and rebaudioside A, but they differ by virtue of the glucose moieties present. The results of stability testing revealed that rebaudioside A is stable in various food matrices following several days or weeks of storage. The extent and rate of degradation is dependent on ph, temperature, and time. When placed in beverages, rebaudioside A is more stable in the ph range 4 to 6, and at temperatures from 5 C to 25 C (Cargill, 2008). Photostability studies of the dry powder and mock beverages were performed to ascertain rebaudioside A behavior under defined conditions of fluorescent and near UV light exposure. Rebaudioside A was found to be photostable under the defined conditions of analysis (Clos et al., 2008). In addition to the above-described stability reports for purified rebaudioside A, in a GRAS notification by Sunwin and WILD Flavors (2010)---regarding purified steviol glycosides with rebaudioside A and stevioside as the principal components---stability was investigated using a 0.04% solution of Reb A 80% in acidic solutions between ph 2.81 and In this study, the solutions were stored at 32 C for 4 weeks, and the Reb A content was determined at 1, 2, and 4 weeks. Reb A 80% was found to be very stable at ph 3.17 and above. At ph 2.81, after 4 weeks of storage under accelerated conditions, only a 7% loss of Reb A was noted. Sunwin and WILD Flavors also studied the stability of Reb A 80% in simulated beverages using 0.1% citric acid (ph 3.2). The solutions were pasteurized and stored for 8 weeks at 4 C and 32 C, and little difference in sweetness perception was found under these conditions. 2. Stability Data for Stevia Pure Preparations Due to the favorable stability profile for products of similar composition as noted in Section III.G.1., PAS conducted a shelf-stability test study on its Stevia Pure. Over the course of 24 months, samples were stored in the warehouse at ambient temperatures ranging from 15 C- 27 C. The stability samples were sampled every 6 months and then tested for steviol glycosides--- including rebaudioside A---moisture, and microbial parameters. A summary of the shelf-stability results is presented in Table 4, and a detailed stability report is provided in Appendix G. GRAS ASSOCIATES, LLC Page 21 of 45

29 Duration Table 4. PAS Stevia Pure Storage Stability Data of Steviol Glycosides Reb A (% dry weight) Stevie Pure Lot# 220A Total Steviol Glycosides (%dry weight) Moisture (%w/w) Total Aerobic Count Yeast t= <5 cfu/g < 5 cfu/g <5 cfu/g 6 months ND ND ND ND 12 months ND ND ND 18 months ND ND ND ND 24 months < 5 cfu/g < 5 cfu/g < 5 cfu/g ND = Not determined The stability data in the scientific literature for stevioside, the JECFA report, and the extensive stability testing for rebaudioside A as presented by Merisant, Cargill, and Sunwin & WILD Flavors, along with PAS s stability testing results, support the finding that PAS s high purity steviol glycosides preparations are well-suited for the intended food uses. Mold H. Sweetness Equivalence of Stevia Pure PAS conducted a sweetness equivalence evaluation to compare Stevia Pure to sucrose at various concentrations. The results of this comparison show that Stevia Pure, at a concentration of %, is estimated to be equivalent to a 5.0% sucrose solution. This suggests a sweetness intensity of approximately 235 times the sweetness of sucrose. The sweetness equivalence report is provided in Appendix H. IV. INTENDED FOOD USES & ESTIMATED DIETARY INTAKE A. Intended Uses The subject Stevia Pure preparations with steviol glycosides ( 95%), containing rebaudioside A as the principal component, are intended to be used as a table top sweetener and general purpose non-nutritive sweetener in various foods other than infant formulas and meat and poultry products The intended use will be as a non-nutritive sweetener as defined in 21 CFR 170.3(o)(19). 8 The intended use levels will vary by actual food category, but the actual levels are self-limiting due to organoleptic factors and consumer taste considerations. However, the amount of Stevia Pure to be added to foods will not exceed the amounts reasonably required to accomplish its intended technical effect in foods as required by FDA regulation. 9 8 Non-nutritive sweeteners: Substances having less than 2 percent of the caloric value of sucrose per equivalent unit of sweetening capacity. 9 See 21 CFR 182.1(b)(1). GRAS ASSOCIATES, LLC Page 22 of 45

30 B. Estimated Daily Intake of Rebaudioside A Preparations There have been many scholarly estimates of potential dietary intake of replacement sweeteners--- including steviol glycosides---that have been published (FSANZ, 2008; Renwick, 2008; WHO, 2003) or submitted to FDA (Merisant, 2008). These are summarized in Appendix I. In GRAS notification 301, a simplified estimate was proposed to and accepted by FDA based on the estimates of exposure in sucrose equivalents (Renwick, 2008) and the sweetness intensity of any particular sweetener (BioVittoria, 2009). As summarized in GRN 301, the 90 th percentile consumer of a sweetener that is 100 times as sweet as sucrose when used as a total sugar replacement would be a maximum of 9.9 mg/kg bw/day for any population subgroup. Using the WHO GEMS/Food database assumption that steviol glycosides, in general, exhibit a minimum relative sweetness intensity of 200:1 when compared to sucrose, one can assume that high purity rebaudioside A also shows a minimal 200-fold sweetness to that of sucrose (WHO, 2006). The estimated sweetness intensity for Stevia Pure is 235-fold that of sucrose as reported in Section III.H. Therefore, the highest 90 th percentile consumption by any population subgroup of PAS s high purity steviol glycosides preparation would be approximately 4.21 mg/kg bw/day. A weighted sum estimate was used to determine the steviol equivalency factor on a worst-case scenario basis. For example, PAS s Stevia Pure steviol equivalence factor was calculated from the molecular weight ratios of steviol to rebaudioside A and the remaining steviol glycosides (as steviolbioside), on a percent composition basis, as follows: SteviolEqu ivalencefactor MW Steviol MWSteviol = MWRebA MWSteviolbioside Based on a weighted sum estimate for steviol equivalents, 10 the consumption would be less than 1.66 mg/kg bw/day on a steviol equivalents basis for any population group, on a worst-case scenario basis. These calculations are summarized in Table 5. Table 5. Daily Intake of Sweeteners (In Sucrose Equivalents) & Estimated Daily Intakes of High Purity Stevia Pure Preparations Population Group Intakes of Sweeteners Intake of Stevia Pure (g sucrose/kg bw/day) a (mg/kg bw/day) b Intake of Stevia Pure as Steviol Equivalents c Low High Low High Low High Healthy Population Diabetic Adults Healthy Children Diabetic Children a Source: Renwick (2008). b Calculated by dividing the sucrose intake by the average relative sweetness value, 235. c Calculated based on the ratio of molecular weights of rebaudioside A and steviol glycosides, as described above. 10 Calculated by the Expert Panel as a percent of molecular weight of steviol to the molecular weight of rebaudioside A and steviol glycosides (as steviolbioside), on a percent composition basis. The total steviol glycosides content was assumed to be 100%. All steviol glycosides, with the exception of Reb A, were treated as steviolbioside for calculation purposes. GRAS ASSOCIATES, LLC Page 23 of 45

31 Based on the totality of dietary intake considerations presented above, the intake estimates are viewed as being conservative. When comparing these EDI assessments for steviol glycosides, we see that total daily consumption of the steviol glycosides and Reb A for defined food uses and as a general purpose sweetener is expected to be substantially less than the acceptable daily intake values discussed at length in Section VI.B. C. Other Information on Human Exposure to Stevia: Use as Food Ingredient & Other Uses For about 25 years, consumers in Japan and Brazil, where stevia has long been approved as a food additive, have been using stevia extracts as non-caloric sweeteners. 11 It was previously reported that 40% of the artificial sweetener market in Japan is stevia based and that stevia is commonly used in processed foods in Japan (Lester, 1999). Although there are no reported uses of rebaudioside A as a dietary supplement, use of steviol glycoside as a dietary supplement is presently permitted in the US, Canada, Australia, and New Zealand, and as a natural health product in Canada. It has wide use in China and Japan in food and in dietary supplements. In 2005, it was estimated that sales of stevia in the US reached $45 million (The Food Institute Report, 2006). More recent reports of consumption figures for stevia reveal pronounced increases in global consumption. Worldwide, Zenith International estimates stevia sales of 3,500 metric tons in 2010, which represents a 27% increase over 2009 figures. The market value is estimated to have increased to $285 million (Zenith, 2011). In 2013, worldwide sales of stevia were reported to reach 4,100 tons which represents a 6.5% increase over 2011 figures, and this corresponds to an overall market value of $304 million (Zenith, 2013). In October 2014, Zenith International reported that worldwide stevia sales were on course to increase 14% to 4,670 tons, associated with a market value of $336 million (Zenith, 2014). Furthermore, it has been projected that the total market for stevia in 2017 will be 7,150 tons with an associated market value of $578 million (Zenith, 2014). Hawke (2003) reported that stevia is commonly used as a treatment for type 2 diabetes in South America. However, for its therapeutic effects, elevated doses in the range of 1 g/person/day or more were reported to be necessary (Gregersen et al., 2004). V. SAFETY INVESTIGATIONS FOR STEVIOL GLYCOSIDES A. Safety Considerations for Steviol Glycosides: Reports & Reviews by Expert Bodies & Other Scientists PAS s high purity steviol glycosides preparations contain rebaudioside A as the major component, with not less than 95% total steviol glycosides. The extensive toxicology and clinical data that exist for preparations of purified steviol glycoside and purified Reb A are applicable to this safety assessment. This is further supported by the fact that EFSA (2010) considers that the results of 11 See Raintree Nutrition Tropical Plant Database. At: -.U6Bl-aiIuTs. Accessed June 30, GRAS ASSOCIATES, LLC Page 24 of 45

32 toxicology studies on either stevioside or rebaudioside A are applicable to the safety assessment of steviol glycosides, as both rebaudioside A and stevioside are metabolized and excreted by similar pathways, with steviol being the common metabolite for both. Much of the supporting evidence with respect to the safety of purified steviol glycoside is derived from pharmacokinetic studies on purified steviol glycosides, which were largely composed of mixtures that were predominately stevioside and Reb A. These studies include a complete battery of toxicology and clinical studies on steviol glycosides, and evidence of poor GI absorption (Gardana et al., 2003; Geuns and Pietta, 2004; Koyama et al., 2003a) of steviol glycosides in the upper GI tract in concert with the conversion of the steviol glycosides to steviol by normal flora of the lower GI tract (Koyama et al., 2003b, Renwick and Tarka, 2008). Additional studies (Hutapea et al., 1997; Geuns et al., 2004) report that human digestive enzymes are not capable of hydrolyzing β-glycosidic bonds, and, thus, steviol glycosides are not digested in the upper gastrointestinal tract. Steviol is absorbed, but is rapidly converted to glucuronides that are subsequently excreted in the urine or eliminated by the enterohepatic circulation. Because of the structural similarity, it is reasonable to expect that Reb A is not absorbed in the GI tract but is similarly converted to steviol by the normal floral of the lower GI tract. Stevia and steviol glycosides have been extensively investigated for their biological, toxicological, and clinical effects (Carakostas et al., 2008; Geuns, 2003; Huxtable, 2002). Additionally, the national and international regulatory agencies have thoroughly reviewed the safety of stevia and its glycosides. Most notably, over the years, JECFA has evaluated purified steviol glycosides multiple times (WHO, 2000, 2006, 2007, 2008), and this has been summarized in Section II.C. FSANZ (2008) also evaluated steviol glycosides for use in food. The JECFA reviews, as well as the other reviews completed before 2008, primarily focused on mixtures of steviol glycosides typically and were not specific for purified rebaudioside A. From the safety perspective, some of the earliest studies on steviol glycosides were of limited value as the actual compositions of materials investigated and their questionable purities undermined drawing firm toxicological conclusions. These early studies reported a decrease in fertility with crude stevia preparations and increased mutagenic activity of the principal metabolite, steviol. Based on these and other questions raised about safety by studies with materials of lesser purity and by studies with unusual protocols in in vivo and in in vitro systems usually employing high doses or high concentrations of test materials, FDA was reluctant to authorize the food use of stevia. These concerns included renal toxicity, effects on glucose metabolism, and inhibition of mitochondrial enzymes. Over the last decade and a half, the safety of steviol glycosides and rebaudioside A in particular have been extensively investigated employing comprehensive and modern toxicology protocols using scientifically accepted dosing regimens of purified and standardized test substances. In the most recent and well-documented chronic toxicity study (additional study details were presented to JECFA in 2006), the apparent no observed adverse effect level (NOAEL) in F344 rats was the dietary level of 2.5% (test sample purity 96%, Toyoda et al., 1997). At 5% of the diet, statistically significant decreases in body weight, percent survival and kidney weight were noted. The author attributed these effects to various factors. The decrease in body weight was attributed to an inhibition of glucose utilization. The decrease in survival seemed to have been caused by an unusual late onset of large granular lymphocyte leukemia in high dose males. The authors reported that this tumor is rather common in F344 rats and that the overall incidence in male rats was actually within the historical control range experienced in the laboratory where studies were GRAS ASSOCIATES, LLC Page 25 of 45

33 conducted. The authors attributed the decrease in kidney weight as probably due to a decrease in chronic inflammation found in the histopathological examination relative to control animals. Reproductive toxicity studies were also conducted with orally administered purified stevioside. No effect on fertility or reproductive parameters was seen in a three-generation study in hamsters at doses up to 2500 mg/kg/day (Yodyingyuad and Bunyawong, 1991). There was an absence of statistically significant effects at doses up to 3% (equivalent to 3000 mg/kg bw/day; sample purity 96%; Mori et al., 1981). Similar results were observed in an additional rat study that was reviewed by Geuns (2003) where limited information is available in English (Usami et al., 1995). In a more recent study, no effects on pregnancy or developmental parameters were observed in Swiss albino mice with stevioside or aqueous stevia extract at doses up to 800 mg/kg bw/day in female mice (Kumar and Oommen, 2008). JECFA encouraged the further elucidation of clinical effects on blood pressure and glucose metabolism on hypertensive and diabetic individuals, respectively, in parallel with normal human subjects. Barriocanal et al. (2006, 2008) investigated the effect of steviol glycosides on subjects with diabetes in a double-blind, placebo-controlled study. Subjects with type 1 or type 2 diabetes were given 250 mg of a product containing 91.7% total steviol glycosides (64.5% stevioside and 18.9% reb A) three times daily for 3 months. There were no significant changes in systolic or diastolic blood pressure, glycated haemoblobin (HbA1c), blood lipids, or renal or hepatic function at the conclusion of the study (Barriocanal et al., 2006, 2008). By 2006, sufficient data were generated for JECFA to satisfactorily establish a temporary ADI, which was finalized in Since the JECFA evaluation (WHO, 2008), over 30 GRAS notifications for steviol glycosides or enzyme modified steviol glycosides have been submitted to FDA, all of which were determined to be GRAS based largely on the ADI established by JECFA. To date, 32 of the submitted notifications have had "no questions" letters of response from FDA (see Table 1). More detailed discussion of reviews on safety of steviol glycosides by expert bodies such as JECFA, FSANZ and EFSA are summarized in Appendix J. A more detailed review of the toxicology studies on steviol, the principal metabolite of steviol glycosides, can be found in Appendix K. A more detailed review of the toxicology studies on steviol glycosides appears in Appendix L. B. Safety Information for Rebaudioside A Since 2008, several well-designed toxicology studies that followed the current regulatory and scientific guidelines for such studies have been reported on purified rebaudioside A, although it is uncertain whether or not these studies were considered by JECFA during its 2008 deliberations. These recent investigations included additional subchronic studies in rats and one in dogs, mutagenicity studies, reproduction and developmental studies in rats, and comparative pharmacokinetic studies with stevioside in rats and humans, as well as additional clinical studies. These studies confirm that rebaudioside A is metabolized similarly to other steviol glycosides, and they exhibited an absence of toxicological effects in the key studies reviewed by JECFA. It should be noted that rebaudioside A, as the steviol glycoside with high sweetness intensity and relatively high prevalence in the stevia leaves, remains an active topic of scientific research. For example, two studies found in a recent literature search examined the anti-hyperglycemic activity of GRAS ASSOCIATES, LLC Page 26 of 45

34 rebaudioside A in diabetic rats (Saravanan et al., 2012; Saravanan and Ramachandran, 2013). These investigators found that the effects of streptozotocin-induced diabetes on glucose and insulin levels were at least partially reversed in a dose-dependent manner with oral administration of rebaudioside A at doses in the range of mg/kg bw. In the second study, the administration of 200 mg/kg bw Reb A to diabetic rats normalized levels of plasma glucose, insulin, lipid peroxidation products, enzymatic, non-enzymatic antioxidants and lipids (Saravanan and Ramachandran, 2013). The doses used are times higher than expected from the use of rebaudioside A as a sweetener (Saravanan et al., 2012). The known anti-hyperglycemic activity of steviol glycosides led JECFA to require clinical studies at reasonably high doses to show that at levels used in food there would be no effect on glucose homeostasis or blood pressure in human consumers. The clinical studies described below on rebaudioside A (Maki et al., 2008a, b) demonstrate the lack of these pharmacological effects of rebaudioside A at expected levels of consumption. Based on the presumption that rebaudioside A is not appreciably absorbed from the GI tract and it is similarly converted to steviol by intestinal flora, the safety review of purified rebaudioside A can be further supported by the large body of published evidence supporting the safety of purified steviol glycosides extracts. Steviol is absorbed from the colon, subjected to glucuronidation in the liver, and excreted via bile primarily as steviol glucuronide in the feces of rats or the urine of humans. The differences in the route of elimination are due to the lower molecular weight thresholds for biliary excretion in rats (325 Da) compared to humans (500 to 600 Da). Although the primary routes of elimination of steviol glucuronide differ between rats and humans, the metabolisms of modified and non-modified steviol glycosides and pharmacokinetics are quite similar, which confirms that the rat is an acceptable model for risk assessment in humans (Roberts and Renwick, 2008; Wheeler, et al., 2008). There is an extensive database of literature on steviol glycosides extracts already in the published literature, along with in-depth reviews in numerous GRAS submissions. JECFA (WHO, 2008), after several years of review, established an ADI of 4 mg/kg bw expressed as steviol equivalents, based on studies on test samples of steviol glycosides with a minimum purity of 95% expressed on a dry weight basis. The critical studies leading to this decision included a chronic study in rats (Toyoda, et al. 1997), which indicated a lack of effects or carcinogenic activity at the highest doses, along with a series of clinical studies conducted at 11 mg/kg bw, which ruled out effects on blood glucose and blood pressure. A more detailed review of the studies conducted on rebaudioside A can be found in Appendix M. VI. GRAS CRITERIA & PANEL SAFETY FINDINGS A. GRAS Criteria FDA defines safe or safety as it applies to food ingredients as: reasonable certainty in the minds of competent scientists that the substance is not harmful under the intended conditions of use. It is impossible in the present state of scientific GRAS ASSOCIATES, LLC Page 27 of 45

35 knowledge to establish with complete certainty the absolute harmlessness of the use of any substance. 12 Amplification is provided in that the determination of safety is to include probable consumption of the substance in question, the cumulative effect of the substance and appropriate safety factors. It is FDA s operational definition of safety that serves as the framework against which this evaluation is provided. Furthermore, in discussing GRAS criteria, FDA notes that: General recognition of safety requires common knowledge about the substance throughout the scientific community knowledgeable about the safety of substances directly or indirectly added to food. General recognition of safety through experience based on common use in food prior to January 1, 1958, shall be based solely on food use of the substance prior to January 1, 1958, and shall ordinarily be based upon generally available data and information. 13 FDA discusses in more detail what is meant by the requirement of general knowledge and acceptance of pertinent information within the scientific community, i.e., the so-called common knowledge element, in terms of the two following component elements: 14 Data and information relied upon to establish safety must be generally available, and this is most commonly established by utilizing published, peer-reviewed scientific journals; and There must be a basis to conclude that there is consensus (but not unanimity) among qualified scientists about the safety of the substance for its intended use, and this is established by relying upon secondary scientific literature such as published review articles, textbooks, or compendia, or by obtaining opinions of expert panels or opinions from authoritative bodies, such as JECFA and the National Academy of Sciences. The apparent imprecision of the terms appreciable, at the time, and reasonable certainty demonstrates that the FDA recognizes the impossibility of providing absolute safety in this or any other area (Lu, 1988; Renwick, 1990; Rulis and Levitt, 2009). As noted below, this safety assessment to ascertain GRAS status for high purity steviol glycosides for the specified food uses meets FDA criteria for reasonable certainty of no harm by considering both the technical and common knowledge elements. 12 See 21 CFR 170.3(i). 13 See 21 CFR (a). 14 See Footnote 1. GRAS ASSOCIATES, LLC Page 28 of 45

36 B. Safety Studies of High Purity Steviol Glycosides Because of their sweetness characteristics, steviol glycosides have viable uses as a non-nutritive sweetener in foods. 15 Periodic reviews by JECFA over the years indicate the progress of knowledge on the toxicology of steviol glycosides. As noted above, several early safety-related studies on these compounds were performed on crude extracts of stevia. These studies also included multiple investigations with in vivo and in vitro models which explored the biological activity of stevia extracts at high doses or high concentrations. These early investigations raised several concerns, including impairment of fertility, renal effects, interference with glucose metabolism, and inhibition of mitochondrial enzymes. In recent years, as more and more studies were performed on purified glycosides, the toxicology profile of steviol glycosides eventually proved to be rather unremarkable. The purified materials that were tested largely consisted of stevioside and rebaudioside A which included a number of subchronic, chronic, and reproductive studies conducted on laboratory animals. These studies were well designed with appropriate dosing regimens and adequate numbers of animals to maximize the probability of detection of important effects. Notably, the initially reported concerns related to the effects of stevia leaves or crude extracts on fertility were refuted by the well-designed reproductive studies with purified steviol glycosides. All other concerns failed to manifest themselves at the doses employed in the long-term rat studies. As discussed in Section V and addressed more fully in Appendix J at its 51 st meeting, JECFA determined that there were adequate chronic studies in rats, particularly the study by Toyoda et al. (1997), to establish a temporary ADI of 0-2 mg/kg bw/day with an adequate margin of safety. The committee also critically reviewed the lack of carcinogenic response in well-conducted studies. These studies justified the Committee conclusion that the in vitro mutagenic activity of steviol did not present a risk of carcinogenic effects in vivo and, therefore, all common steviol glycosides that likely share the same basic metabolic and excretory pathway, and that the use of high purity preparations of various steviol glycosides, are safe to use as a sugar substitute. Subsequently, the additional clinical data reviewed by JECFA allowed the Committee to establish a permanent ADI of 0-4 mg/kg bw/day (based on steviol equivalents). The GRAS Expert Panel critically reviewed the JECFA assessment and agrees with the calculation of the ADI for steviol glycosides. Subsequently, several published and unpublished studies (summarized in Appendix M) on purified preparations of rebaudioside A showed an absence of toxicological effects in rats (Curry and Roberts, 2008; Nikiforov and Eapen, 2008) and dogs (Eapen, 2008) in subchronic studies and an absence of reproductive (Curry et al., 2008, Sloter 2008a) and developmental effects (Sloter, 2008b) in rats. Most notably, pharmacokinetic studies in rats (Roberts and Renwick, 2008) and humans (Wheeler et al., 2008) on purified rebaudioside A follow the same pathway of being degraded to steviol by intestinal bacteria with subsequent rapid glucosylation and elimination in urine and feces. The Panel concludes that these studies on rebaudioside A strengthen the 15 It has also been reported that steviol glycosides may have pharmacological properties, which can be used to treat certain disease conditions such as hypertension and type 2 diabetes. Chatsudthipong and Muanprasat (2009) published a comprehensive review where they note that such therapeutic applications have not been firmly established as being due to steviol glycosides. The reviewers point out that the effects occur at higher doses than would be used for sweetening purposes. Furthermore, many effects noted in older studies may have been due to impurities in preparations that do not meet the contemporary purity specifications established by JECFA for use as a sweetener. If oral doses of steviol glycosides impart pharmacological effects, such effects would undoubtedly occur due to actions of the principal metabolite, steviol, but the pharmacological effects of steviol have not been comprehensively investigated. GRAS ASSOCIATES, LLC Page 29 of 45

37 argument that all steviol glycosides which follow the same metabolic pathway are safe at the JECFA established ADI. The Panel has reviewed the findings from human clinical studies. The Panel noted that, regarding the clinical effects reported in humans, in order to corroborate the observations in these studies that these effects of steviol glycosides only occur in patients with either elevated blood glucose or blood pressure (or both), JECFA called for studies in individuals that are neither hypertensive nor diabetic (WHO, 2006). The supplemental data presented to JECFA and also published by Barriocanal et al. (2008) demonstrate the lack of pharmacological effects of steviol glycosides at 11 mg/kg bw/day in normal individuals, or approximately slightly more than 4 mg/kg bw on the basis of steviol equivalents. Clinical studies on purified rebaudioside A showed an absence of effects on blood pressure (Maki, et al., 2008a) and blood glucose levels (Maki et al., 2008b) at doses comparable to the exposure expected in food. The Panel concludes that there will be no effects on blood pressure and glucose metabolism in humans from the use of Stevia Pure in food as a non-nutritive sweetener. JECFA s review also included anticipated dietary patterns and the use concentrations expected in various foods in order to calculate an estimated daily intake (EDI) (WHO, 2003, 2006). Based on the assumption of 100% substitution of steviol glycosides for sucrose, an EDI of 5 mg/kg bw/day of steviol was calculated for US consumption. JECFA noted that the replacement estimates were highly conservative and that this calculated intake of steviol glycosides (as steviol) would more likely be 20% 30% of these values. As summarized in Table 5, the panel used the simplified method to calculate maximum consumption of Stevia Pure. The highest estimated consumption was for healthy children at 4.21 mg/kg bw/day corresponding to 1.66 mg/kg bw/day based on steviol equivalents. The Panel agrees with the JECFA ADI of 4 mg/kg bw/day based on steviol equivalents and notes that the estimates of anticipated dietary intake, as contained in Table 5, are below the ADI. The Panel also noted from a study that DNA damage was seen in a variety of organs as assessed by Comet assay in rats given drinking water containing 4 mg/ml steviol glycosides for up to 45 days (Nunes et al., 2007a). This study is summarized in Appendix L. Several experts in the field have since questioned the methodology used in this study (Geuns, 2007; Williams, 2007; Brusick, 2008). The Panel has reviewed the cited publications, along with the responses made by the authors (Nunes et al., 2007b; Nunes et al., 2007c), and concurs with the challenges to the methodology utilized by Nunes et al., 2007a, thereby discounting the validity and importance of this study. In a recent review, Urban et al. (2013) examined the extensive genotoxicity database on steviol glycosides because some concern has been expressed in two recent publications (Brahmachari et al., 2011 and Tandel, 2011) in which the authors concluded that additional testing is necessary to adequately address the genotoxicity profile. The review aimed to address this matter by evaluating the specific genotoxicity studies of concern, while evaluating the adequacy of the database that includes more recent genotoxicity data not noted in these publications. The results of this literature review showed that the current database of in vitro and in vivo studies for steviol glycosides is robust and does not indicate that either stevioside or rebaudioside A are genotoxic. This finding, combined with a paucity of evidence for neoplasm development in rat bioassays, establishes the safety of all steviol glycosides with respect to their genotoxic/carcinogenic potential. GRAS ASSOCIATES, LLC Page 30 of 45

38 In another study with stevioside in rats, tartrate-resistant alkaline phosphatase (TRAP) levels were measured and found to be significantly decreased at doses as low as 15 mg/kg bw (Awney et al., 2011). TRAP is an enzyme that is expressed by bone-resorbing osteoclasts, inflammatory macrophages, and dendritic cells. This enzyme was not measured in any previous toxicology studies on steviol glycosides, nor has it been adequately vetted for application in toxicological studies. Critical reviews of the this study by Carakostas (2012) and Waddell (2011) revealed a poor study design that included: insufficient numbers of animals; group-housing with the potential for stress-related changes; unreliable access to steviol via drinking water resulting in suspect dosing calculations in group-housed cages; no indication of fasting prior to blood collection (which affects many chemistry and hematological values); no urine collection; and no histopathological evaluations for confirmation of findings beyond the controls. Additionally, the report did not adequately describe mean or individual organ weight data, and it lacked comparison of study findings against laboratory historical control data. In summary, the Expert Panel agrees with the safety conclusions of the 32 GRAS Expert Panels in the notifications previously submitted to FDA that resulted in "no questions" responses from FDA (as summarized in Table 1), JECFA (WHO, 2006; WHO, 2008), and Renwick (2008) that there are a sufficient number of good quality health and safety studies to support the determination that the intended use of purified preparations of steviol glycosides, including rebaudioside A, when added to food at levels up to full replacement of sucrose on a sweetness equivalency basis, meets FDA s definition of safe. In addition, the Panel has compared the specifications of PAS s high purity Stevia Pure steviol glycosides preparation to the composition of the test materials used in all the published studies. The Panel agrees that PAS s high purity steviol glycosides preparation is sufficiently similar to those used in all key studies reviewed by JECFA, and those on rebaudioside A subsequently reviewed by FDA, and there is no need for further studies to be conducted on the Stevia Pure product. The Panel has also reviewed the expected levels of dietary intake (see Table 5) and agrees that there is sufficient information to conclude that the subject high purity Stevia Pure steviol glycoside product can be safely used as a table top sweetener and as a general purpose non-nutritive sweetener in various foods other than infant formulas and meat and poultry products. C. Common Knowledge Elements of GRAS Determinations The first common knowledge element for a GRAS determination is that data and information relied upon to establish safety must be generally available; this is most commonly established by utilizing published, peer-reviewed scientific journals. The majority of studies reviewed as part of this safety assessment have been published in the scientific literature as reported in Section V. The majority of the literature relied upon by JECFA has also been published---most importantly the chronic rat studies on steviol glycosides. JECFA did make limited use of unpublished studies, and they were summarized in the two JECFA monographs. Moreover, JECFA publicly releases the results of their safety reviews, and their meeting summaries and monographs are readily available on their website. Thus, these studies become generally available to the scientific community. JECFA reviewed only a limited number of studies conducted specifically on rebaudioside A. The collection of supporting data on rebaudioside A has been enhanced by a series of studies published during 2008 and cited earlier. The clinical studies that address JECFA s concern on unwanted pharmacological effects with steviol glycosides (Barriocanal et al., 2008) and with rebaudioside A (Maki et al., 2008a, b) are also published in the peer-reviewed scientific literature. GRAS ASSOCIATES, LLC Page 31 of 45

39 The Panel recognizes that the safety of steviol glycosides in human foods has been the subject of interest for many years. In addition to the reported substantial history of consumption of stevia, especially in South America and Asia, many scientific studies have been conducted and published. Some of the earlier studies have raised concerns about the safety, and the Expert Panel has given careful attention to such concerns. The overriding evidence has diminished the Panel s concerns based on improved study designs, better study execution, or simply updated investigations that better reflect state-of-the art toxicological principles and findings. The remaining common knowledge element for a GRAS determination is that there must be a basis to conclude that there is consensus among qualified scientists about the safety of the substance with its intended food use. The JECFA opinion largely meets the common knowledge test on its own. The Panel is cognizant of the scientific rigor and broad base of scientific expertise that resides with the prestigious JECFA. JECFA is composed of expert scientists from various regulatory agencies around the world, as well as other scientists chosen because of their specific expertise on various classes of food ingredients. In addition, FDA participated in the JECFA deliberations. The JECFA conclusion has been reviewed and validated by other respected regulatory agencies including FSANZ, the Switzerland Federal Office of Public Health, France s Agence Francais De Securite Sanitaire Des Alimenta, Health Canada, and others (FSANZ, 2008; Switzerland Federal Office of Public Health, 2008; AFSSA, 2009; Health Canada, 2012; Republic of South Africa Department of Health, 2012a, b; AVA, 2013; FSSAI, 2012; Republic of the Philippines, 2013). Furthermore, the favorable scientific opinion on the safety of steviol glycosides use as a sweetener in foods as issued by EFSA in 2010 reinforces the safety determinations of many other qualified organizations (EFSA, 2010). In addition, a number of individual well-respected scientists have indicated that steviol glycosides are safe for human consumption at doses in the range of the JECFA ADI (Xili et al., 1992; Toyoda et al., 1997; Geuns, 2003; Williams, 2007). The common knowledge element has been embellished by the many respected scientists that participated in the Cargill-sponsored research conducted on rebaudioside A, most notably Brusick (2008) and Renwick (2008). An assertion of general recognition of safety was made by Carakostas et al. (2008). The authors of a recent review of the genetic toxicology database of steviol glycosides concluded that the available data "establish the safety of all steviol glycosides with respect to their genotoxic/carcinogenic potential" (Urban et al., 2013). We also note that, since December 2008, nearly three dozen GRAS notifications have been submitted to FDA for stevia-derived sweetener products, and FDA s detailed reviews have yielded no questions letters in 32 cases to date. In summary, there are many diverse groups of scientists from all corners of the globe that together provide strong fulfillment of the consensus requirement. Of particular significance, from the perspective of establishing consensus for the safety of high purity steviol glycosides, are the 32 GRAS notifications with no questions determinations by FDA since 2008 (see Table 1). While the scientific conclusions are not unanimous regarding the safe human food uses of steviol glycosides, the Expert Panel believes that a wide consensus does exist in the scientific community to support the GRAS conclusion on high purity rebaudioside A and high purity steviol glycosides, as described in this notification. The broader scientific community has concluded that past concerns expressed by others over the years (Huxtable, 2002), and earlier safety issues noted by FDA, have been resolved by newer data on more purified test materials and the rigid specifications for purity published by JECFA for steviol glycosides, including rebaudioside A. Indeed, scientists from FDA are members of JECFA and have not objected to the safety decision on steviol glycosides. There GRAS ASSOCIATES, LLC Page 32 of 45

40 is also a wider consensus that the body of new research on rebaudioside A is sufficient, as opposed to the small group of scientists that argue that more studies need to be done before the sweetener is made available in the US. VII. CONCLUSIONS 16 In consideration of the aggregate safety information available on Reb A and the naturally occurring steviol glycosides, the Panel concludes that Stevia Pure, a high purity steviol glycoside preparation with Reb A as the primary component, is safe for use as a general purpose non-nutritive sweetener in foods other than infant formulas and meat and poultry products. Based on the information that Reb A displays similar pharmacokinetics compared with the other naturally occurring steviol glycosides, the JECFA ADI for steviol glycosides of 4 mg/kg bw/day (as steviol equivalents) can be applied to Reb A. Based on published dietary exposure data for other approved sweeteners and adjusting for relative sweetness intensity, the intake of rebaudioside A was estimated for healthy non-diabetic children and adults, and diabetic children and adults. The estimated intakes of Stevia Pure for several population groups summarized in Table 5 are no greater than 4.21 mg/kg bw/day or 1.66 mg/kg bw/day as steviol equivalents. This calculated daily intake is below the ADI of 4 mg/kg bw expressed as steviol equivalents as established by JECFA. The Panel finds that the dietary levels from anticipated food consumption will not exceed the ADI when Stevia Pure is used as a general non-nutritive sweetener. The Panel also finds that the 95% purity specification for steviol glycosides is sufficient in view of the accepted JECFA specification for 95% purity for naturally occurring steviol glycosides. The Panel concludes that high purity steviol glycosides consisting primarily of Reb A, as described throughout the subject notification and as manufactured by PAS, are appropriate food grade substances, and that adverse pharmacological effects are not likely to occur at this designated ADI level. Furthermore, even high consumers of steviol glycosides are not likely to exceed this specified ADI. Therefore, the Panel concludes that Stevia Pure, when consumed in foods as described within this GRAS notification, is generally recognized as safe (GRAS) within the meaning of the Food, Drug, and Cosmetic Act. 16 The detailed educational and professional credentials for two of the individuals serving on the Expert Panel can be found on the GRAS Associates website at Drs. Kraska and McQuate worked on GRAS and food additive safety issues within FDA s GRAS Review Branch earlier in their careers and subsequently continued working within this area in the private sector. Dr. Kapp s curriculum vitae can be accessed at All three panelists have extensive technical backgrounds in the evaluation of food ingredient safety. Each individual has previously served on multiple GRAS Expert Panels. Dr. Kraska served as Chair of the Panel. GRAS ASSOCIATES, LLC Page 33 of 45

41 Productora Alysa SpA s high purity steviol glycosides ( 95%), referred to as Stevia Pure---primarily containing rebaudioside A---when produced in accordance with FDA Good Manufacturing Practices requirements and when meeting at a minimum the JECFA purity specifications for steviol glycosides, are Generally Recognized As Safe when consumed as a nonnutritive sweetener in foods other than infant formulas and meat and poultry products within the JECFA ADI of 4 mg/kg bw/day on a steviol equivalent basis. In order to remain within the designated ADI, it is important to observe good manufacturing practices principles in that the quantity of a substance added to food should not exceed the amount reasonably required to accomplish its intended technical effect. This declaration has been made in accordance with FDA s standard for food ingredient safety, i.e., reasonable certainty of no harm under the intended conditions of use. (b) (6) (b) (6) Richard C. Kraska, Ph.D., DABT Chair (b) (6) Robert S. McQuate, Ph.D. Robert W. Kapp, Jr., Ph.D., Fellow ATS, ERT (UK) Date: November 5, 2014 GRAS ASSOCIATES, LLC Page 34 of 45

42 VIII. REFERENCES THIS IS A COMPREHENSIVE REFERENCE LIST WHICH ALSO ENCOMPASSES REFERENCES CITED IN VOLUME 2 - APPENDICES I, J, K, L & M AFSSA, Agence Francais De Securite Sanitaire Des Aliments, See AFSSA website at: Accessed 6/30/14. Also see: Accessed 6/30/14. Akashi, H., Yokoyama, Y., Security of dried-leaf extracts of Stevia. Toxicological tests. Food Industry 18, Anonymous, 2004a. Evaluation of the ingestion of stevioside, orally, in humans through a randomized clinical study of the type blind double. Subproject 1: Investigation of the hypolipidemic and hepatotoxic potential of the stevioside using doses usually consumed of the stevioside as sweetener. Unpublished report of a study conducted by the State University of Maringá and the Academical Hospital of Maringá. Submitted to WHO by State University of Campinas, Brazil. Anonymous, 2004b. Evaluation of the ingestion of stevioside, orally, in humans through a randomized clinical study of the type blind double. Subproject 2: Investigation of the antihypertensive potential, insulintropic, hypolipidemic and toxic (hepatotoxic potential, nephrotoxic and of interference in the endocrine system) of the stevioside using doses above the usually consumed, but previously respecting values used in humans. Unpublished report of a study conducted by the State University of Maringá and the Academical Hospital of Maringá. Submitted to WHO by State University of Campinas, Brazil. AVA, Agri-Food & Veterinary Authority of Singapore, Food Additives Permitted Under the Singapore Food Regulations. Available online: Accessed June 30, Awney, H.A., Massoud, M.I. El-Maghrabi, S., Long-term feeding effect of stevioside sweetener on some toxicological parameters of growing male rats. Journal of Applied Toxicology, Online Publication: 19 NOV 2010; DOI: /jat Aze, Y., Toyoda K., Imaida, K., Hayashi, S., Imazawa, T., Hayashi, Y., Takahashi, M., Subchronic oral toxicity study of stevioside in F344 rats. Bull Natl Inst Hyg, In Japanese. Barriocanal, L., Palacios, M., Benitez, S., Canete, F., Jimenez, J.T., Jimenez, N. & Rojas, V., Lack of pharmacological effect of steviol glycosides as a sweetener in humans. Studies on repeated exposures in normotensive and hypotensive individuals and Type 1 and Type 2 diabetes. Presented at the 2nd International Symposium on Stevia, November Barriocanal, L.A., Palacios, M., Benitez, G., Benitez, S., Jimenez, J.T., Jimenez, N., Rojas, V., Apparent lack of pharmacological effect of steviol glycosides used as sweeteners in humans. A pilot study of repeated exposures in some normotensive and hypotensive individuals and in Type 1 and Type 2 diabetics. Regul Toxicol Pharmacol 51, BioVittoria, GRAS Notification for Luo Han Guo Extract Submitted to the US Food and Drug Administration, Washington, DC as GRAS Notification 301. See FDA website at Accessed June 30, GRAS ASSOCIATES, LLC Page 35 of 45

43 Brahmachari, G., Mandal, L.C., Roy, R., Mondal, S., Brahmachari, A.K., Stevioside and related compounds molecules of pharmaceutical promise: a critical overview. Arch Pharm (Weinheim); 344(1):5-19. Brandle, J.E., Starratt, A.N., Gijzen, M., Stevia rebaudiana: its agricultural, biological and chemical properties. Can J of Plant Sci 78, Bridel, M., Lavielle, R., Le principe a saveur sucre e du Kaa -he -e (Stevia rebaudiana) Bertoni. Bull Soc Chim Biol, 13, Brusick, D.J., A critical review of the genetic toxicity of steviol and steviol glycosides. Food Chem Toxicol, 46(7)(Suppl.1), S83-S91. Carakostas, M.C., Curry, L.L., Boileau, A.C., Brusick, D.J., Overview: the history, technical function and safety of rebaudioside A, a naturally occurring steviol glycoside, for use in food and beverages. Food Chem Toxicol, 46(7)(Suppl.1), S1-S10. Carakostas, M., Letter to the editor: Long-term feeding effects of stevioside sweetener on some toxicological parameters of growing rats. J Appl Toxicol 32(2): Cargill, GRAS Notification for Rebaudioside A, Submitted to the US Food and Drug Administration, Washington, DC. Identified as GRAS Notification 253. See FDA website at (Accessed June 30, 2014). Cavalcante da Silva, G.E., Assef, A.H., Albino, C.C., Ferri, L.A.F., Tasin, G., Takahashi, M.H., Filho, W.E.,Bazotte, R.B., Investigation of the tolerability of oral stevioside in Brazilian hyperlipidemic patients. Braz Arch Biol Technol, 49(4), CCFA, Codex Committee on Food Additives, Proposals for Additions and Changes to the Priority List of Food Additive Proposed for Evaluation by JECFA (CL 2008/26-FA). Codex Alimentarius Commission E, FAO/WHO/JECFA CX/FA 09/41/11. Chan, P., Tomlinson, B., Chen, Y., Liu, J., Hsieh, M., Cheng, J., A double-blind placebo-controlled study of the effectiveness and tolerability of oral stevioside in human hypertension. Br J Clin Pharmacol, 50, Chang, S.S., Cook, J.M., Stability studies of stevioside and rebaudioside A in carbonated beverages. J Agric Food Chem 31, Chatsudthipong, V., Muanprasat, C., Stevioside and related compounds: therapeutic benefits beyond sweetness. Pharmacol Therapeutics, Jan: 121: Clarke, J.J., In Vitro Mammalian Cell Gene Mutation Test (L5178Y/TK+/- Mouse Lymphoma Assay) [with Rebaudioside A]. BioReliance, Rockville, MD. Unpublished Report,Study Number AB21TG.704.BTL. Clos, J.F., DuBois, G.E., Prakash, I., Photostability of rebaudioside A and stevioside in beverages. J Agric Food Chem, 56, Compadre, C.M., Hussain, R.A., Nanayakkara, N.P., Pezzuto, J.M., Kinghorn, A.D., Mass spectral analysis of some derivatives and in vitro metabolites of steviol, the aglycone of the natural sweeteners, stevioside, rebaudioside A, and rubusoside. Biomed Environ Mass Spectrom, 15, GRAS ASSOCIATES, LLC Page 36 of 45

44 Curi, R., Alvarez, M., Bazotte, R.B., Botion, L.M., Godoy, J.L., Bracht, A., Effect of Stevia rebaudiana on glucose tolerance in normal adult humans. Braz J Med Biol Res 19, In Portuguese, English abstract only) Curry, L.L., Roberts, A., Subchronic toxicity of rebaudioside A. Food Chem Toxicol, 46(7)(Suppl. 1), S11-S20. Curry, L.L., Roberts, A., Brown, N., Rebaudioside A: two-generation reproductive toxicity study in rats. Food Chem Toxicol, 46(7)(Suppl. 1), S21-S30. Eapen, A.K., A 90-Day Oral (Dietary) Toxicity Study of Rebaudioside A in Rats. WIL Research Laboratories, LLC. Unpublished Report, Study Number WIL Eapen, A.K., A 6-Month Oral (Dietary) Toxicity Study of Chrysanta 99-P in Beagle Dogs. WIL Research Laboratories, LLC. Unpublished Report, Study Number WIL European Commission, 1999a. Opinion on Stevia Rebaudiana Bertoni plants and leaves. Scientific Committee on Food (CS/NF/STEV/3 Final, 17 June 1999). European Commission, 1999b. Opinion on stevioside as a sweetener. Scientific Committee on Food (CS/ADD/EDUL/167Final, 17 June 1999). EFSA, European Food Safety Authority, Scientific Opinion on the safety of steviol glycosides for the proposed uses as a food additive. EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS). EFSA Journal 8 (4), 1537, pp EFSA, European Food Safety Authority, 2011a. Scientific Opinion on Flavouring Group Evaluation 310 (FGE.310): Rebaudioside A from chemical group 30. EFSA Journal 9(5):2181, pp1-37. EFSA, European Food Safety Authority, 2011b. Revised exposure assessment for steviol glycosides for the proposed uses as a food additive. EFSA Journal 9 (1), 1972, pp EU, Official Journal of the European Union. Commission Regulation (EU) No. 1131/2011 of November 11, Previously available at: FAO, Food and Agriculture Organization, 2007a. Steviol Glycosides. FAO JECFA Monographs 4. FAO, Food and Agriculture Organization, 2007b. Chemical and Technical Assessment: Steviol Glycosides. Revised by Paul M. Kuznesof, PhD for the 68 th JECFA Meeting. FAO, Food and Agriculture Organization, Steviol Glycosides. FAO JECFA Monographs 5. FAO, Food and Agriculture Organization, Steviol Glycosides. FAO JECFA Monographs 10; also see Accessed June 30, FAO/WHO, List of Substances Scheduled for Evaluation and Request for Data. Food and Agriculture Organization of the United Nations/ World Health Organization, Joint FAO/WHO Expert Committee on Food Additives. Seventy-third meeting Food additives and Contaminants.Geneva, 8 to 17 June Published 14 September Previously available at: GRAS ASSOCIATES, LLC Page 37 of 45

45 FCC, Rebaudioside A monograph. Food Chemicals Codex (7th Ed.) First Supplement, National Academy Press (NAP); Washington, DC, pp Ferri, L.A.F., Alves-Do-Prado, W., Yamada, S.S., Gazola, S., Batista, M.R., Bazotte, R.B., Investigation of the antihypertensive effect of oral crude stevioside in patients with mild hypertension. Phytother Res, 20, FoodNavigator, Codex approval will open new stevia markets: PureCircle. See Accessed June 30, FoodNavigator, Thailand s stevia approval poses challenge for Indonesian Regulators. See Accessed June 30, FSANZ, Food Standards Australia New Zealand, Final Assessment Report, Application A540, Steviol Glycosides as Intense Sweeteners. FSANZ, Food Standards Australia New Zealand, Assessment Report. Application A1037. Steviol Glycosides Increase in Permitted Use Levels. FSANZ, Food Standards Australia New Zealand, Approval report Application A1037 Steviol Glycosides Increase in Permitted Use Levels. (13 May 2011). df. Accessed June 30, FSSAI, Food Safety and Standards Authority of India, Minutes of the Tenth Meeting of Food Authority held on 20 th September, 2012 at 1100 hrs at FDA Bhavan, New Delhi. Available online: Accessed June 30, Gardana, C., Simonetti, P., Canzi, E., Zanchi, R., Pieta, P., Metabolism of stevioside and rebaudioside A from Stevia rebaudiana extracts by human microflora. J Agri Food Chem, 51, Geuns, J.M.C., Molecules of interest stevioside. Phytochemistry 64, Geuns, J.M.C., Augustijns, P., Mols, R., Buyse, J.G., Driessen, B., 2003a. Metabolism of stevioside in pigs and intestinal absorption characteristics of stevioside, rebaudioside A and steviol. Food Chem Toxicol, 41, Geuns, J.M.C., Malheiros, R.D., Moraes, V.M.B., Decuypere, E.M.P., Compernolle, F. Buyse, J.G., 2003b. Metabolism of stevioside by chickens. J Agri Food Chem 51, Geuns, J.M.C., Pietta, P., Stevioside metabolism by human volunteers. Unpublished report from Laboratory Functional Biology, Kuleuven, Leuven Belgium and ITB-CNR, Segrate (MI), Italy. Submitted to WHO by the Federal Ministry of Social Affairs, Public Health and the Environment; Belgium. Cited in WHO, Geuns, J.M., Buyse, J., Vankeirsbilck, A., Temme, E.H., Compernolle, F., Toppet, S., Identification of steviol glucuronide in human urine. J Agric Food Chem 5: Geuns, J.M.C., Letter to the Editor: Comments to the paper by Nunes et al., Analysis of genotoxic potentiality of stevioside by comet assay. Food Chem Toxicol 45 (2007) GRAS ASSOCIATES, LLC Page 38 of 45

46 Gregersen, S., Jeppensen, P.B., Holst, J.J., Hermansen, K., Antihyperglycemic effects of stevioside in Type 2 diabetic subjects. Metabolism, 53, Hanson, J.R., De Oliveira, B.H., Stevioside and related sweetditerpenoid glycosides. Nat Prod Rep, 10, Hawke, J., The Bittersweet Story of the Stevia Herb. Nexus Magazine, Vol.10, No. 2. Available at: Assessed July 23, Health Canada, Revised Guidelines for the Use of Stevia in Natural Health Products. Available at: Accessed June 30, Health Canada, Information and Consultation Document on Health Canada s Proposal to Allow the Use of the Food Additive Steviol Glycosides as a Table-Top Sweetener and as a Sweetener in Certain Food Categories. Bureau of Chemical Safety, Food Directorate, Health Products and Food Branch. Health Canada, List of Permitted Sweeteners (Lists of Permitted Food Additives). Available at: (Accessed on 9/16/14). Hong Kong Centre for Food Safety, Legislative council brief. Sweeteners in food (amendment) regulation. May. egco_brief.pdf. Accessed June 30, Hsieh, M., Chan, P., Sue, Y., Liu, J., Liang, T., Huang, T., Tomlinson, B., Chow, M.S., Kao, P., Chen, Y., Efficacy and tolerability of oral stevioside in patients with mild essential hypertension: A two-year, randomized, placebocontrolled study. Clin. Therap. 25, Hutapea, A.M., Toskulkao, C., Buddahasukh, D., Wilairat, P., Glinsukon, T., Digestion of stevioside, a natural sweetener, by various digestive enzymes. J Clin Biochem Nutr 23, Hutapea, A.M., Tolskulkao, C., Wilairat, P., Buddhasukh, D., High-performance liquid chromatographic separation and quantitation of stevioside and its metabolites. J Liq Chromatogr & Rel Technol 22, Huxtable, R.J., Pharmacology and toxicology of stevioside, rabaudioside A, and steviol. In: Kinghorn, A.D., (Ed.), Stevia: The Genus of Stevia, Taylor and Francis Inc., New York. Ishidate, M., Sofuni, T., Yoshikawa, K., Hayashi, M., Nohmi, T., Sawada, M., Matsuoka, A., Primary mutagenicity screening of food additives currently used in Japan. Food Chem Toxicol 22, Jeppesen, P.B., Barriocanal, L., Meyer, M.T., Palacios, M., Canete, F., Benitez, S., Logwin, S., Schupmann, Y., Benitez, G. & Jimenez, J.T., Efficacy and tolerability of oral stevioside in patients with type 2 diabetes: a long-term, randomized, doubleblinded, placebo-controlled study. Diabetologia, 49(Suppl. 1), , Abstract No Kennelly, E.J., Sweet and non-sweet constituents of Stevia rebaudiana (Bertoni). In: Kinghorn, A.D. (Ed.), Stevia, The Genus Stevia. Medicinal and Aromatic Plants Industrial Profiles, Vol. 19. Taylor and Francis Inc., London and New York, pp Kerr, W.E., Mello, M.L.S., Bonadio, E., Mutagenicity tests on the stevioside from Stevia rebaudiana (Bert.) Bertoni. Brazil. J Genetics 1, GRAS ASSOCIATES, LLC Page 39 of 45

47 Kinghorn, A.D., Overview. In: Kinghorn, A.D., (Ed.), Stevia: The Genus Stevia. Medicinal and Aromatic Plants Industrial Profies, Vol. 19. Taylor and Francis, London and NY, pp Kinghorn, A.D., Soejarto, D.D., Intensely Sweet Compounds of Natural Origin. Med Res Rev 9(1), Klongpanichpak, S., Toskulkao, D., Temcharoen, P., Apibal, S., Glinsukon, T., Lack of mutagenicity of stevioside and steviol in Salmonella typhimurium TA98 and TA100. J Med Assoc Thailand 80, Kobayashi, M., Horikawa, S., Degrandi, I. H., Ueno, J. and Mitsuhashi, H Dulcosides A and B, new diterpene glycosides from Stevia rebaudiana. Phytochemistry 16: Kobylewski, S., Eckhert, C.D., Toxicology of Rebaudioside A: A Review. University of California at Los Angeles, unpublished. Available at: Accessed June 30, Koyama, E., Sakai, N., Ohori, Y., Kitazawa, K., Izawa, O., Kakegawa, K., Fujino, A., Ui, M., 2003a. Absorption and metabolism of glycosidic sweeteners of Stevia mixture and their aglycone, steviol in rats and humans. Food Chem Toxicol 41, Koyama, E., Ohori, Y., Kitazawa, K., Izawa, O., Kakegawa, K., Fujino, A., Ui, M. 2003b. In vitro metabolism of theglycosidic sweeteners, Stevia mixture and enzymically modified Stevia in human intestinal microflora. Food Chem Toxicol 41, Krsmanovic, L., Huston, T., Rebaudioside A: Mammalian Erythrocyte Micronucleus Test. BioReliance, Rockville, MD. Unpublished Report, Study Number AB21TG.123.BTL. Kumar, R.D., Oommen, O.V., Stevia rebudiana Bertoni does not produce female reproductive toxic effect: Study in Swiss albino mouse. J Endocrinol Reprod 12, Lester, T., Stevia rebaudiana. The Australian New Crops Newsletter, Issue 11, January Previously available at: Lu, F.C., Acceptable daily intake: inception, evolution and application. Regul Toxicol Pharmacol 8, Maki, K.C., Curry, L.L., Carakostas, M.C., Tarka, S.M., Reeves, M.S., Farmer, M.V., McKenney, J.M., Toth, P.D., Schwartz, S.L., Lubin, B.C., Dicklin, M.R., Boileau, A.C., Bisognano, J.D., 2008a. The hemodynamic effects of rebaudioside A in healthy adults with normal and low-normal blood pressure. Food Chem Toxicol, 46(7)(Suppl.1), S40-S46. Maki, K.C., Curry, L.L., Reeves, M.S., Toth, P.D., McKenney, J.M., Farmer, M.V., Schwartz, S.L., Lubin, B.C., Boileau, A.C., Dicklin, M.R., Carakostas, M.C., Tarka, S.M., 2008b. Chronic consumption of rebaudioside A, a steviol glycoside, in men and women with type 2 diabetes mellitus. Food Chem Toxicol, 46(7)(Suppl.1), S47-S53. Matsui, M., Matsui, K., Kawasaki, Y., Oda, Y., Noguchi, T., Kitagawa, Y., Sawada, M., Hayashi, M., Nohmi, T., Yoshihira, K., Ishidate, M. & Sofuni, T., Evaluation of the genotoxicity of stevioside and steviol using six in vitro and one in vivo mutagenicity assays. Mutagenesis 11, McQuate, R.S., Ensuring the safety of sweeteners from stevia. Food Technology: 65(4). Accessed June 30, GRAS ASSOCIATES, LLC Page 40 of 45

48 Medon, P.J., Pezzuto, J.M., Hovanec-Brown, J.M., Nanayakkara, N.P., Soejarto, D.D., Kamath, S.K., Kinghorn, A.D., Safety assessment of some Stevia rebaudiana sweet principles. Fed Proc 41, Merisant GRAS Notification for Rebaudioside A, Submitted to the US Food and Drug Administration, Washington, DC under the name Whole Earth Sweetener Company LLC. Identified as GRAS Notification 252. Access: Accessed June 30, Mitsuhashi, H., Safety of Stevioside. In: Tama Biochemical Co. Ltd. Report on Safety of Stevia, pp Mori, N., Sakanoue, M., Takeuchi, M., Shimpo, K., Tanabe, T., Effect of stevioside on fertility in rats. J Food Hyg So. Jpn 22, In Japanese. Nakajima, (initials unknown), 2000a. Chromosome aberration assay of rebaudioside A in cultured mammaliancells. Test number 5001 ( ). Unpublished report of a study conducted at the Biosafety Research Center, Japan. Submitted to WHO by Ministry of Health and Welfare, Japan. Nakajima, (initials unknown), 2000b. Micronucleus test of rebaudioside A in mice. Test numer 5002 ( ). Unpublished report of a study conducted at the Biosafety Research Center, Japan. Submitted to WHO by Ministry of Health and Welfare, Japan. Nakayama, K., Kasahara, D., Yamamoto, F., Absorption, distribution, metabolism and excretion of stevioside in rats. J Food Hyg So. Jpn, 27, 1-8. Nanayakkara, N.P., Klocke, J.A., Compadre, C.M., Hussain, R.A., Pezzuto, J.M., Kinghorn, A.D., Characterization and feeding deterrent effects on the aphid, Schizaphis graminum, of some derivatives of the sweet compounds, stevioside and rebaudioside A. J Nat Prod 50, NewHope360, IADSA welcomes Codex adoption of steviol glycosides. Nikiforov, A.I., Eapen, A.K., A 90-day (oral) toxicity study of rebaudioside A in Sprague-Dawley rats. Int J Toxicol 27, Nunes, A.P., Ferreira-Machado, S.C., Nunes, R.M., Dantas, F.J., De Mattos, J.C., Caldiera de Araujo, A., 2007a. Analysis of genotoxic potentiality of stevioside by comet assay. Food Chem Toxicol 45, Nunes, A.P., Ferreira-Machado, S.C., Nunes, R.M., Dantas, F.J., De Mattos, J.C., Caldiera de Araujo, A., 2007b. Response. Food Chem Toxicol 45, Nunes, A.P., Ferreira-Machado, S.C., Nunes, R.M., Dantas, F.J., De Mattos, J.C., Caldiera de Araujo, A., 2007c. Response. Food Chem Toxicol 45, NutraIngredients, EFSA Opinion paves way for EU approval of stevia-based sweeteners. Available online: Accessed on June 30, Ogawa, T., Nozaki, M., Matsui, M., Total synthesis of stevioside. Tetrahedron 36, GRAS ASSOCIATES, LLC Page 41 of 45

49 Oh, H., Han, E., Choi, D., Kim, J., Eom, M., Kang, I., Kang, H., Ha, K., In vitro and in vivo evaluation of genotoxicity of stevioside and steviol, natural sweetener. J Pharm Soc Korea 43, Oliveira-Filho, R.M., Uehara, O.A., Minett, C.A.S.A., Valle, L.B.S., Chronic administration of aqueous extract of Stevia rebaudiana (Bert.) Bertoni in rats: endocrine effects. Gen Pharma 20, Pezzuto, J.M., Compadre, C.M., Swanson, S.M., Nanayakkara, D., Kinghorn, A.D., Metabolically activated steviol, the aglycone of stevioside, is mutagenic. Proc Nat Acad Sci USA 82, Planas, G. M., Kuc, J., Contraceptive properties of Stevia rebaudiana. Science 162, Procinska, E., Bridges, B.A., Hanson, J.R., Interpretation of results with the 8-azaguanine resistance systemin Salmonella typhimurium: No evidence for direct acting mutagenesis by 15-oxosteviol, a possible metabolite of steviol. Mutagenesis 6, Renwick A.G., Acceptable daily intake and the regulation of intense sweeteners. Food Addit Contam. 7, Renwick, A.G., The use of a sweetener substitution method to predict dietary exposures for the intense sweetener rebaudioside A. Food Chem Toxicol 6(Suppl.1), S61-S69. Renwick, A.G., Tarka, S.M., Microbial hydrolyis of steviol glycosides. Food and Chemical Toxicology 46, S70-S74. Republic of the Philippines, Food and Drug Administration, Available online: Accessed June 30, Republic of South Africa Department of Health, 2012a. No. R. 733 Foodstuffs, Cosmetics and Disinfectants Act, 1972 (Act No. 54 of 1972): Regulations Relating to the Use of Sweeteners in Foodstuffs, 10 September See: ners_in_foodstuffs_sep12.pdf. Accessed June 30, Republic of South Africa Department of Health, 2012b. Foodstuffs, Cosmetics and Disinfectants Act, 1972 (Act 52 of 1972): List of Permissable Sweeteners Referred to in Regulation 4 of the Regulations Relating to the Use of Sweeteners in Foodstuffs (R.733 of 10 September 2012). Originally accessed at: Roberts, A., Renwick, A.G., Comparative toxicokinetics and metabolism of rebaudioside A, stevioside, and steviol in rats. Food Chem Toxicol, 46(Suppl.1), S70-S74. Roberts, A., Munro, I., Letter to the Editor: Stevioside and related compounds: Therapeutic benefits beyond sweetness. Pharmacology & Therapeutics, 122(3), e1-e2. Rulis, A.M., Levitt, J.A., FDA's food ingredient approval process: Safety assurance based on scientific assessment. Reg Tox Pharm 53, Saravanan, R., Ramachandran, V., Modulating efficacy of Rebaudioside A, a diterpenoid on antioxidant and circulatory lipids in experimental rats. Environ Toxicol Pharmacol 36, GRAS ASSOCIATES, LLC Page 42 of 45

50 Saravanan, R., Vengatashbabu, K., Ramachandran, V., Effect of Rebaudioside A, a diterpenoid on glucose homeostasis in STZ-induced diabetic rats. J Physiol Biochem 68, Sasaki, Y.F., Kawaguchi, S., Kamaya, A., Ohshita, M., Kabasawa, K., Iwama, K., Taniguchi, K., Tsuda, S., The comet assay with 8 mouse organs: results with 39 currently used food additives. Mutat Res 519, Schvartzman, J.B., Krimer, D.B., Moreno-Azorero, R., Cytological effects of some medicinal plants used in the control of fertility. Experientia 33, Sekihashi, K., Saitoh, H., Sasaki, Y., Genotoxicity studies of Stevia extract and steviol by the comet assay. J. Toxicol. Sci. 27 (suppl. 1), 1-8. Sloter, E.D., 2008a. A dietary two-generation reproductive toxicity study of Chrysanta 99-P in rats. WIL Research Laboratories, LLC. Unpublished Report, Study Number WIL Sloter, E.D., 2008b. Oral (Gavage) study of Chrysanta 99-P on embryo/fetal development in rats. WIL Research Laboratories, LLC. Unpublished Report, Study Number WIL Soejarto, D.D., Kinghorn, A.D., Farnsworth, N.R., Potential sweetening agents of plant origin. III.Organoleptic evaluation of stevia leaf herbarium samples for sweetness. J Nat Prod, 45, Sung, L.H., Report on pharmacokinetic (PK) studies of T100 sunstevia 95% stevioside in rats. Unpublished report from Sunlabel Pte Ltd, Singapore. Submitted to WHO by the Ministry of Health and Welfare, Japan. Sunwin and WILD Flavors GRAS Notification for purified steviol glycosides with rebaudioside A and stevioside as the principal components, Submitted to the US Food and Drug Administration, Washington, DC and identified as GRAS Notification 304. Access: Accessed June 30, Suttajit, M., Vinitketkaumnuen, U., Meevatee U., Buddhasukh, D., Mutagenicity and human chromosomal effect of stevioside, a sweetener from Stevia rebaudiana Bertoni. Environ Health Perspec.101, Switzerland Federal Office of Public Health, index.html?lang=fr. Accessed June 30, Tandel, K.R., Sugar substitues: Health controversy over perceived benefits. J Pharmacol Pharmacother 2(4): Temcharoen, P., Pimbua, J., Glinsukon, T., Rojanapo, W., Apibal, S., Mutagenic activity of steviol to Salmonella typhimurium TM 677: Comparison of the activity of S9 liver fractions from five laboratory animal species. Bull Health Sci & Tech 1, Temme, E.H.M., Vankeirsbilck, A., Buyse, J. & Geuns, J.M.C., A short term study of stevioside in healthy subjects. In: Geuns, J.M.C. & Buyse, J., eds. Safety of stevioside. Proceedings of the first symposium sponsored by KULeuven, 16 April 2004, Leuven, Belgium. Heverlee, Belgium, EU print, pp Terai, T., Ren, H., Mori, G., Yamaguchi, Y., Hayashi, T., Mutagenicity of steviol and its oxidative derivatives in Salmonella typhimurium TM677. Chem Pharm Bull, GRAS ASSOCIATES, LLC Page 43 of 45

51 The Food Institute Report, FDA News May 15, From Newsday, May 2, Toskulkao, C., Chaturat, L., Temcharoen, P., Glinsukon, T., Acute toxicity of stevioside, a natural sweetener, and its metabolite, steviol, in several animal species. Drug ChemToxicol 20, Toyoda, K., Matsui, H., Shoda, T., Uneyama, C., Takahashi, M., Assessment of the carcinogenicity of stevioside in F344 rats. Food Chem Toxicol 35, Urban, J.D., Carakostas, M.C., Brusick, D.J., Steviol glycoside safety: Is the genotoxicity database sufficient? Food Chem Toxicol 51: Usami, M., Sakemi, K., Kawashima, K., Tsuda, M., Ohno, Y., Teratogenicity study of stevioside in rats. Bull Natl Inst Hyg Sci 113, In Japanese. Waddell, W.J., Letter to the editor re: Long-term feeding effects of stevioside sweetener on some toxicological parameters of growing rats. J Appl Toxicol 31(6): Wagner, V.O., Van Dyke, M.R., Bacterial Reverse Mutation Assay of Rebaudioside A. BioReliance, Rockville, MD. Study Number AB21TG.503.BTL. Wang, L.Z., Goh, B.C., Fan, L., Lee, H.S., Sensitive high-performance liquid chromatography/ mass spectrometry method for determination of steviol in rat plasma. Rapid Commun Mass Spectrom 18, Wasuntarawat, C., Temcharoen, P., Toskulkao, C., Mungkornkarn, P., Suttajit, M., Glinsukon, T., Developmental toxicity of steviol, a metabolite of stevioside, in the hamster. Drug ChemToxicol 21, Wheeler, A., Boileau, A.C., Winkler, P.C., Compton, J.C., Prakash, I., Jiang, X., Mandarino, D.A., Pharmacokinetics of rebaudioside A and stevioside after single oral doses in healthy men. Food Chem Toxicol, 46(7)(Suppl.), S54-S60. WHO, Joint FAO/WHO Expert Committee on Food Additives. WHO Food Additive Series; 42. Safety evaluation of certain food additives. Stevioside. WHO, GEMS/Food regional diets (regional per capita consumption of raw and semi processed agricultural commodities). Geneva: Global Environment Monitoring System 144 steviol glycosides K2 Food Contamination Monitoring and Assessment Programme and Food Safety Department, World Health Organization. WHO, Joint FAO/WHO Expert Committee on Food Additives. WHO Food Additive Series; 54. Safety evaluation of certain food additives, Steviol Glycosides, pp WHO, Joint FAO/WHO Expert Committee on Food Additives. Sixty-eighth meeting, Summary and Conclusions, Steviol Glycosides. Issued July 12, WHO, Joint FAO/WHO Expert Committee on Food Additives. Sixty-ninth meeting: Summary and Conclusions, Steviol Glycosides. Issued July 4, WHO, Joint FAO/WHO Expert Committee on Food Additives. WHO Food Additive Series: 60. Safety evaluation of certain food additives. Steviol Glycosides (addendum). GRAS ASSOCIATES, LLC Page 44 of 45

52 Williams, G. M., Letter to the Editor, Food Chem Toxicol, 45 (2007) Williams, L.D., Burdock, G.A., Genotoxicity studies on a high-purity rebaudioside A preparation. Food Chem Toxicology, 47, Wingard, R.E., Brown, J.P., Enderlin, F.E., Dale, J.A., Hale, R.L., Seitz, C.T., Intestinal degradation and absorption of the glycosidic sweeteners stevioside and rebaudioside A. Experientia 36, Wood, H. B., Jr., Allerton, R., Diehl, H. W., & Fletcher, H. G., Jr. (1955).Stevioside. I. The structure of the glucose moieties. J Org Chem 20, Xili, L., Chengjiany, B., Eryi, X., Reiming, S., Yuengming, W., Haodong, S., Zhiyian, H., Chronic oral toxicity and carcinogenicity study of stevioside in rats. Food Chem Toxicol 30, Yamada, A., Ohgaki, S., Noda, T., Shimizu, M., Chronic toxicity of dietary stevia extracts. Shokuin Eiseigaku Zasshi. 2, Yodyingyuad, V., Bunyawong, S., Effect of stevioside on growth and reproduction. Hum Reprod 6, Zenith International, Stevia Sales Increased 27% Last Year, Says Zenith. Originally accessed at: news/stevia-sales-increased-27-last-year-says-zenith. Zenith International, Global Stevia Market Passes $300 Million. million/? _uid=81ad8cbddc/list_id=396c Accessed on June 30, Zenith International, Global Stevia Market up 14% in Accessed on October 27, See Volume 2 of 2 for Appendices GRAS ASSOCIATES, LLC Page 45 of 45

53 GRAS ASSESSMENT of HIGH PURITY STEVIOL GLYCOSIDES ( 95%) Food Usage Conditions for General Recognition of Safety for Productora Alysa SpA Calle Tres No. 600 Belloto Norte, Quilpué Chile VOLUME 2 OF 2 Evaluation by GRAS Expert Panel Richard C. Kraska, Ph.D., DABT Robert S. McQuate, Ph.D. Robert W. Kapp, Jr., Ph.D., Fellow ATS, ERT (UK) November 5, 2014

54 TABLE OF CONTENTS APPENDIX A Certificates of cgmp Compliance 3 APPENDIX A-1 Bureau Veritas Certificate of Compliance.. 4 APPENDIX A-2 Bureau Veritas ISO 22000:2005 Certificate... 5 APPENDIX A-3 Bureau Veritas HACCP Certificate.. 6 APPENDIX B Specifications & Certificates of Analysis for Production Processing Aids. 7 APPENDIX B-1 Dow Chemical Documentation for Food Additive Status for Cation Exchange Resins 8 APPENDIX B-2 Dow Chemical Documentation for Food Additive Status for Anion APPENDIX B-3 Exchange Resins 9 Dow Chemical Documentation for Food Additive Status for Anion Exchange Resins & Adsorbent. 10 APPENDIX B-4 Supplier Letter of Regulatory Compliance for Spiral Wound Element APPENDIX C Analytical Method for Steviol Glycosides Quantitation. 12 APPENDIX D HPLC Analysis Report for Five Lots of Stevia Pure.. 27 APPENDIX E Certificates of Analysis for Multiple Production Batches of Stevia Pure 43 APPENDIX E-1 Certificate of Analysis for Stevia Pure 289A.. 44 APPENDIX E-2 Certificate of Analysis for Stevia Pure 292A.. 46 APPENDIX E-3 Certificate of Analysis for Stevia Pure 297B.. 48 APPENDIX E-4 Certificate of Analysis for Stevia Pure 299B.. 50 APPENDIX E-5 Certificate of Analysis for Stevia Pure 310A.. 52 APPENDIX F Pesticide Analytical Reports for Stevia Pure from Analab APPENDIX F-1 Test Report for Pesticides for Stevia Pure APPENDIX F-2 Test Report for Pesticides for Stevia Pure APPENDIX G Shelf Stability Testing Report for Stevia Pure 60 APPENDIX H Sweetness Intensity Test Report for Stevia Pure.. 64 APPENDIX I Estimated Daily Intake Levels of Steviol Glycosides. 68 APPENDIX J Summary of Published Safety Reviews.. 73 APPENDIX K Studies on Principal Metabolite: Steviol. 78 APPENDIX L Studies on Steviol Glycosides Preparations that are Primarily Mixtures of Stevioside & Rebaudioside A 81 APPENDIX M Studies on Steviol Glycosides Preparations that are Primarily Rebaudioside A.. 93 GRAS ASSOCIATES, LLC Page 2 of 99

55 APPENDIX A Certificates of cgmp Compliance A-1 Bureau Veritas Certificate of Compliance A-2 Bureau Veritas ISO 22000:2005 Certificate A-3 Bureau Veritas HACCP Certificate GRAS ASSOCIATES, LLC Page 3 of 99

56 A-1 Bureau Veritas Certificate of Compliance CERTIFICATE OF COMPLIANCE No. 002 CON/ The Center for Studies, Measurement, and Quality Certification, CESMEC S.A., hereby certifies that the company PRODUCTORA ALYSA SpA (PRODALYSA SpA), located at 600 Calle Tres, Belloto Norte, Quilpue, is the Holder of the CESMEC (ISO CASCO 5 Model) Confonnity Mark, as of August 1, The following product is certified under this system, and complies with the specified technjcal specification listed below: Powder and liquid Stevia extracts, extracted and purified exclusively through the usc of water as solvent from the leaves ofthe Stevia Rebaudania Bertoni spt.>cie, to be used as sweetener. This is to be used in the brands STEVIA PURE POWDER and STEVJA J>URE LIQUID The Certification is given in accordance with: "PRODALYSA'S PROCESS CONTROL PROCEDURES" The applied certification system ensures that the products have been subjected to a Quality Control System by PRODALYSA SpA., which has been approved and audited regularly by CESMEC to make sure they comply with the requirements established in the aforementioned procedure. The certificate extended to the product guarantees that it complies with the requirements of the standards and/or technical specifications given exclusively. CESMEC is not liable for the product's non-compliance in matters not included in the standards/teclmical specifications. Thjs Certificate of Compliance is issued at the reque (PRODALYSA SpA) for all purposes deemed necessar.(b) (6) (b) (6) Santiago, April4, 2014 THIS CERTIFICATE IS VALID FOR SIX MONTHS STARTING FROM THE DATE/TWAS ISSUED. Certif No. 002 GRAS ASSOCIATES, LLC Page 4 of 99

57 GRAS Assessment Productora Alysa SpA A-2 Bureau Veritas ISO 22000:2005 Certificate PRODUCTORA ALYSA SpA PRODALYSA SpA RUT : Calle Tres N 600, Belloto Norte, Quilpue- Va lpara iso C HI LE Bureau Veritas Certification, certify that the Management System ofthe above organisation has been assessed and found to be in accordance with the requirements ofthe standards detailed below. STANDARD ISO 22000:2005 SCOPE OF SUPPLY PROD I CTIO!\ OF Ill(; IlL\ ' PURint:D POWDER Al\0 UQLIO \<\11JiitALS'JVVL\ R &AUDIA~A BE.RT0!\ 1 EXTRACTS ATni Qf.~UIUt PLA..,'T. STAitn'C FRO\i 51" \"IA R 8Al 01A.'A U'..A\~t"..SA~ O PROOUCt:DAS I"VJ'ER.\I DIATE N.An.ltAL.S\\C T \I:lt PMOOl.CTS t'or n n ; H)()D 1'01 \I ltv. TA8L T'OP SW t'f" 111LR PRODUC" RS AS PO\\OtR.S. UQtfi0SA~DTA8U:TS.. for I..OC..\1 \.~0 I''TUtltATIO'CALMARJa:T'S. I'C'l.A!OI\C: PROCESSIXCSTACF..S FRO-M RA~ \tat RlALA'0 P1tODUC110X MATt:IUAl.S R C t:p110-"'. nn:.\tictreadll\t. UOUD PACI'AC lsc. S PRA'\o' OR\'l"iC. P'C)WDt~R PRI\t \ltv PAC"KACI,C. stco!\dary PAo.:ACNC.STORACE A \O S-UIP\1C.'n"OF rl""lsll 0 PRODlCT. PROOLCTIO:> OFCARMU:LS. LC MO L.ICHTCARA~HI. Mtrn: WITH STEVIA IWIAUDIANA 8 RTO'~ OTliACT IJ<QOILPUt PI.A~,. AS 1\ C Mt' Oit~ST tom 'I ttt: t()()o I"VDlJSTR\'.I"iCUfDI\C nn: STAGES OF RAW MATERIAL R CF.P110,, P'OROO~Il\C:, 81. ~01'-G.COOKL~C.. rackaci.nc, STORACI!A!.~D SIUPPIXC OF t"""l'istl t:d PNOOt ("'1'. t'l..akoraoos U o:x -rkactos NATIJRAI.I:S I DOS Y 0:X POLYO ALTAMt:.VT PIJRIFICADOS tn PLAN la no; VUII, PIIt. A PARTIR DE Slt;VlA RKOAUOIANA BERTONI COMO PRODUCTO IN'rt:RMEOIO ENOC1..2.ANT&NATURAL PARA o:~ipiu:sas UE,\I,IMENTOS. P RODUCTORAS Dt EMIUO..MNT S I~STOS PARA CONSUMO IIIIMASO CIII OLVO. LfQUIDOS V PAS'I'lLLAS, PARA MEitCAOOS ~ACIONALES E IN' It'MJ'ACIONAI... ES, COMPRtL"<iOLt:NOO I..AS t!tapas DE MOCEPC10N Ot: MATt:RlA PRIMAE INSUMOS, PHO('t.:SAMIENTO. 'ra.atami f.:n'ro rtrmico. r.kvasado EN LiQmOO.SECADO SPRAY, EI<VASAOO F.N POLVO. EMRALAJf. ALMACt'NMIIENTO V O&SPACIIO DE PRODUCTO 1T..IitMI.NAOO. II.AIIOR.\00' OE CARMI 1.0 V CA~I 1.0 UGIIT CO STEVIA RJ:IIAUDI-'""!A 8 RTOSI E' PI.A" A lie QUII PI f C0\10 IJ<CREDIJ:NTE PARA LAS E\IPRESAS DE Al.lM NTOS. 00\IPA.ENDILWQ LAS ETA PAS ot: RECEPCIO DY. MATI'RIAS rlmias. FMACCIONAMTENTO. M ZCI.A..COC00S. t:..was.\00, Al.MA("t ~A\tlt_,,.O V DF.SPACHO DE rroolc1'0sttr\4lnados. Cmtji~attOtt ~ycle start date: 26 January Subje~tto the continued satisfactory operation oftlw organisation 's Manogemem System. 25 January This certificate expires on: Ortginal certification date: 26 January Certificate Number: IND U Certificate Serial N : BVCSG Version Number.- 0 I Revision Date: December ~ (b) (6) U KAS MANAGEMENT SYSTEMS 008 Patricio Testa Oliva Technical Manager.4o.rt.j r.-u ' ---«"'IX',~... IJ,.,,, 4Jfll,,,_,,_,;,,.,...,lHI, GRAS ASSOCIATES, LLC IJrunda_wH W"' /'UI/Wittlftr:/tHIJlll UWIIOMJ.>~t"t}/1/Jf IWih.-.11\ f'j:.j,iiif AflN uh ( '"'" Nh I,., AIL~It.ml.ll \I.JnlllltAI..,.X fu.w J,IJI,,r,,,l( I~.,,~ {ljl "-''' J 1/J.111f. -.V A "l/"'11 tl.nl,, Alum ft.~ <~!) u : /n./fji _J,J.... ~,- ff1r #oftf "''lllttw~it 111/, J r,h,.~\i'u \<f'o~wifr1t \ 1ilfl 1111 lflllt/lfl/ /'t".h.llt.ll.t,.\r.mi/1+1\.~i C h1/e Page 5 of 99

58 GRAS Assessment Productora Alysa SpA A-3 Bureau Veritas HACCP Certificate PRODUCTORA AL YSA SpA PRODALYSA SpA RUT: Calle Tres N 600, Belloto Norte, Quilpue - Valparaiso C HILE Bureau Veritas Certification. certifica que el Sistema de Gestion de Ia organizacion mencionada ha sido evaluado y se muestra acorde con los requerimientos de las normas detalladas a continuacion. NORMA NCh Of 2004 HACCP basado en CODEX ALIMENTARIUS Codex Alimentarius CAC-RCP , Rev ALCI\NCE DEL SISTEMA EI..AIIORACI6 ' DE EXTRACTOS 'ATI RALF..S tiquioos Y E~ POLVOALTAME,"l E P l RIFICAOOS E.'< PLA 'ITA DE QllllP UE. A PARTIR DE STEVIA REBAt:DIANA BERTONI C0 \10 PROD I CTO INTE~\IEDIO '1DIJLZAN"fE 'IATlJRAL PARA EI\1 PRESAS m; ALI\I E ITOS. PRODUCTORAS DE t:'idul.zan"fes LISl'OS PARA CONSU:\10 II UMANO EN I'OI.VO. I.IQIJIOOS V PASTILI..AS, PARA MERCAOOS NACIO'ALES E I NTF:RNACIONAI.ES, COMP RENDIENDO l.as [TAPAS DE RECEPCI6N DE MATt:RIA I'RIMA E INSIJMOS, PROCESAMlENTO, TRATAMIENTO TERMICO, ENVASADO EN LiQUIOO. SECAOO SPRA V. ENVASADO EN I'OLVO, t:i\1bai..aje, ALMAC:F.NAMI ENTO V DF..SPACHO DE PRODUCTO TERJ\IINAI>O. f:i.ailoraci ON OE CARAMELO Y CARA~U:LO L.ICIIT CON STEVIA R : naij OIANA RERTONI E~ PLANTA DE Q UI LPUE COMO ING REDIENT I'ARA L.AS EMI'RF..SAS DE ALIM ENTOS, <'01\IPRENDI ENDO LAS t: TAPAS DE RECEPCION DE MATERIAS PRI\1AS. FRACC10NA.\ fi NTO,I\1EZCI..A, COCCI6N. t:.'vasado. ALMACENA.\ II ENTO V I>ESPACIIO DE PRODUCl"OS T ERMI I\AI>OS. 1 C1clo de Cuttficacl6n comien:a en laftclur 26 [ nero 2014 Sujt lo o una con1mua y salisfacloria operaci6n del Sislema de Ges1i611 de Ia organi:ac16n E.s1e cerltficado es wilido has/a: 25 [nero 2017 Fccha Origitwl de Centficaci6n: 26 [nero 2011 N1imero de l?egislro INN: D- 546 Cmtficado Serie N : B VCSG 1623 Versi!ln N1imero: 00 Fecha de Revision: nero 13, 2014 (b) (6) II fii) tru \!!J\!!JII INH CH/l SISTOIA JW:I(Nl DE ACREDITACIOII SC011 Cierenle 1ecnico (, ~110 c. lmpr~si(>" /1ur~.\tliiii(JI.!II GRAS ASSOCIATES, LLC /II '~TIIo..h r rii/icniunt ( hllr Frunao;,tJ ~oguc ra \ Jf!t_J, Oficuru /311/ Prt.J\uknctu Chtl Page 6 of 99

59 APPENDIX B Specifications & Certificates of Analysis for Production Processing Aids B-1 Dow Chemical Documentation for Food Additive Status for Cation Exchange Resins B-2 Dow Chemical Documentation for Food Additive Status for Anion Exchange Resin B-3 Dow Chemical Documentation for Food Additive Status for Anion Exchange Resins and Adsorbent B-4 Ultura Spiral Wound Membrane Element U.S. FDA Certificate GRAS ASSOCIATES, LLC Page 7 of 99

60 B-1 Dow Chemical Documentation for Food Additive Status for Cation Exchange Resins The Dow Chemical Company U.S.A Effective: April2, 2012 Supersedes: June 12, 2009 FOOD ADDITIVE STATUS Product DOWEX 88 (H) Cation Exchange Resin DOWEX 88MB (H) Cation E xchange Resin DOWEX MONOSPHERE 88 (H) Cation Exchange Resin DOWEX N406 (H) Cation Exchange Resin DOWEX N 606 (H) Cation Exchange Resin Food and Drug Administration (FDA) These products comply with the U.S. Food and Drug Administration's Food Additive Regulation 2 1 CFR l 73.25(a)(l ). Use of these products is subject to good manufacturing practices and any limitations which are part of the regulations. The regulations should be consulted for complete details. If you have any questions or require further information, please contact us. S incere ly, (b) (6) Wendy W. Bingaman EH&S Global Product Leader Dow Water and Process Solutions The Dow Chemical Company Phone : (2 15) wbingaman@dow.com This information is considered accurate and reliable as of the date appearing above and is presented in good faith. Because use conditions and applicable laws may differ from one location to another and may change with time, Recipient is responsible for determining whether the information in this document is appropriate for Recipient's use. Since Dow has no control over how this information may be ultimately used, all liability is expressly disclaimed and Dow assumes no obligation or liability therefore. No warranty, express or implied, is given nor is freedom from any patent owned by Dow or others to be inferred. Trademark of The Dow Chemical Company ("Dow") or an affiliated company of Dow Form No GRAS ASSOCIATES, LLC Page 8 of 99

61 B-2 Dow Chemical Documentation for Food Additive Status for Anion Exchange Resin The Dow Chemical Company U.S.A. Effective: April2, 2012 Supersedes: December 20,2010 FOOD ADDITIVE STATUS Product DOWEX 22 (Cl) Anion Exchange Resin Food and Drug Administration (FDA) This product complies with the U.S. Food and Drug Administration's Food Additive Regulation 21 CFR (a)(5). Use of this product is subject to good manufacturing practices and any limitations which are part of the regulations. The regulations should be consulted for complete details. If you have any questions or require further information, please contact us. Sincerely, (b) (6) W endy W. Bingaman EH&S Global Product Leader Dow Water and Process Solutions The Dow Chemical Company Phone: (215) wbingaman@ dow.com This information is considered accura te and reliable as of the date appearing above and is presented in good faith. Because use conditions and applicable la ws may differ from o n e location to another and may change with time, Recipient is responsible for determining whether the information in this document is appropriate for Recipient's use. Since Dow has no control over how this information may be ultimately used, a!! liability is expressly disclaimed and Dow assumes no obligation or liability therefore. No warranty, express or implied, is given nor is freedom from any patent owned by D ow or others to be inferred. Trademark of The Dow Chemical Company ("Dow") or an affiliated company of Dow Form No GRAS ASSOCIATES, LLC Page 9 of 99

62 B-3 Dow Chemical Documentation for Food Additive Status for Anion Exchange Resins and Adsorbent The Dow Chemical Company U.S.A Effective Date: February 17, 2012 Supersedes: December 20, 2010 FOOD ADDITIVE STATUS Product DOWEX 66 Anion Exchange Resin DOWEX MARATHON WBA Anion Exchange Resin DOWEX MONOSPHERE 66 Anion Exchange Resin DOWEX MONOSPHERE 77 Anion Exchange Resin DOW EX OPT!PORE SD-2 Polymeric Adsorbent Food and Drug Administration (FDA) This product complies with the U.S. Food and Drug Administration's Food Additive Regulation 21 CFR (a)(5). Use of these products is subject to good manufacturing practices and any limitations which are part of the regulations. The regulations should be consulted for complete details. If you have any questions or require further information, please contact us. Sincerely, (b) (6) Wendy W. Bingaman EH&S Global Product Leader Dow Water and Process Solutions The Dow Chemical Company Phone: (215) This informal ion is considered accurate and reliable as of the dare appearing above and is presented in good faith. Because use conditions and applicable laws may differ from one location to another and may change with time, Recipient is responsible for determining whether the informal ion in this document is appropriate f or Recipient's use. Since Dow has no control over how this information may be ultimately used, all liability is expressly disclaimed and Dow assumes no obligation or liability therefore. No warranty, express or implied. is given nor is freedom from any patent owned by Dow or others to be inferred. Trademark of The Dow Chemical Company ("Dow") or an affiliated company of Dow Form No GRAS ASSOCIATES, LLC Page 10 of 99

63 B-4 Ultura Spiral Wound Membrane Element U.S. FDA Certificate March, 20th 2014 ULTURA (Oceanside) Inc Avenida de Ia Plata Oceanside, CA USA T: F: SPIRAL WOUND ELEMENT U.S. FDA CERTIFICATE This letter certifies that all the materials of construction used by UL TURA, Inc. for Its net-wrap and fiberglass membrane elements comply with applicable regulations of the U.S. Food and Drug Administration (FDA). Element Description: Element Membrane: Spiral Wound Element Polyethersulfone (PES). Polysulfone {PS) Polyfluoride (PVDF). Nanofiltration (NF) Reverse Osmosis (RO). Polyacrylonitrile (PAN) Element Construction: Spiral wound with net outer wrap Spiral wound with fiberglass wrap Element conform to USDA 3-A standards and FDA regulation CFR Title 21 Element construction FDA compliant code Membrane 21 CFR Permeate Tube & ATD 21 CFR Permeate carrier 21 CFR Feed Spacer 21 CFR Glue 21 CFR Resin Fiber Glass 21 CFR Net Outer Wrap 21 CFR Sophie Luchini (b) (6) QC Manager Our brands.aptwater' [t]l!ttt:hil \\ IOW!!li!J [::JIPOX' GRAS ASSOCIATES, LLC Page 11 of 99

64 APPENDIX C Analytical Method for Steviol Glycosides Quantitation Note: Productora Alysa SpA has developed a method for steviol glycosides quantitation that combines the analytical methods detailed in the JECFA 2008 and JECFA 2010 steviol glycoside monographs, with the addition of a sample-drying step. The Productora Alysa SpA method is provided below. Retain lots of Stevia Pure were analyzed per JECFA 2010 steviol glycosides monograph. GRAS ASSOCIATES, LLC Page 12 of 99

65 Stevia Pure next generauon sweetener Specifications for Pure Steviol Glycosides SUMMARY The monograph has been designed to supply the standards for ensuring the quality of water extracted and purified steviol glycoside extracts, where both stevioside and rebaudioside A coexist as major glycosides. This monograph should be valid for all extracts containing more than 95% of the seven glycosides described later in this document and no other produced as a result of the chemical processing of Stevia extracts. In addition, the purification of steviol glycosides should ensure both the absence of chemical residues from the purification process and the maintenance of the water solubility of the steviol glycosides even at high concentrations. This method provides tools to confirm the complete avoidance of use of of organic solvents in the purification of the glycosides, as well as the safety in terms of heavy metals and pesticides residues. Special attention is given to the fact that steviol glycosides are highly hygroscopic compounds, specially when purified in an aqueous process. In order to avoid any bias in the quantification of glycosides, a drying complementary step for the injected samples is provided in order to correct any hygroscopic derived error occurred in the gravimetric steps. Stevioside and rebaudioside A are the component glycosides of principal interest for their sweetening property. In those samples where both glycosides coexist, coelution and subestimation of the predominant glycoside could ocurr. In order to provide an accurate method, both qualitative and quantitative in terms of glycoside determination, both a principal method based in the JECFA 2010 procedures is included, as well as a verification method for those products where a major proportion of glycosides is composed both by Stevioside and Rebaudioside A. Associated glycosides, also naturally occurring, include rebaudioside C, rebaudioside D, rebaudioside F, dulcoside A, rubusoside, steviolbioside, and rebaudioside B, which are generally present in preparations of steviol glycosides at levels lower than stevioside or rebaudioside A are determined by the method. Description The product is obtained from the leaves of Stevia rebaudiana Bertoni. The leaves are extracted with cold de-ionized water and the aqueous extract is purified with a combination of food grade membrane systems to a total purity above 95% of glycosides, where a final removal of colored compounds native to Stevia can be removed by water eluted food grade ion-exchange resin systems. During the process, the product should not be exposed to organic solvents, fining agents or crystallizations affecting the natural condition of the product, by product formation or water solubility. The result is a GRAS ASSOCIATES, LLC Page 13 of 99

66 Stevia Pure next generauon sweetener product free of organic solvents, water soluble at concentrations above 35% w/w and free of chemical fining agent residues. Function: Multi purpose sweetener; sugar substitute. Storage: Keep dry and store in tight containers at ambient temperature. Identification Chemical name Stevioside: 13-[(2-0-B-D-glucopyranosyi-B-D-glucopyranosyl)oxy] kaur-16-en-18-oic acid, B-D-glucopyranosyl ester Rebaudioside A 13-[(2-0-B-D-glucopyranosyi-3-0-B-Dglucopyranosyi-B-Dglucopyranosyl)oxy]kaur-6-en-8-oic acid, B-Dglucopyranosyl ester Structural Formula The nine named steviol glycosides: 2 GRAS ASSOCIATES, LLC Page 14 of 99

67 Stevia Pure next generauon sweetener Comeouncl name R1 R2 Stevtostde,11-Gic,11-Gic-,11-Gic(2 >1) Rebaudiosicle A,11-Gic,11-Gic-,8-Gic(2 1) I,8-Gic(3 1) Rebaudioside 8 H f1-gic-p-gic(2-71) I,8-Gic(3-71) Rebaudioside C,8-Gic,8-Gic-a-Rha(2 1) I f1-gic(3 1) Rebaudtostde 0,8-G i c-,8-g ic(2~1),8-gic-,8-gic(2-7 1) I,8-Gic(3-71) Rebaudioside F,11-Gic,11-Gic-p-Xyl(2-71) I,8-Gic(3-71} Ou/coside A,8-Gic,8-Gic-a-Rha(2 1} Rubusoside,8-Gic,8-Gic Steviolbioside H,11-Gic-,11-Gic(2~ 1) Steviol (R1 = R2 = H) is the aglycone of the steviol glycosides. Glc and Rha represent, respectively, glucose and rhamnose sugar moieties. Formula weight: Stevioside: Rebaudioside A: GRAS ASSOCIATES, LLC Page 15 of 99

68 Stevia Pure next generauon sweetener I. ACTIVE PRINCIPLE DETERMINATION The active principle determination includes the simultaneous determination of 9 of the naturally occurring glycosides in the stevia extract. It must be noted, that while the method described in section A is appropriate for the identification of all steviol glycosides, it may under estimate the major occurring glycoside within steviol glycosides Rebaudioside A and Stevioside in samples where both glycosides coelute. For those cases, a quantitative complementary method is provided in section B, where both glycosides are clearly resolved. Determine the percentages of the individual steviol glycosides by HPLC (Vol. 4) under the following conditions. Reagents Acetonitrile: more than 95% transmittance at 210 nm. Standards Stevioside: more than 99.0% purity on the dried basis. Rebaudioside A: more than 99.0% purity on the dried basis. Mixture of nine steviol glycosides standard solution: Containing stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, dulcoside A, rubusoside and steviolbioside. This Solution is diluted with water accordingly and is used for the confirmation of retention times. Standards are available from Wako Pure Chemical Industries, ltd. Japan and ChromaDex, USA. A) Qualitative and Quantitative Determination of Steviol Glycosides Standard solution Accurately weigh 50 mg of stevioside and rebaudioside A standard into each of two 50- ml volumetric flasks. Dissolve and make up to volume with water. Sample solution Accurately weigh mg of sample into a 50-ml volumetric flask. Dissolve and make up to volume with water-acetonitrile (7:3). 4 GRAS ASSOCIATES, LLC Page 16 of 99

69 Stevia Pure next generauon sweetener Weight Verification: With regard to the sample, after its injection, prepare for each sample 3 dishes with 10 g of injection solution in triplicate. Dry for 5 h at 105 c. Remove caps from stove, let the dish cool down in a dessiccator for 10 m inutes and weight immediately after removing from dissicator. Correct sample real injection weight with this determination. Procedure Inject 5 t.tl of sample solution under the following conditions. Column: Capcell pak C18 MG II (Shiseido Co. ltd) or Luna 5t.t C18(2) 100A (Phenomenex) or equivalent (length: 250 mm; inner diameter: 4.6 mm, particle size: 5t.tm). Mobile phase: 32:68 mixture of acetonitrile and 10 mmoi/l sodium phosphate buffer (ph 2.6) Flow rate: 0. 7 ml/min Detector: UV at 210 nm Column temperature: 40 Record the chromatogram for about 30 min. Identification of the peaks and Calculation Identify the peaks from the sample solution by comparing the retention time with the peaks from the mixture of nine steviol glycosides standard solution (see under figure). Measure the peak areas for the nine steviol glycosides from the samp le solution. Measure the peak area for stevioside and rebaudios ide A from their standard solutions. Calculate the percentage of each of the steviol glycosides except rebaudioside A in the sample from the formula: %X= [WS/W] X [fxax/as] X 100 Calculate the percentage of rebaudioside A in the sample from the formula: %Rebaudioside A= [WR/W] x [AX/AR] x 100 Where : X is each steviol glycoside; WS is the amount (mg) calculated on the dried basis of stevioside in the standard solution; WR is the amount (mg) calculated on the dried basis of rebaudioside A in the standard solution; 5 GRAS ASSOCIATES, LLC Page 17 of 99

70 Stevia Pure next generauon sweetener W is the amount (mg) calculated on the dried basis of sample in the sample solution; AS is the peak area for stevioside from the standard solution; AR is the peak area for rebaudioside from the standard solution; AX is the peak area of X for the sample solution; and fx is the ratio of the formula weight of X to the formula weight of stevioside: 1.00 (stevioside), 1.20 (rebaudioside A), l.oo(rebaudioside B), 1.18 (rebaudioside C), 1.40 (rebaudioside D ),1.16 (rebaudioside F), 0.98 (dulcoside A), 0.80 (rubusoside)and 0.80 (steviolbioside). After verifying WS, WR and W have been verified through the Weight Verification method described above, calculate the total purity of the sample through the sum of the nine percentages. Acceptance criteria: NLT 95% ofthe sum of nine glycosides. Note: If the sample contains both Rebaudioside A and Stevioside, there my by coelution and over/under quantification of the major occurring glycoside. If any coelution exists, quantification of these two glycosides should be adjusted through the method described in the following section, and adapted fomr the JECFA 2008 method. B) Major Glycosides Quantification Verification High Performance Liquid Chromatography (HPLC) is used for the quantification of Rebaudioside A, and Stevioside as a complementary mehtod. The qualitative and quantitative analysis of the major glycosides verification in stevia extract is performed using HPLC (High Performance Liquid Chromatography) and a NH2 column (normal phase mode), in the following manner. About 60 to 120 mg of the sample is accurately weighed and dissolved in water to make 100 ml of a test solution. About 50 mg each of stevioside and rebaudioside A, dried previously for 2 hours at 105" C, are accurately weighed, and the standard solutions of both compounds are prepared in the same way as the test solution. The test solution and standard solutions are injected into the HPLC in the following conditions of flow rate and retention times: HPLC Method 6 GRAS ASSOCIATES, LLC Page 18 of 99

71 Stevia Pure next generauon sweetener Mobile Phase: 80% v/v of HPLC Acetonitrile (Merck, KGaA. Darmstadt, Germany) and 20% v/v of HPLC water (Merck, KGaA. Darmstadt, Germany) Flow: 1 ml/minute Column:Kromasii100-SNH2. Column Amino. 250 * 4.6 mm (Akzo Nobel) Wavelength: 210 nm Injection Volume: 5 ~I Dilution: Within 2-4 g/1. Samples are analyzed for weight injection correction by drying (see Weight Verification in next section), both for standards (Stevioside and Rebaudioside A). C) Assay Determine the percentages of the individual steviol glycosides by high pressure liquid chromatography. Standards: Stevioside, >99.0% purity and rebaudioside A, >97% purity (available from Wako pure Chemical Industries, Ltd. Japan). Mobile phase: Mix HPLC-grade acetonitrile and water (80:20). Adjust the ph to 3.0 with phosphoric acid (85% reagent grade). Filter through 0.22 ~m Millipore filter or equivalent. Standard solutions: (a) Accurately weigh 50 mg of dried (105", 2 hours) stevioside standard into a 100-ml volumetric flask. Dissolve with mobile phase and dilute to volume with mobile phase. (b) Repeat with previously dried rebaudioside A standard. Sample solution: Accurately weight mg of dried (105", 2 hours) sample into a 100-ml volumetric flask. Dissolve with mobile phase and dilute to volume with the mobile phase. Weight Verification: Both for standards and sample, after sample injection, prepare for each 3 dishes with 10 g of solution in triplicate. Dry for 8 h at 10S"C. Remove caps f rom stove, let the dish cool down in a dessiccator for 10 minutes and weight immediately 7 GRAS ASSOCIATES, LLC Page 19 of 99

72 Stevia Pure next generauon sweetener after removing from dissicator. Correct both sample and standard sample weights with this determination. Chromatography Conditions Column: Supelcosil LC-NH or equivalent (length: em; inner diameter: mm) Mobile phase: A 80:20 mixture of acetonitrile and water (see above) Flow rate: Adjust so that the retention time of rebaudioside A is about 21 min. Injection volume: 5-10 ~-tl Detector: UV at 210 nm Column temperature: 40 Procedure: Equilibrate the instrument by pumping mobile phase through it until a driftfree baseline is obtained. Record the chromatograms of the sample solution and the standard solutions. Measure the peak areas for the seven steviol glycosides from the sample solution (the minor components might not be detected). Measure the peak area for stevioside for the standard solution. Calculate the percentage of each of the seven steviol glycosides, X, in the sample from the formula: %X= (W S/W] x (fxax/ AS] x 100 Where: WS is the amount (mg) of stevioside in the standard solution; W is the amount (mg) of sample in the sample solution; AS is the peak area for stevioside from the standard solution; A X is the peak area of X for the sample solution; and f X is the ratio of the formula weight of X to the formula weight of stevioside: 1.00 (stevioside), 1.20 (rebaudioside A). After verifying WS, WR and W have been verified through the Weight Verification method described above, calculate the total purity of the sample through the sum of the nine percentages. Calculate the percentage of the two main steviol glycosides. Acceptance criteria: NLT 75% of the sum of the two main glycosides. 8 GRAS ASSOCIATES, LLC Page 20 of 99

73 Stevia Pure next generauon sweetener II. Physical Properties: Water Solubility: Water solubility is a characteristic factor of naturally extracted and purified steviol glycosides extracts. Purified steviol glycoside extracts should be completely water soluble at least at 35% wfw water solutions. Preparation of a 35% w/w solution: Weigh accurately 3.5g of the sample and dissolve into 6.5g of deionized water. The sample should be dried previously for 2 hours at 105" C. Leave the sample at 4"C overnight for 12 hours. No deposits or turbidity increase should be formed. Turbidity determination: Should be done by using Hanna Instruments Turbidimeter, Model HI 93703, multi range or equivalent. [Note: This equipment must be monthly calibrated using Calibration Solution HI Hanna Instruments and Calibration Solution HI Hanna Instruments and according to the equipment's calibration procedure included in the Turbidimeter Operation Manual]. Check the calibrated condition of the nephelometer by reading and calibrating with a 0 NTU standard solution. After the calibrated condition of the equipment is verified, the sample left overnight is shaken vigorously, and fed into the sample tube of the equipment. Acceptance Criteria: NMT 35 NTU. loss on Drying: Moisture determination for pure steviol glycosides is done at 105"C for 3 hours A.O.A.C Acceptance Criteria: NMT 6% (105", 2 hours) Thermal Stability: Thermostability is a characteristic factor of pure stevia extracts. Pure Steviol Glycoside Extracts should keep sweetness and water solubility when heated to 95" C during 2 hours. This remains an easy method for detecting adulteration. 9 GRAS ASSOCIATES, LLC Page 21 of 99

74 Stevia Pure next generauon sweetener Ill. Chemical Properties: A) Natural Impurities Ash Weigh three to five well-mixed samples into a shallow, relatively broad ashing dish that has been ignited, cooled in desiccators, and weighed soon after reaching room temperature. Ignite in furnace at ca ssoo ( dull red) until light gray ash results, or to constant weight. Cool in desiccators and weigh soon after reaching room temperature. Reignited CaO is satisfactory drying agent for desiccator. Acceptance Criteria: Not more than 1% Arsenic Note: When water is specified as a diluent, deionized reverse osmosis filtered water must be used. When nitric acid is specified, use nitric acid of HPLC grade for trace element analysis with as low a content of arsenic as is practical. Dilute nitric acid: Dilute 2.0 ml of nitric acid with water to 100 mi. Yttrium Internal standard solution: Use a commercially available 1000!Jg/kg yttrium ICP standard solution. [NOTE: the internal standard should be 20!Jg/kg in all blanks, standards and samples.] Standard stock solution: Dilute a 1000 mg/kg commercially available arsenic ICP standard solution to 1000!Jg/kg with Dilute nitric acid, transfer 10.0 ml of this solution to a 100-ml volumetric flask, add 2.0 ml of Dilute nitric acid, and dilute water to volume (100!Jg/kg). [ NOTE: Prepare this solution fresh every two weeks or before if contamination is suspected.] Standard solution: 10!Jg/kg lead prepared as follows., transfer 5.0 ml of the Standard stock solution to a 50-ml volumetric flask, add 3.0 ml of Dilute nitric acid, add 1.0 ml of Yttrium Internal standard solution, and dilute with water to volume (10!Jg/kg). [ NOTE: Prepare this solution fresh weekly]. Standard blank solution: Transfer 1.0 ml of the Yttrium internal solution to a 50-ml volumetric flask, add 3.0 ml of Dilute nitric acid, and dilute with water to volume. 10 GRAS ASSOCIATES, LLC Page 22 of 99

75 Stevia Pure next generauon sweetener Sample Solution [ CAUTION: Wear proper eye protection, protective clothing, and gloves during sample preparation. Closely follow the manufacturer's safety instructions for use of the microwave digestion apparatus.] Transfer 500 mg of sample into a Teflon digestion vessel liner. Prepare samples in duplicate. Add 10 ml of nitric acid, and swirl gently. Cover the vessels with lids, leaving the vent fitting off. Predigest for at least 1 hour under a hood. Place the rupture membrane in the vent fitting, and tighten the lid. Place all vessels on the turntable of a microwave oven with a magnetron frequency of about 2455 MHz and a selectable output power of 0 to 950 watts in 1% increments, equipped with advanced composite vessels with 100-ml poly Teflon liners (ref 2). Connect the vent tube to the vent trap, and connect the pressure-sensing line to the appropriate vessel. Initiate a two-stage digestion procedure by heating the microwave at 15% power for 10 min, followed by 25% power for 10 min. Remove the turntable of vessels from the oven, and allow the vessels to cool to room temperature (a cool water bath may be used to speed the cooling process). Vent the vessels when they reach room temperature. Transfer the cooled digests into 50-ml volumetric flasks, add 1.0 ml of the Yttrium internal standard solution, and dilute with deionized water to volume. Spectrophotometric system, Plasma Spectrochemistry, Appendix IIC Mode: Inductively coupled plasma-mass spectrometer (ICP-MS) ICP-MS: use a system equipped with a quadropole mass spectrometer and an ion detector maintained under vacuum; the system may include a suppression system to mitigate interference from the 40 Ar 35 Cl' ion. If not, correction for this interference must be determined by a suitable method, such as that recommended by the instrument manufacturer. The isotope ratio of 40 Ar 35 0 Cl+/ Ar 37 Cl+ in the Standard blank solution may be used to correct this interference. Analysis: [ Note: Instrument performance must be verified to conform to the manufacturer's specifications for resolution and sensitivity. Before analyzing samples, the instrument must pass a suitable performance check.] Aspirate from the Standard blank solution should not yield a significant intensity for arsenic. Calculate the internal standard ratios for the Sample solution and Standard solution as ratio of the arsenic to yttrium intensities. Calculate the concentration (mg/kg) of arsenic in the sample taken using the following formula: (Ru/Rs) x Cs x (50/S) Ru: Internal standard (arsenic response/yttrium response) from the Sample solution. Rs: Internal standard ratio (arsenic response /yttrium response) from the Standard solution. Cs: Concentration of arsenic in the Standard solution (J.tg/kg) 11 GRAS ASSOCIATES, LLC Page 23 of 99

76 Stevia Pure next generauon sweetener 50: Sample dilution factor S: Weight of sample used to prepare the Sample solution (mg) Acceptance criteria: NMT than 1 mg/kg lead Note: When water is specified as a diluent, deionized reverse osmosis filtered water must be used. When nitric acid is specified, use nitric acid of HPLC grade for trace element analysis with as low a content of lead as is practical. Dilute nitric acid: Dilute 2.0 ml of nitric acid with water to 100 mi. Thalium Internal standard solution: Use a commercially available 1000 ~g/kg thallium ICP standard solution. [NOTE: The internal standard should be 20 ~g/kg in all blanks, standards and samples.] Standard stock solution: Dilute a 1000 mg/kg commercially available lead ICP standard solution to 1000 ~g/kg with Dilute nitric acid, transfer 10.0 ml of this solution to a 100- m I volumetric flask, add 2.0 ml of Dilute nitric acid, and dilute water to volume (100 ~g/kg). [ NOTE: Prepare this solution fresh every two weeks or earlier, if contamination is suspected.] Standard solution: 10 ~g/kg lead prepared as follows., transfer 5.0 ml of the Standard stock solution to a 50-ml volumetric flask, add 3.0 ml of Dilute nitric acid, add 1.0 ml of Thallium Internal standard solution, and dilute with water to volume (10 ~g/kg). [ NOTE: Prepare this solution fresh weekly]. Standard blank solution: Transfer 1.0 ml of the Thallium internal solution to a 50-ml volumetric flask, add 3.0 ml of Dilute nitric acid, and dilute with water to volume. Sample Solution [ CAUTION: Wear proper eye protection, protective clothing, and gloves during sample preparation. Closely follow the manufacturer's safety instructions for use of the microwave digestion apparatus.] Transfer 500 mg of sample into a Teflon digestion vessel liner. Prepare samples in duplicate. Add 10 ml of nitric acid, and swirl gently. Cover the vessels with lids, leaving the vent fitting off. Predigest for at least 1 hour under a hood. Place the rupture membrane in the vent fitting, and tighten the lid. Place all vessels on the turntable of a microwave oven with a magnetron frequency of about 2455 MHz and a selectable 12 GRAS ASSOCIATES, LLC Page 24 of 99

77 Stevia Pure next generauon sweetener output power of 0 to 950 watts in 1% increments, equipped with advanced composite vessels with 100-ml poly Teflon liners (ref 2). Connect the vent tube to the vent trap, and connect the pressure-sensing line to the appropriate vessel. Initiate a two-stage digestion procedure by heating the microwave at 15% power for 10 min, followed by 25% power for 10 min. Remove the turntable of vessels from the oven, and allow the vessels to cool to room temperature (a cool water bath may be used to speed the cooling process). Vent the vessels when they reach room temperature. Transfer the cooled digests into 50-ml volumetric flasks, add 1.0 ml of the Thallium internal standard solution, and dilute with deionized water to volume. Spectrophotometric System, Plasma Spectrochemistry, Appendix IIC Mode: Inductively coupled plasma-mass spectrometer (ICP-MS) ICP-MS: Use a system equipped with a quadropole mass spectrometer and an ion detector maintained under vacuum; the instrument should read all the isotopes for lead (206, 207, and 208 amu) and the thallium internal standard (205 amu), and should report the total lead content using the most naturally abundant isotope at 208 amu. Analysis: [ Note: Instrument performance must be verified to conform to the manufacturer's specifications for resolution and sensitivity. Before analyzing samples, the instrument must pass a suitable performance check.] Aspirate from the Standard lank solution should not yield a significant intensity for arsenic. Calculate the internal standard ratios for the Sample solution and Standard solution as ratio of the lead to the thallium intensities. Calculate the concentration (mg/kg) of arsenic in the sample taken using the following formula: (Ru/Rs) x Cs x (50/5) Ru: Internal standard (lead response/thallium response) from the Sample solution. Rs: Internal standard ratio (lead response /thallium response) from the Standard solution. Cs: Concentration of lead in the Standard solution (llg/kg) 50: Sample dilution factor S: Weight of sample used to prepare the Sample solution (mg) Acceptance criteria: NMT 1 mg/kg, calculated on the anhydrous basis. B) Added Impurities: Organic Impurities: 13 GRAS ASSOCIATES, LLC Page 25 of 99

78 Stevia Pure next generauon sweetener Ethanol and Methanol: No ethanol or methanol should be used in the production of naturally purified steviol glycoside extracts. IV. REFERENCES AOAC Official Method Japan Food Additive Association. Stevia extract. In Voluntary Specifications of Non Chemically Synthesized Food Additives (2nd Ed.), Japan Food Additive Association (Ed.), Tokyo,pp N. Kolb, J. L. Herrera, D. J. Ferreyra, and R. F. Uliana. Analysis of Sweet Diterpene Glycosides from Stevia rebaudiana: Improved HPLC Method. J. Agric. Food Chern., 49 (10), , Mizutani, K and Tanaka, 0. Use of Stevia rebaudiana sweeteners in Japan. In Stevia. The genus Stevia. Edited by Kinghorn, Douglas. A, Departament Medicinal Chemistry and Pharmacognosy. Un iversity of Illinois at Ch icago. USA. Taylor and Francis Group. New York, Moore, Jeffrey. Rebaudioside A Monograph, US Pharmacopeia, JECFA Standards. Monographs 5 (2008), supersed ing specifications prepared at the 68th JECFA (2007), published in FAO JECFA Monographs 5 (2008). Prepared at the 69th JECFA (2008), published in FAO JECFA JECFA Standards Monograph 10 (2010), prepared at the 73rd JECFA (2010) and published in FAO JECFA Monographs 10 (2010). 14 GRAS ASSOCIATES, LLC Page 26 of 99

79 APPENDIX D HPLC Analysis Report for Five Lots of Stevia Pure GRAS ASSOCIATES, LLC Page 27 of 99

80 .. 1) Proprietary Process for Stevia Pure Water Purified Steviol Glycosides Extract Stevia Pure extract, produced by Productora Alysa SPA, is a steviol glycosides purified extract containing> 95% of glycoside content on a weight to weight basis. Within glycosides, approximately 65% is Rebaudioside A and the rest is a combination of Stevioside, Rebaudioside C, Rebaudioside D and other native steviol glycosides considered in the JECFA 2010 analytical methodology. The process starts with an extraction of leaves with membrane deionized water only, yielding a an extract with 35% of purity in glycosides and a profile of glycosides with about 65% w/w on Rebaudioside A (variable depending on distribution of glycoside content of the leaves). The aqueous extract is purified to 65-70"/o using membrane filtration system only. Mechanical separation takes place at this stage, and steviol glycosides, mainly Rebaudioside A and Stevioside, are screened from large molecular weight and low molecular weight compounds. The resulting extract is finally polished with water eluted food grade ion exchange resin to achieve minimum glycoside specifications. At this stage, nothing other than membrane deionized water is used in the process. The polished extract is then concentrated to 30" Brix for spray drying. Following pasteurization at 85"C for 55 minutes, the pasteurized product is spray dried. During the spray drying process, an inlet temperature of 210"C is utilized, and the outlet temperature is 95"C to reduce moisture of the powdered product to below 5%. The process is produced under an HACCP, ISO 2000 Certified process, under Good Manufacturing Practices. Moreover, the fact that our process is not using any organic solvent in the product is certified and permanently audited by Bureau Veritas under the ISO CASCO 5 Certification. Calle Tres N 600, Quilpue, Chile Telephone: GRAS ASSOCIATES, LLC Page 28 of 99

81 .. Table 1: Process Stages, Operation Ranges, Ingredients, Equipments and Process Aids. Process Stage Operation Ranges Ingredients Equipment Process Aids Raw Material Moisture < 8% Stevia Leaves No Process Aids Selection Rebaudioside Content> 60% Water Extraction Temperature 20 C Reverse Osmosis Deionized Water Temperature > 10 C, < Membrane 4o c. Purification Steviol Glycosides purified 60 to 90% 2o c Deionized Water Eluted No Process Aids Food Grade PES (poly-ethylene Stevia Extract sulphide) spiral No Process Aids wound membranes. Water treatment food lon exchange Process Stevia Extract grade ion No Process Aids Steviol Glycosides Purified exchange above 9S% resins. Food Grade PES Membrane Solid Content> 30% Stevia Extract spiral wound Concentration membranes. No Process Aids Particles and all suspended Food Grade PES Microfiltration solids > 0,2 micrometers Stevia extract spiral wound No Process Aids removed. membranes. Time 55 min Pasteurization Temperature 852C Stevia Extract No Process Aids Spray Drying Stevia Extract Food Grade Temperature: 2102C Plastic Liners Moisture < 8% PEAD inside No Process Aids cardboard boxes. Product Release Full Product Analysis Stevia Pure No Process Aids Flow Diagram 1: Process flow diagram of Stevia Pure production. Calle Tres N 600, Quilpue, Chile Telephone: GRAS ASSOCIATES, LLC Page 29 of 99

82 I Process Operation Process Ingredient Calle Tres N 600, Quilpue, Chile Telephone: GRAS ASSOCIATES, LLC Page 30 of 99

83 2) Analytic Methodology for Stevia Pure 95% Sweetener Steviol Glycosides High Performance Liquid Chromatography (HPlC) is used for glycoside determination both for the raw materials (leaves) and the final product {free flowing white powder). There are several methods used for the determination of steviol glycosides (Mizutani, JECFA 2008, JECFA 2010). While Jecfa 2008 is precise for the determination of Rebaudioside A and Stevioside, JECFA 2010 is good for the determination of minor glycosides as well as for the quantification of Rebaudioside A, whenever the extract does not contain a relevant concentration of Stevioside. While we propose JECFA 2010 as an official method for this GRAS assessment, we also recommend to have JECFA 2008 as a backup method for determinations whenever coelution of Rebaudioside A and Stevioside make the areas of these two compounds difficult to integrate. The proposed combination of methods is attached as Appendix A. Calle Tres N 600, Quilpue, Chile Telephone: GRAS ASSOCIATES, LLC Page 31 of 99

84 .. 3) HPLC Analysis for retained lots. a) Lot (b) (6) Lot(b) (6) (1l (2) (Sl (4) (6) (7) = IW (1)11131' 12l'l10ll' 161 Stand. Area Cone. Component Retention Area Weight (g/kg) Stand [(ppm) (c) fx % RebD 3, ,0450 1,84 1,40 2,95 Reb A (a) 7, ,6905 1, , ,80 Stev (b) 8, ,5780 1, , ,00 15,77 RebF 10, ,9040 1,84 1,16 1,09 RebC 11, , ,84 1,18 7,01 DulcA 11,933 53,7475 1,84 0,98 0,38 Rubusoside 15, ,4565 1,84 0,80 0,80 RebB 23,050 74,5530 1,84 1,00 0,54 Steviolbioside - NO 1,84 0,80 ND 97,34 * Note: Stevioside, Rebaudioside D, F, C and B, Dulcoside A, RubllSoside and Steviolbioside cale~liated by using Stevio~ide Standard and Rebaudioside A calculated by using Rebaudioside A Standard, as in accordance to JECFA Where fx is the ratio of the formula weight of glycoside to the fonmlia weight ofstevioside. a) Standard Obtained from Chromadex, Rebaudioside A Lot I. Purity - 95,60 b) Standard Obtained from Chromadex, Stevioside Lot Purity = 95,39 c) Sll:mdard Concentration take in to account standard purity Calle Tres N 600, Quilpue, Chile Telephone: GRAS ASSOCIATES, LLC Page 32 of 99

85 .. Figure 2: Lot 292 A b) Lot (b) (6) Lot (b) (6) ( 1) (2) (3) (4 ) (6) (7) = (4 ). (1)/((3). (2) ' (10)). (6) Stand. Area Cone. Component Retention Area Weight (g/kg) Stand (ppm) (c) fx % RebD 3, ,3395 1,79 1,40 2,99 Reb A (a) 8, ,0160 1, ,48 Stev (b) , ,00 15,33 Reb F 10, ,0390 1,79 1,16 1,03 RebC 11, ,4140 1,79 1,18 6,72 Dulc A 12,166 48,6630 1,79 0,98 0,35 Rubusoside 16, ,1955 1,79 0,80 0,79 Reb B 23,250 77,6140 1,79 1,00 0,57 Steviolbioside - NO 1,79 0,80 NO 97,26 Note: Stevioside, Rebaud ioside D, F, C and B, Dulcoside A, Rubusoside and Steviolbioside calculated by using Stevioside Standard and Rebaud ioside A calculated by using Rebaudioside A Standard, as in accordance to JECFA Where fx is the ratio of the formula weight of glycoside to the formula weight of stevioside. a) Standard Obtained from Chromadex, Rebaudioside A lot Purity= 9S,60 b) Standard Obtained from Chromadex, Stevioside lot Purity = 95,39 c) Standard Concentration take in to account standard purity Calle Tres N 600, Quilpue, Chile Telephone: GRAS ASSOCIATES, LLC Page 33 of 99

86 .. Figure 3: Lot 297 B HPLC analysis. cl Lot Lot(b) (6) (b) (6) (1l (2) (3 ) {4) (6) (7) = (4)' {11/{(3)' f2l'{10ll' (6) Stand. Cone. Area (ppm) Component Retention Area Weight (g/kg) Stand (C) fx % RebD 5, ,0000 1,82 1,40 3,46 Reb A (a) 8, ,0000 1, , ,07 Stev (b) 8, ,0000 1, , ,00 14,69 Reb F 10, ,0000 1,82 1,16 1,04 RebC 10, ,0000 1,82 1,18 7,03 DulcA 12,116 51,6700 1,82 0,98 0,37 Rubusoside 16, ,0000 1,82 0,80 2,31 Reb 8 22,466 85,0000 1,82 1,00 0,62 Steviolbioside - NO 1,82 0,80 NO 96,59 Note: St evioside, Rebaudioside D, F, C and B, Dulcoside A, Rubusoside and Stev iolbioside ca lculated by using Stevioside Standard and Rebaudioside A calculated by using RebaudiosideA Standard, as in accordancetojecfa Where fx is the ratio of t he formula weight of glycoside to the f ormula weight of stevioside. a) Standard Obtained from Chromadex, Rebaudioside A lot Purity= 95,60 Calle Tres N 600, Quilpue, Chile Telephone: GRAS ASSOCIATES, LLC Page 34 of 99

87 b) Standard Obtained from Chromadex, Stevioside Lot Purity= 95,39 c) Standard Concentration take in to account standard purity ~- "' "' (!) Figure 4: Lot (b) (6) HPLC Analysis d)lot (b) (6) Lot (b) (6) Component 1(1) Retention Area Reb D 4, ,0000 Reb A (a) 8, ,2000 Stev (b) 8, ,0000 Reb F 9, ,0000 Reb C 10, ,0000 DulcA 11,816 72,0000 Rubusoside 15, ,0000 Reb 8 21,416 45,0000 St eviolbioside - ND (2) Weight (g/kg) (3) Area Stand 1,85 1, ,5 1, ,8 1,85 1,85 1,85 1,85 1,85 1,85 (4) (6) Stand. Cone. (ppm) (c) fx 1, ,00 1,16 1,18 0,98 0,80 1,00 0,80 17) = (4) ' (1)1((.3)' (2) ' (10)) (6) % 3,80 65,58 15,15 1,11 8,72 0,50 1,16 0,32 ND 96,35 GRAS ASSOCIATES, LLC Page 35 of 99

88 "Note: Stevioside, Rebaudioside D, F, C and B, Dulcoside A, Rubusoside and Steviolbioside calculated by using Stevioside Standard and Rebaudioside A calculated by using Rebaudioside A Standard, as in accordance to JECFA Where fx is the ratio of the formula weight of glycoside t o the formula weight of stevioside. a) Standard Obtained from Chromadex, Rebaudioside A Lot Purity= 95,60 b) Standard Obtained from Chromadex, Stevioside Lot Purity = 95,39 c) Standard Concentration take in to account standard purity rg_ co., > Ui 0>.0., LL cr: ~~ "' co ""- a "' cr;_ (.) 0 "' o;_ "'- ~ "' <{ "'..'!.0 0 " cr: " A "' :::;:_ r::; m.0., cr: Figure 5: Lot 299 e) Lot (b) (6) Lot (b) (6) Component Retention Reb D 5,033 Reb A (a) 8,016 Stev (b) 8450 Reb F 9,616 Reb C 10,283 DulcA Rubusoside 15,266 Reb B 20,683 Steviolbioside - (1) Area 323, , , , , ,0000 ND (2) (3) Area Weight (g/kg) Stand 1, 78 1, , ,8 1, 78 1, , 78 1, 78 1, 78 (4) Stand. Cone. (ppm) (c) (6) fx 1,40-1,00 1,16 1,18 0,98 0,80 1,00 0,80 (7) = (4)' (1 ) I ((3) ' (2) ' (1 0 )) ' (6) % 3,36 63,81 17,71 1,07 6,41 0,50 1,66 0,61 ND Calle Tres N 600, Ouilpue, Chile Telephone: vwwv.prodalysa.cl GRAS ASSOCIATES, LLC Page 36 of 99

89 95,14 * Note: Stevioside, Rebaudioside D, F, C and B, Dulcoside A, Rubusoside and Steviolbioside calrulated by using Stevioside Standard and Rebaudioside A calculated by using Rebaudioside A Standard, as in accordance to JECFA Where fx is the ratio of the formula weight of glycoside to the formula weight of stevio side. a) Standard Obtained from Chromadex, Rebaudioside A lot Purity= 95,60 b) Standard Obtained from Chromadex, Stevioside Lot Purity= 95,39 c) Standard Concentration take in to account standard purity 0...,._ "' ro.. > (') Ui (') a _ "" ro N _ ld 0 <D (DO <D 0 N _ "' "" ro!1 <D_ <D_ ~ :::: ;'? 0 (]).<> N u... <.: "' => m ~~ ".<>.<> :; =>.. 0 0:: 0:: A A. (b) (6) Fig 6. HPLC for Lot f) Rebaudioside A Standard Standard Obtained from Chromadex, Rebaudioside A Lot Purity= 95,60 Component Retention Area Height Reb D 3,383 78,914 3,157 RebA 7, , ,731 Stev 8, ,992 12,444 Reb B 22,616 93,294 3,021 GRAS ASSOCIATES, LLC Page 37 of 99

90 .. Figure 6. Rebaudioside A Standard g) Standard Obtained from Chromadex, Stevioside Lot Purity= 95,39 Component Retention Area Height RebA 7, , ,502 Stev 8, , ,184 Steviolb 24,133 86,614 2,584 Figure 7. Stevioside Standard a) Phenolics Phenolic compounds are found commonly in fruits, coffee, green tea, chocolate and wine. During the process of purification of steviol glycosides, most phenolics are permeated out of Calle Tres N 600, Quilpue. Chile Telephone: www prodalysa cl GRAS ASSOCIATES, LLC Page 38 of 99

91 .. the extract. The remammg phenolics can be quantified precisely by the Folin Cicalteau method (referenced and attached below) and commonly used for determining the finest quality of wines. As an example, phenolic maturity is a critical indicator of good wine quality. The Folin Cicalteau reagent method is as follows for total phenolics measurements: 1) Get aliquots of extract in test tubes (between 0.02 and 0.1 ml of extract), filling to 0,5 ml with distilled water, 0,25 ml of Folin Cicalteau reagent and 1,25 ml of a sodium carbonate solution. 2) Measure absorbance after 40 min at 725 nm and record value. 3) Calculate phenolics as total tannic acid equivalents from the calibration table described in the method. Phenolic levels in lots 273 A, 273 B, 289 are below 1% w/w, as our process is validated to produce lots with phenolic contents below a 1%, when getting purities above 95%. All of our lots achieving the steviol glycosides purities fulfill this requirement. During the process validation all residual phenolic contents were determined in final products, and examples of this are the following lots: Lot (b) 0,42% Lot (6) 0,46% Lot 0,76% Lot 0,54% Lot 0, 65% Moreover, some specific phenolic fractions normally linked to health benefits can be determined using UV Vis detector HPLC methods (Donovan et al, 1998 ) by using specific standards for these components. We determine phenolic indicators such as Gallic Acid, Epichatechins and Catechins not as standard quality control, but only upon request of customers. It can be observed qualitatively in the HPLC below that phenolics have been removed from the product during the purification process (Figures 4 and 5). Example of finished product: Calle Tres N 600, Quilpue, Chile Telephone: GRAS ASSOCIATES, LLC Page 39 of 99

92 .. 4.1) Standard Measurement: Component Areas Retention Time Code Gallic Acid 5812,95 13,25 (1) Catechin 1368,69 35,883 Caffeic Acid 7343,28 37,983 E pichateq ui n 1576,80 41,433 Standard Concentration m InJection: All at 50 ppm (2). Gallic Ac1d Standard from Riedel de Haen (99 %, GmbH), Catechin from Fluka (96%, Switzerland), caffeic Acid from Fluka (95 %, Switzerland) and Epichatequin from Sigma (96 %, Switzerland) Figure 4: Standard Phenolic Chromatogram for solution of 4 standards: Gallic Acid (Retention Time 13,250), Catechin (Retention Time 35,883), Caffeic Acid (Retention Time 37,983) and Epichatequin (Retention Time 41,433) 4.2) Final Product Specific Phenolic Fraction Determination Component Areas Retention Area Code Gallic Acid 24,021 13,450 (3) Catechin 0 35,85 Caffeic Acid 0 37,95 Epichatequin 0 41,5 Dry Weight of Injection: 2130 ppm (4), Gallic Acid Determination (5) = (2) I (1) x (3) /(4) x 100 = 0,0096% (96 ppm) Calle Tres N 600, Quilpue, Chile Telephone: GRAS ASSOCIATES, LLC Page 40 of 99

93 .. Figure 5: Final Product Flat Phenolic Chromatogram Steviol Glycosides Methods: Reference JECFA 2010 JECFA 2008 Stevia Pure Integration of JECFA Methods with Dry Weight Determination Adjustement Polyphenol methods Reference Folin Ciocalteu: Makkar, H.P.S., Bluemmel, M., Borowy, N.K., Becker, K. Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods. J. Sci. Food Agric. 61: Makkar, H. Quantification of Tannins in Tree and Shrub Foliage. A Laboratory Manual. Kluwer Academic Publishers. The Netherlands. pp Reference Specific Phenolic Method: Gallic Acid, Catechins, Epicatechins, Caffeic Acid, Quercetin, within others. Donovan, J. L., McCauley, J.C., Tobella Nieto, N., Waterhouse, A. Effects of Small-scale tinning on the phenolic composition and antioxidant activity of merlot wine. In Chemistry of Wine Flavor. Calle Tres N 600, Quilpue, Chile Telephone: GRAS ASSOCIATES, LLC Page 41 of 99

94 .. Edited by Waterhouse, Andrew and Ebeler, Susan. American Chemical Society. Washington DC, (b) (6) (b) (6) Gene:~:gber M.Sc. Chemical Engineering Prodalysa SPA Calle Tres N 600, Quilpue, Chile Telephone: GRAS ASSOCIATES, LLC Page 42 of 99

95 APPENDIX E Certificates of Analysis for Multiple Production Batches of Stevia Pure E-1 Certificate of Analysis for Stevia Pure E-2 Certificate of Analysis for Stevia Pure E-3 Certificate of Analysis for Stevia Pure E-4 Certificate of Analysis for Stevia Pure E-5 Certificate of Analysis for Stevia Pure (b) (6) GRAS ASSOCIATES, LLC Page 43 of 99

96 E-1 Certificate of Analysis for Stevia Pure 289A STEVIA PURE next generation sweetener Product Certificate of Analysis Product Manufacturer Lot : Stevia Pure in Powder : Productora Alysa SPA : (b) (6) Properties Appearance Moisture Specification White to Light yellow < 6% > 60% BATCH W: White to light yellow 2,86 67,07 (b) (6) ph (of 1% solution) Ash Arsenic Lead > 95% 4,5-7,0 < 1% < 5000 m < 1 mg/kg < 1 mg/kg No Additives 96,59 5,33 < 1 N/A* < 1 < 1 No Additives c solvents are used during the processing of Stevia Pure. The full process is run under itions. (b) (6) (b) (6) Javi re oductora Alysa SPA. Calle Tres N 600, Belloto Norte, Quilpue, Chile Phone I Fax: GRAS ASSOCIATES, LLC Page 44 of 99

97 STEVIA PURE next generation sweetener MICROBIOLOGY (b) (6) Analysis Specification BATCH W: Methodology Aerobic Plate Count (ufclg) < 2000 < 10* NCh 2659 Of.2002 Mold < 100 NCh 2734 < 10* (u f c I g) Of Yeast <100 NCh 2734 < 10* (u f c I g) Of Total Coliforms <3 NCh 2635/1 < 3* NMP/g Of Escherichia coli <3 < 3* NCh 2636 NMPig Of Salmonella NCh 2675 Negative/Positive Negative Negative Of (2,f?g} U.S.P. XXII Pseudomonas Microbiological aeruginosa Negative Negative Test I Negative/Positive Microbiological (10 g) Limit Test (61) Staphylococcus < 3 < 3* NCh 2828 au reus, NMP I g Of * < 1nd1cate absence, below l1m1t of detection. Calle Tres N 600, Belloto Norte, Quilpue, Chile Phone I Fax: GRAS ASSOCIATES, LLC Page 45 of 99

98 E-2 Certificate of Analysis for Stevia Pure 292A STEVIA PURE next generation sweetener Product Certificate of Analysis Product Manufacturer Lot : Stevia Pure in Powder : Productora Alysa SPA : (b) (6) Laborato (b) (6) Appearance Moisture Rebaudioside A (%of dry weight) Total Steviol Glycosi (%of dry weight) ph (of 1% solution) Ash Residual Solvents Arsenic Lead Additives > 60% > 95% 4,5-7,0 < 1% < 200 mg/kg Methanol < 5000 mg/kg Ethanol < 1 mg/kg < 1 mg/kg No Additives 68,8 97,34 5,49 < 1 N/A* < 1 < 1 No Additives (b) (6) c solvents are used during the processing of Stevia Pure. The full process is run under itions. (b) (6) Javi ure Productora Alysa SPA. Calle Tres N 600, Belloto Norte, Quilpue, Chile Phone I Fax: GRAS ASSOCIATES, LLC Page 46 of 99

99 STEVIA PURE next generation sweetener MICROBIOLOGY (b) (6) Analysis Specification BATCH W: Methodology Aerobic Plate Count (ufclg) < 2000 < 10* NCh 2659 Of.2002 Mold < 100 NCh 2734 < 10* (u f c I g) Of Yeast <100 NCh 2734 < 10* (u f c I g) Of Total Coliforms <3 NCh 2635/1 < 3* NMP/g Of Escherichia coli <3 < 3* NCh 2636 NMPig Of Salmonella NCh 2675 Negative/Positive Negative Negative Of (2,f?g} U.S.P. XXII Pseudomonas Microbiological aeruginosa Negative Negative Test I Negative/Positive Microbiological (10 g) Limit Test (61) Staphylococcus < 3 < 3* NCh 2828 au reus, NMP I g Of * < 1nd1cate absence, below l1m1t of detection. Calle Tres N 600, Belloto Norte, Quilpue, Chile Phone I Fax: GRAS ASSOCIATES, LLC Page 47 of 99

100 E-3 Certificate of Analysis for Stevia Pure 297B STEVIA PURE next generation sweetener Product Certificate of Analysis Product Manufacturer Lot : Stevia Pure in Powder : Productora Alysa SPA :(b) (6) Laborato Properties sis Specification BATCH W: (b) (6) Appearance White to Light yellow < 6% > 60% > 95% 4,5-7,0 < 1% White to light yellow 3,36 69,48 97,26 5,08 < 1 N/A* < 1 mg/kg No Additives < 1 < 1 No Additives (b) (6) (b) (6) c solvents are used during the processing of Stevia Pure. The full process is run under itions. re roductora Alysa SPA. Calle Tres N 600, Belloto Norte, Quilpue, Chile Phone I Fax: GRAS ASSOCIATES, LLC Page 48 of 99

101 STEVIA PURE next generation sweetener MICROBIOLOGY Analysis S pacification BATCH W:297 B Methodology Aerobic Plate Count (ufclg) < 2000 < 10* NCh 2659 Of.2002 Mold < 100 NCh 2734 < 10* (u f c I g) Of Yeast <100 NCh 2734 < 10* (u f c I g) Of Total Coliforms <3 NCh 2635/1 < 3* NMP/g Of Escherichia coli <3 < 3* NCh 2636 NMPig Of Salmonella NCh 2675 Negative/Positive Negative Negative Of (25 g) U.S.P. XXII Pseudomonas Microbiological aeruginosa Negative Negative Test I Negative/Positive Microbiological (10 g)... ~ir!jit I~?!J JJ Staphylococcus <3 < 3* NCh 2828 au reus, NMP I g Of * < 1nd1cate absence, below l1m1t of detection. Calle Tres N 600, Belloto Norte, Quilpue, Chile Phone I Fax: GRAS ASSOCIATES, LLC Page 49 of 99

102 E-4 Certificate of Analysis for Stevia Pure 299B STEVIA PURE next genera tion sweetener Product Certificate of Analysis Product Manufacturer Lot : Stevia Pure in Powder : Productora Alysa SPA. (b) (6).~.~~~!.~~~. r:y.. A!'!.~.I.Y.~!~..."!,!,!, BATCH N" (b) (6)!,.'...:.~~ ~ ~~~~~...".... ~ ~~~.;~~~~~~~...:... :! Appearance White to Light yellow! White to fight yellow! r ;.;~~~~~~... T... ~ 6 %... T... 3:5o... i ;... -i... ;... ; : Rebaudioside A : : :! (%of dry weight)! > 60%! 65,58! > <( ~ :! Total Steviol Glycosides!! 96,35!! (%of dry weight)! > 95%!! r ~;; i~i.1%. ~i~;i ~~i... T... 4:;; ::. :r:a... T... 4:7o... i C:f~~~~~~: ~~~e~ts:::::::::t: :::: :<:~~~ :i;,t :~:et~~n~i ::::::J:::: :::: ::::::::::::~~> ::: ::::::::::::::J ;. < 5000 mg/kg Ethanol ; ; t: :::~~~~<::: :::: :::: :::::::::::r :::: :::: ::::::~:~::~:~~~ ::: :::::::::::::1: :::: :::: ::::::::::::: ~ :~:: :::: ::::::::::::::: J! Lead! < 1 mg/kg! < 1! r Adciitives... y ;;~t.:cid~~~~.. -r.. N ~:;.:ci;;~i~~~...! ' ! : No Org c solvents are used during the processing of Stevia Pure. The full process is run under aqueous o ditions. (b) (6) (b) (6) ure roductora Alysa SPA. Calle Tres N 600, Belloto Norte, Quilpue, Chile Phone I Fax: GRAS ASSOCIATES, LLC Page 50 of 99

103 STEVIA PURE next generation sweetener MICROBIOLOGY ~ (b) (6) Analysis Specification BATCH W: Methodology... (.... Aerobic Plate Count! NCh 2659 < 2000 < 10 (u f c /g)! Of ~ Mold (u f c / g) < 100 < 10 ' ' ' <1 00 < 10. NCh 2734 Of ( ooo 0000 o ooo ooo Yeast (u f c / g) NCh 2734 Of ooo o ooo ooo ooo o(o ooo ooo ooo ooo ooooo ooo ooo ooo ooo ooo Total Coliforms NMP / g < 3 < 3 NCh 2635/1 Of '"" "'""'""'""' "" "" "'""'"'!'""' "" "" "'""'""'""' ""' ""'""'""'""' "" "" "'"" ""'""' "" "" "'""'""'"" Escherichia coli! < 3 < 3 NCh 2636 NMP / g : Of sa; iiioiieii<i T" -~ ~ ~-~~ ~ ~... ~1;t~~~~~:.~~~--- ]... :~~~~~~ ~-~~~~~~ ~~:-~~~~ --- U.SP XXII Pseudomonas aeruginosa Microbiological. 'P.ti Negative Negative Test/ N ega t tve,, ost ve M. b. 1 1 (10 g) ICrO 10 OQICa... J!~.11 I~!?t(f?.U.... Staph_vlococcus! NCh 2828 < 3 < 3 aureus, NMP I g! Of <. iii<iicaie.a&seice: i,-eiow. i"ri i c.tcieiecii-.;ii: GRAS ASSOCIATES, LLC Page 51 of 99

104 E-5 Certificate of Analysis for Stevia Pure (b) (6) STEVIA PURE next generation sweetener Product Certificate of Analysis Product Man ufa ctu re r Lot : Stevia Pure in Powder : Productora Alysa SPA (b) (6) BATCH W: (b) (6) Rebaudioside A (%of dry weight) Total Steviol Glycosides (%of dry weight) > 60% > 95% ph (of 1% solution) 4,5-7,0 Ash < 1% Residual Solvents < 200 mg/kg Methanol < 5000 mg/kg Ethanol 63,81 95,14 4,80 < 1 N/A* Arsenic Lead ves < 1 mg/kg < 1 mg/kg No Additives < 1 < 1 No Additives (b) (6) (b) (6) c solvents are used during the processing of Stevia Pure. The full process is run under itions. re roductora Alysa SPA. Calle Tres N 600, Belloto Norte, Quilpue, Chile Phone I Fax: GRAS ASSOCIATES, LLC Page 52 of 99

105 STEVIA PURE next generation sweetener MICROBIOLOGY Analysis Specification BATCH W(b) (6) Methodology Aerobic Plate Count (ufclg) < 2000 < 10* NCh 2659 Of.2002 Mold < 100 NCh 2734 < 10* (u f c I g) Of Yeast <100 NCh 2734 < 10* (u f c I g) Of Total Coliforms <3 NCh 2635/1 < 3* NMP/g Of Escherichia coli <3 < 3* NCh 2636 NMPig Of Salmonella NCh 2675 Negative/Positive Negative Negative Of (2,f?g} U.S.P. XXII Pseudomonas Microbiological aeruginosa Negative Negative Test I Negative/Positive Microbiological (10 g) Limit Test (61) Staphylococcus < 3 < 3* NCh 2828 au reus, NMP I g Of * < 1nd1cate absence, below l1m1t of detection. Calle Tres N 600, Belloto Norte, Quilpue, Chile Phone I Fax: GRAS ASSOCIATES, LLC Page 53 of 99

106 APPENDIX F Pesticide Analytical Reports for Stevia Pure from Analab F-1 Test Report for Pesticides for Stevia Pure 360 F-2 Test Report for Pesticides for Stevia Pure 373 GRAS ASSOCIATES, LLC Page 54 of 99

107 F-1 Test Report for Pesticides for Stevia Pure 360 Analab ldenrincaci6n del C liente Nombre Atenci6n Direcci6n Ciudad TeiCfono Analab- Informc Resultados N Este lnfonnc consla de I mue>lm(s) RUT del Chente: Este lnfonnc fuc emitido cl II de enc de 2014 a las 10:49:07 am. PRODUCTORA ALYSA SPA : SRA. ANDREA BELANCIC : CALLE TRES N 600 : QUILPUE : Dato ~ de Ia Solicitud Condicion de Pago : CONTRA FACTURA Plazo de Entrega : Comuno Ematl I de 2 Lab.: RESIDUOS DE PESTICIOAS Original. Clii!ntl! Muestra : Clave Tipo Subtipo LOD LOQ ANAL/SIS SOLICIT ADOS: MUL TIRES/DUOS : STEVIA PURE IN POWDER LOTE: 360 : STEVIA Mucstrcado por Fccha de Mucstreo RES/DUOS NO DETECT ADOS (LOD) 0 01 ABAMECTIN 0.01 ACEPHATE 0.01 ALACHLOR ALOICARB 0.01 ALDRIN 0.01 ATRAZINE \ZOXYSTRO!!lN 0.02 BENOMYL 0.01 GAMMA BHC 0.01 BIFENTRIN 0.01 BROI'.ACILO 0.01 BROMOPHOS ETHYL 0.01 BUPIRli'.ATO BUPROFEZI:< CAA3ARYt CARBE:l\'OAZII' HIDROXI~~PURAN KETO CARBOFURAN 0.01 At.PHA CHLOiWANE 0 01 GAMMA CHLORDANE 0.01 CHLORPYRIFOS-ETHYL 0.01 CHLORPYRIPOS-METHYL 0.01 GAMMA CYHALOTRINA 0.01 LAMBDA-CYHALOTHRIN 0.02 CYPROCONAZOLE 0.01 CYPRODINIL 0.01 op DOE 0.01 pp - ODE 0.02 OELTAMETHRIN 0.01 DIAZINON 0.01 OICLORA.~ 0.01 DICHLORVOS 0.01 OIPE'SILAMINA 0.02 DIFESOCONAZOL 0.03 DI T IOCARBAI'.ATO (COMO CSliO.OZ Eh'DOSUt.FAN I 0.01 Elo'DRIN 0.01 ESFE:t.'\/At.ERATO 0.02 PE.~AMIPHOS 0.01 FENARI!~OL 0.01 PENHEXAMID FENITROTHION FBNTHION 0.01 FENVALERATO 0.01 PLUAZINAM FLUOIOXONJ L 0.01 PLUOUINCONAZOLE 0.01 FLUSII..AZOL 0.01 HEPTACHLOR 0.01 HEPTACHLOR BPOXIOE 0.01 HBXAZINCNE 0.01 IMAZA.:.IL 0.01 IPROOIO:\"E: 0.01 KRESOXYM-METHYL 0.02 MECAABAI' 0.01 MEPANIPYRIM 0.03 METHIOCAR METHOXYCHLOR 0.02 METHOMYL 0.01 METRIBUZJNE 0.01 MICLOBUTANIL 0.01 MONOCROTOPHOS 0.01 OXJ\MYL 0.02 OXYF't.OORPEN 0.01 PARATHION-METHYL 0.02 PENCONAZOLE : ELCLIENTE :NO INDICA RESULTADOS mg/kg (ppm) ACETAMlPIUO ALOICARB SULPHONE AZ!NPHOS ETHYL 0.01 ACRINATHRIN 0.02 ALDICARB SULPHOXIDE 0.01 AZINPHOS METHYt At.PHA BHC BETA BHC BITERTANOL BRo.-.oPHOS MFT!Nt. CADUSAI"'S 0.02 BOSCALIO 0.01 BROMOPROPILATO 0.01 CAPT A!\' 0.01 CARBOFl!NO'I'HION C. 02 CARBOFURAN O.Ol CHI NOMETil TON ATE 0.03 CLOPENTEZI:\"E 0.01 Clr..ORFENVINPHOS O.C2 CHLOROTHALOliiL CYANACINA CYHEXI\TTN 0.01 CYPLUTHRII' 0.01 CYPERMETHRIN 0.01 op DOD 0.01 pp - DOD 0.01 op - DDT pp - DOT OICLOBUTRAZOL DICOI"':. :JIMETHOATE 0.01 OICHLOPLUANID 0.01 DIELDRIN 0.01 DISUt.FO'l'ON 0.02 E:NDOSU:.PAN II 0.01 B~'DOSUt.P~~ SULFATE 0.01 ET.-IION 0.01 ETHOPROPHOS 0.01 f'enbuconazole 0.01 FENCHLORFOS PENPROPATHRIN 0.02 FENOXYCARB 0. OS PF.R9AM 0.01 t'lpronil LUI'ENOXURON 0.02 PLUMlOXAZIN 0.02 PLUCYTRINATE 0.01 FLWALINATO 0.01 HEXACHLOR09ENZENE HEXACONAZOLE C.02 IMI:JACLOPRIO 0.02 lnooxacarb c 02 LUFE.':URON 0.01 MALATHIO." C.C2 1-!ETAt.AXY: METHAHIDOPHOS C.02 METOXYFI!!NOZIOE 0.01 METHIOATI!ION 0.01 MEVINPHOS MlREX NAFTOL 0.01 OMETHOIITB 0.01 PACLOBUTRAZOL PARJITHION-ETHYL 0.01 PENOIMETHALIN PENTACt.ORANIZOL A'IALAB CHILE S.A cs labora10no Acrednado par cl NN bajo Nonna 'ICH ISO LE ~ Analob cs adcmis labor..,ono Ofte al ckl Scmcio A&ncol.t y Ganackro en Vmos y Ak:oholcs. Productos Pecuarios. Pl.tgu c das y Fcruhzanlcs dcsdc 1988 y 5" cnc:ucntra rcaioij'>do como l..oborotono Of1C111I d.: Conformldad ck Ia Cali<bd ck Produetos de E.'p0113C16n au1onzado par ciin'., Rcg,.trado con cl 'l'32..pn resolucion 967 del MtniStcno de Economia. F<'<llCflto y RoconstrucciOn de Cluk Euqo o1 Fema'ldez3592 ~.~.. Fono ' ' ' ( '7.,..,.. M? >11 ' ~.alao.d ea.."' S'9 -eo.r.o 1 -San"ago GRAS ASSOCIATES, LLC Page 55 of 99

108 Analab- Informe Resultados N de Este In forme consta de I muestra(s) - RUT del Cliente: Ana lab Este In forme fuc emitido el II de ene de 2014 a las 10:49:07 am - Lab.: RESIDUOS DE PESTICIDAS 0.01 PERMETHRIN PHOSMBT 0.01 PHOLPET 0.02 PIPERONYL BUTOXIOE 0.02 PYMETROZINE 0.02 PYRACLOSTROBIN 0.02 PIRIMICARB 0.01 PIRIMIPHOS-ETHYL 0.01 PIRIMIPHOS-METHYL 0.01 PROCYMIOONE 0.01 PROFENOPHOS 0.01 PROPACHLOR 0.02 PROPICONAZOLE 0.02 PROPOXUR 0.02 PROTHIOPHOS 0.01 PYRIMETANIL PYRIPROXIFEN 0.01 QUINAI.FOS QUINTOZENE 0.02 s SIMAZINE 0.01 TEBUCONAZOLE 0.01 TEBtJFENOZIDE TECNAZENE 0.01 TBRBUTRINE TETRADIFON 0.03 THIABENDAZOLE THIOPHANATE-METHYL 0.01 TOLYLFLUAN TRIADIME!'ON 0.01 TRIAZOPHOS TRICHLORFON 0.01 TRIFLOXISTROB!N TRIFLURALINE 0.02 TRIFORINE 0.01 VAMIDOTHION Original: Cliente PHOSALONE 0.01 PYRIOABEN 0.01 PROCLORAZ 0.20 PROPARGITE 0.02 PYRETHRUM 0.05 QUINOXYFEN 0.01 SPIROOICLOFEN 0.01 TERBUTILAZINE 0.01 THIACLOPRID 0.02 TRIADIMENOL 0.02 TRIPLUMIZOLB 0.01 VINCLOZOLIN Observaciones : FECHA DE RECEPC!ON DE MUESTRA: flora FECHA DE IN/ClO ANAL/SIS: 03101/2014 HORA FECHA DE TtRMJNO ANI.L!SJS: HORA 10:24 Hrs 11:00 Hrs 18:00Hrs - Metodologfas Empleadas: P-002/Luke (para todos los compuestos. salvo Mancozcb) Metoda basado en AOAC 19th edilion , , Pesticides Analysed with HPLC Procedures, Analytical Methods for Peslidde Residues in Foodstuffs. Sixtil edition, Ministry of Public Health. Welfare and Sport. The Netherlands P-009/EBDC Mancozeb (") Expm<;ado como CS2 Metoda basado en Journal of the AOAC (COL. 54, N 3, 1971) - Cromatografia Gaseosa con Detector de Masas (GC-MS) - Cromacograna Liquida (HPLC) con Detector Triple Cuadrupolo (MSIMS) - El porcentaje de recuperaci6n prvmedio para las forllficaciones sigulemes fue: a.- Recuperaci6n compuestos analizados por GC 86 % b.- Recuperaci6n compuestos analizados por LC 75% - L.D: Limite de Delecci6n. ("') Parametros fuera del alcance de acreditacion: Los resultados son validos s6lo para Ia mucstra analizada. Este informe de ana/isis no puedc ser reproducido total o parcialmenre sin Ia autorizaci6n de Ana1ab Chile S.A. (b) (6) (b) (6) JU M. RAMIREZ VARGAS!CENCI ADO EN QUIMICA SUBGERE~TE DE LABORATORIOS JOR QUiMIC DE PESTIC!OA GEREN UNIT A ALIZADO RESIDUOS FDA-USA EGENERAL ANA.U\.6 CHILE S.A. es l..aborotor~o Acn:ditado por ci!nn bajo Nonna NCH-ISO LE: Analab cs adcmas l..abonuorio Oficial del Servicio Agricola y Ganadcro en: Vinos y Alcoholes. Producros Pccuarios, Plaguicidas y Fcnilizantes dcsdc 1988 y sc eneuentm reg1strado co1110 Laboratono Ofic1al de Confonn1dad de Ia Calidad de Productos de Exportacion autorizado por ci!nn, Rcgisrrndo con cl N-32 scgtin rcsolucion 967 del Ministcrio de Economia. Fomcmo y R~X:onsuucci6n de Chile. E.requ>el Fernandez Mac;ul Fono (56 2) Fax (56-2) '7- analab@analat> d Casalla 519 Com10 11 Saotrago GRAS ASSOCIATES, LLC Page 56 of 99

109 F-2 Test Report for Pesticides for Stevia Pure = IV Arzalab l dentificacion dtl Clitntt Nombre A tern: ion Direcci6n Ciudod Telefono Datos de Ia Solicit uri Condici6n de Pogo Plazo de Entrega Anala b - lnforme Resultados N :.\lc lnfonnc consta de I mucslra(~l RUT del Clicntc : UJ-6 lstc lnfonnc fue ermbdo cl20 d.: JUO de 2014 o Ia.~ 4:4 1:08 pm PRODUCTORA ALYSA SPA : SRTA. SUSANA MORALES : CALLE TRES N 600 : QUILPUE : CONTRA FACTURA : Comuna 1 de 3 lab.: RES!DUOS DE PEST ICIDAS Original: Cllente smorales@prodalysa.cl Muesrra: Cla'e lipo Sub11po : STEVIA PURE IN POWDER LOT : 373 : STE~'lA \oluesucado poe Fecha de MucstroO : POLifO EL CLIENT :NO INDICA WD LOQ ANAUSIS SOU CIT ADOS: RESULTADOS MUL TIRESIDUOS mglkg (ppm) RESIDUOS NO DEfECT ADOS (LOD) ABAMEC'TIN 0.01 ACEPHATE 0.02 ACETAAIPRIO 0.01 ACRINATHRIN \LACH.LOR 0.01 AI.DICARB 0.02 AI.DICARB SULPHONE 0.02 ALDICARB SOLPHOXIDE 0.01 AI.DRIN 0.01 ATRAZINE 0.01 AZINPHOS-ETHYL \ZlNPHOS-HETHYL 0.03 AZOXYSTROBIN 0.02 BE.~OMYL 0.01 ALPHA-BHC 0.01 BETA BHC 0.01 GA"'.MA BHC 0.01 BIFENTRIN 0.01 BITERTA~L O.C2 BOSCALID 0.01 BROY.ACILO 0.01 BRCMOPHOS ETHYL 0.01 BROHOPHOS ~~L O.C1 BROHOPROPILI\1' BUPIRJI'.ATO 0.01 BUPROPEZIN 0.01 CA:lUSI\FOS CAPT AI' 0.01 CARBARYL 0.02 CARBc:N!lAZII' 0.01 CARBOPE..'IOTHIOll 0.02 CARBOI'URAII HIOROXICARBOPURAN KETO CARBOFURA.'l 0.01 CHINOHETHIOl/ATE 0.03 CLOF~'TEZI!\'E 0.01 ALP!'.A-CHLOROA'IE 0.01 GAMMA CH:.OROANE 0.01 CHLORPE..WINPHOS 0.02 CHLOROTHALONIL 0.01 CHLORPYRIFOS ETHYL 0.01 CHLORPYRIFOS METHYL 0.01 CYANACHIA 0.01 CYFLUTHRIN G~~ CYHALOTRINA 0.01 LAMBDA CYHALOTHRIN o.os CYHEXATIN 0.01 CYPERMETHRIN 0.02 CYPROCONAZOLE 0.01 CYPRODINIL 0.01 op DOD 0.01 pp op - ODE 0.01 pp - ODE 0.01 op - DDT 0.01 pp DDT 0.02 OELTAM'ETHRIN 0.01 OII\ZINON O.Ol OICLOBUTRAZOL 0.01 DICHLOFLUANID 0.01 DICLOP.AN 0.01 DICHWRVOS 0.01 DICOFOL 0.01 DIELDRIN 0.01 DIPENILAHINA 0.02 OIF.~0COliAZOLE 0.01 OIMETI-iOATE 0.01 DISULFOTON 0.03 ;)ITIOCARBAMATO (COMO CS2JC.02 El/OOSULPA'l I ENOCSULFA>; II 0.01 :endosulfan SULFATE 0.01 ENORIN 0.01 'ggf'ey\'alerato o.o: ETHION 0.01 ETHOPROPHOS 0.02 FENI\MlPHOS 0.01 FES~IMOL O. Ol P1':'."3UCOlll\ZOLE 0.01 P~;CHLORFOS 0.01 PEl\'HexA."! I FENITROTHIO:> 0.02 FEl/PROPATHRI!>I 0,02 P.WXYCA't F'El\"!HIOX 0.01 FE:NALERATO 0.05 i'erbai' O.Cl FIPRONIL 0.01 FWAZINAA 0.01 F~UOIOXONIL 0.02 PLUFENOXURON 0.02 PLUMIOXAZII PLUOUINCONAZOLE 0.01 FLUSILAZOL 0.02 FLUC'l'TRINATE 0.01 PLWALINATO 0.01 HEPTACHLOR 0.01 HEPTAC!-'.LOR EPOXIDE 0.01 HEXAC!'.LOROBENZENE 0.01 HEXACONAZOLE 0.01 HEXAZINONE 0.01 IMAZAI.IL 0.02 IMIDACLOPRIO 0.02 INOOXACARB I PROD I ONE 0.01 KRESOXYM METHYL 0.02 LUPENURON 0.01 MALATHION 0.02 MECAR3AM 0.01 MEPANIPYRIM 0.02 METALAXYL 0.01 METHAHIDOPHOS 0.03 METHIOCARB 0.01 HETHOXYCHLOR 0.02 MBTOXYFENOZIOE 0.01 METHIOI\THION 0.02 METHOMYL 0.01 MEIRIBUZll/1; 0.01 MEVI!n>HOS 0.02 MIREX 0.01 MICLOBUTANlL 0.01 MONOCl~OTOPHOS NAFTOL 0.01 OMETHOATE 0.01 OXAAYL 0.02 OXYFLOORFEN 0.01 PACLOBUTRAZOL 0.01 PARA TH I C.'l- ETHY:. A 'IALAB Clfll.E S.A. es l.abor.~t ono Acrcduado por d J:O.."N bojo 'lonna '\Cif ISO POlS l 2SO - 2& ~ 2~ Analab cs adern:b Labornrono OficaJI dd S<nt<OO Agncola ~ G3nad<m <n. \'mos y,\kobolcs. Productos Pccuanos. P l~tcidas y Fentltmnle> dcm!e 1988 y sc encucntra <egisttado como Labonuono Ofictal de Confomudad de Ia CBhdad de Productos d<: hpo<uct6n autorizado por d IN'. R<itStrado con cl N"32 scgun n:soluci6n 967 dd Mtnostcno de Economia. Fomcnto )' Rcconstruect6n d<: Chile Exeqt.1et Fe'!'lndel Macut - Fonc:I56-2J Fa (56 2) wv.w.1".a'>od analall~analab d Cat~ '<0 <1 -SanNgo GRAS ASSOCIATES, LLC Page 57 of 99

110 - AnaJab -lnforme Resultados N Este lnfonne consta de I muestra(s} le -- RUT del Cliente: 76667JtlJ-fJ Analab Estc lnfonnefueemiridoel20dejunde2014alas 4:41:08pm 2 de 3 Lab.: RFSIDUOS DE PESTTCTDAS Original: CJieme 0.01 PARATHION-METHYL 0.02 PENCONAZOLS 0.01 PENDIMETHAL!N 0.01 PERMETHRIN 0.01 PHOSMET 0.01 PHOLPET 0.02 PIPERONYL BUTOXIDE 0.02 PYMETROZINE 0.02 PYRACLOSTROB ln PIRIMICARB 0.01 PIRIMIPHOS ETHYL 0.01 PIRIMIPHOS METHYL 0.01 PROCYMIOOI,fE 0.01 PROFENOPHOS 0.01 PROPACHLOR 0.02 PROPICONAZOLE 0.02 PROPOXUR 0.02 PROTHIOPHOS 0.01 PYRIMETANIL 0.01 PYRIPROXIFEN 0.01 Q\11Nl\LFOS 0.01 QUlNTOZE:NE 0.02 s SIMAZINE 0.01 TEBUCONAZOLE 0.01 TEBUFENOZIDE 0.02 TECNAZENE 0.01 TERBUTRINE TETRI\DIPON 0.03 THIABENDAZOLE 0.03 THIOPHANATE-~IETHYL 0.01 TOLYLFLUANID 0.02 TRIADIMEFON TRli\ZOPHOS 0.01 TRICHLORFON TRIFLOXISTROBIN TRI!'LURALINE TRIFORINE 0.01 VAMIDOTHION 0.02 PENTACLORANIZOL 0.01 PHOSALCNE 0.01 PYRIDABEN 0.01 PROCLORAZ 0.20 PROPARGITE 0.02 PYRETHRUM QUINOXYFEN 0.01 SPIRODICLOFEN 0.01 TERBUTILAZINE 0.01 THIACLOPRID 0.02 TRIADI MENOL 0.02 TRIFLUMIZOLE 0.01 VINCLOZOLIN Observaciones : PECHA DE RECEPCJON DE MUESTRA: 13/ HORA PECHA DE TNIC!O A NALlS IS: HORA PECHA DE TERM! NO ANAUSIS: 20/ HORA 12:06 Hrs 09:00 Hrs 14:00Hrs ANALAB CHILE S.A. cs laborotorio Acrcditodo por ciinn bajo Nonna NGI-ISO LE: Analab cs ad01n is Laboratorio Oficial del Scrvicio Agricola y Ganadcro en: Vinos y Alcoholcs. Productos Pccuario:;. Pla!,'llicidas y Fcnilizantcs desdc 1988 y sc cncucntra rcgiwndo como laboratorio Oficial de Confonnidad de Ia Calidad de Product()!; de E ponaci6n autorizado por ciinn. Rcgistrado con el N"32 segun rcsoluci6n 967 del Ministerio de Economla. Forncnto y Rcconstrucci6n de Chile. Exequlel Femjndez Macul- Fono(S&-2) Fax (56 2) w.w~.analab d- analab@analab d- Gasllla 519. Correo 11 -Sanuago GRAS ASSOCIATES, LLC Page 58 of 99

111 -- #j Atzalab Analab- Informe Resultados N Esle lnformc cons1a de I muestra(s) RUT del Cbente: f».EJ Es1e lnfonne fueemitidoel20dejunde 2014 a Ia. 4 41:08 pm J de J Lab.: RES!DUOS DE PESTICIDAS Original: Cliente Refcrencia~: Mctodologfas Empleadas: - P 002/Luke Ml!todv ba:.adv en AOAC 19th edition Pesticides Analysed with HPLC Procedures, Analytical Methods for Pesticide Residues In Foodstuffs. Sixth cdiclon. Ministry of Public Health, We/far'(' and Sport. The Netherlands - P-009/EBDC ("') Expresado como CS2 Mctodo basado en Journal of the AOAC (COL. 54. N" } Cromatografla Caseosa con Detector de Masas (CC-MS} - Cromatografia Liquids (HPLC) con Detector Triple Cuadrupolo (MSIMS) - 1 porc.ema)e de recuperacion promedio para las fortlflcac:ionl!!> slguiemcs fuc>. o Recup<'mcidn compuestos anallzados por CC 108 % b. Recuperacidn compuestos analizados por LC 82 % ~ L.D : Ltmlte de Deteccion. ( )- Fuera dt'l alcancc dl' Ia Acreditacion. Parametres fuero del alcance de acredifacion: Acibenzolar Smet/1, Aclonifen. Ametiin. Aminocarb. Azaconazo/e Bendtocarb. Bensulfuron mellf. Benzoximato. Bromuconazole. Butafenacilo. Carfentrazona etil. Clorfluazuron. Cloroxuron. Cletodim. Clcxtinafop propargil. Clorontraniliprol. C/orotoluron. Clotlanldin. Cumafos. Ciazofamlda. Ciclanilida, Dicamba. dlcrotofos. 0/Nofcncarb. Dlflubcnzuron. Dimetomorf. Oiniconazol. Dmotefuran. Diuron. Emameccina benzoato. Etaconazol l Etaconazol/1. Etcfon. tiprol.eiirimol. Etofvmesato. Famoxadona, Fcnamidona. Fcnmedifam. Fenoxaprop p eli/. Fenpropidin. Fenpropimorf. Flonlc,1mid Flubcndlamida. Flufenacet.Fiuometuron. Flutolanlf. Fluoxastrobin. Foramsulfuron. Forclorfenuron. Fuberidazol. Fural&ilo. Furatiocarb. Clifosato. Haloxifop metil. Heptenofos. HidrometJ/nona. lndaziflam. lpconazol. lsoprocarb. /soproturon. Mandlpropamid. Maneb. Mefenacet. mefenoxam, Mc pronil. meptlfdlnocap. Mesosulfuron, melil Metalxmttlaturon. Metaflumlzol. Metamitron. Metconazol. Metobromuron. Metoprotrina. metrafenona. Mexacarbato. Monolinuron. Neburon. Nitenpiram. No, aluron. Oxadixilo. Pencicuron. Picoxistrobin. Promecarb. Prometrina. Promeron. Profam. Propineb. Piracarbolid. Quitalofop etil. Rotanona,Secbumeton, Siduron. Simetrin, Spinctoram A. Spinetoram B. Spiroletramato. Tebufenpirad. Tebutluron, Tefluben7uron. Terbumeton. Tetraconazol. Tiadiazuron, Tlobencarb. Tiram. Triasulfuron. Triciclazol, Triclconazol. Zineb, Ziram. Zoxamida Los re:,u/tado!> son vtj/idos solo para Ia muestra analizada. Estf' lnforrnl' dl' amji/s/s no pucde scr fy.'producldo total o pare/a/mente sin Ia autorlzaclon de Ana lab Chile S.A. (b) (6) (b) (6) JU I CE:-<CIAOO E :-< QL1~11CA SUBGERE:"<. T E DE LABORAT ORJOS ANALAB CIIILE S.A. c> Labor:uorio Acroditado porcl "''N bajo Nonna NCH-ISO LE & Analab cs adcmli.~ Laboratono Oficral del Scr.rcro Agricola)' Ganadcro en Vino\ y Alcoboles. Productos Pccuanos. I'Jagurcrdas y Fcnrliz.antcs dc:sde 1988 y se eneucntra rcgistnldo como Laboratono Olicral de CoMomudad de Ia Cahdad de Productos de Exponru:i6n uutonatdo por ciinn. Rcgrstrado con cl N"32 scgun rcsoluci6n 967 del Mmr>teriu de Economiu. Fomcn1o y Rceonstruccr6n de Chile. Exequlel Ferntndez3 592 Macul Fono (56-2) Fax (56 2) w\im.anarab d analab:q,analab d Casll'a 519 Correa 11 Sanllago GRAS ASSOCIATES, LLC Page 59 of 99