Read GRAS Notice 000375: Enzyme modified steviol glycosides text version

GRI 1111111 111111 HIE GRAS Notice (GRN) No. 375 http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/default.htm

TOYO SUGAR REFINING CO.,LTD

18-20, Nihonbashi,Koami-cho,Chucrku, Tokyo 103-0016, Japan Tel : + 81-(0)3-3668-7871Fax : +81-M-3668-7879

Delivered via Courier

ECEOE.

Division of Biotechnology and GRAS Notice Review

February 23, 2011 Office of Food Additive Safety (HFS-200) GRAS Notification Program Center for Food Safety and Applied Nutrition Food And Drug Administration 5100 Paint Branch Parkway College Park, MD 20740-3835 RE: GRAS Notice for a-Glucosylated Steviol Glycosides Dear Sir or Madam: In accordance with 21 CFR 070.36 [Notice of a claim for exemption based on a Generally Recognized as Safe (GRAS) determination] published in the Federal Register [62 FR 18938 (17 April 1997)1, I am submitting in triplicate, on behalf of the notifiers, Toyo Sugar Refining Co., Ltd., 18-20, NihonbashiKoamicho, Chuo-ku, Tokyo 103-0016, Japan and Nippon Paper Chemicals Co., Ltd., 1-2-2, Hitotsubashi, Chiyoda-ku, Tokyo 100-0003, Japan, a Notice of the determination, on the basis of scientific procedures, that a-glucosylated steviol glycosides, as defined in the enclosed documents, are GRAS under specific conditions of use as a food ingredient, and therefore, are exempt from the premarket approval requirements of the Federal, Food, Drug and Cosmetic Act. The Notice presents information setting forth the basis for the GRAS determination and includes a comprehensive summary of the data available and that has been reviewed by an independent panel of experts (the Expert Panel) in support of the safety of a-glucosylated steviol glycosides under the intended conditions of use. Each copy of the GRAS Notice includes the GRAS Notification dossier (GRAS Exemption Claim for a-Glucosylated Steviol Glycosides), with the signed Expert Panel Consensus Statement comprising Appendix A of the dossier. Should you have any questions or concerns regarding this GRAS Notice, please do not hesitate to contact us at any point during the review process so that we may provide a response in a timely manner. I look forward to receiving acknowledgement of receipt of this notice. Sincerely,

(b) (6)

NAMEYoshihisa lida POSITIONGeneral Manager Toyo Sugar Refining Co.,Ltd.

Encl.

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Division of Biot echnology and GRAS Notice Review

GRAS Exemption Claim for oc-Glucosylated Steviol Glycosides

Submitted to:

Office of Food Additive Safety (HFS-200) GRAS Notification Program Center for Food Safety and Applied Nutrition (CFSAN) Food and Drug Administration 5100 Paint Branch Parkway College Park, MD U.S.A. 20740-3835

Submitted by:Toyo Sugar Refining Co., Ltd.

18-20, Nihonbashi-Koamicho, Chuo-ku, Tokyo 103-0016 Japan

Nippon Paper Chemicals Co., Ltd. 1-2-2, Hitotsubashi, Chiyoda-ku, Tokyo 100-0003 Japan

February 23, 2011

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GRAS Exemption Claim for a-Glucosylated Steviol Glycosides

Table of Contents

I.GRAS Exemption Claim A. B. C. D. E. F. Claim of Exemption from the Requirement for Premarket Approval Pursuant to Proposed 21 CFR §170.36(c)(1) [62 FR 18938 (17 April 1997)] (U.S. FDA, 1997) Name and Address of Notifier Common Name of the Notified Substance Conditions of Intended Use in Food Basis for the GRAS Determination Availability of Information 3 3 4 4 4 5 6 6 8 10 10 11 13 14 14 14 14 14 15 15 3

II.Detailed Information about the Identity of the Substance A. B. C. Identity Method of Manufacture Specifications and Analytical Data for Food Grade Material C.1 Chemical and Microbiological Specifications C.2 Additional Analyses III.Self-Limiting Levels of Use IV.Basis for GRAS Determination A.Introduction B.Conditions of Intended Use in Food and Consumer Exposure B.1 History of Use of Steviol Glycosides B.2 Current Regulatory Status of Steviol Glycosides B.3 Sweetening Potency C.Estimated Intake of the a-Glucosylated Steviol Glycosides

C.1 Intended Use of a-Glucosylated Steviol Glycosides and Levels of Use in Foods 15 C.2 Estimated Consumption of a-Glucosylated Steviol Glycosides from Proposed Food Uses 17 D.Metabolic Fate of a-Glucosylated Steviol Glycosides Toyo Sugar Refining Co., Ltd. Nippon Paper Chemicals Co., Ltd. February 23, 2011 19

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E.Potential Cleavage of a-Glucopyranosyl Moieties F.Toxicological Studies F.1 Pivotal Data to the Safety of a-Glucosylated Steviol Glycosides F.2 Corroborative Data to the Safety of a-Glucosylated Steviol Glycosides G. Summary and Basis for GRAS Conclusion V. References

20 21 22 23 24 27

Appendix AExpert Panel Consensus Statement Concerning the Generally Recognized as Safe (GRAS) Status of a-Glucosylated Steviol Glycosides for use as a General Purpose Sweetening Agent

List of Figures and Tables

Figure II.A-1Structural Formulae of a-Glucosylated Steviol Glycosides Figure II.B-1Schematic Overview of the Manufacturing Process for a-Glucosylated Steviol Glycosides Figure II.C.2-1 Degree of a-Glucosylation of Stevioside and Rebaudioside A in a-Glucosylated Steviol Glycosides 13 10 6

Table II.B-1Raw Materials and Processing Aids Used in the Manufacturing of a-Glucosylated Steviol Glycosides Table II.C.1-1 Chemical Specifications for a-Glucosylated Steviol Glycosides Table II.C.2-1Degree of a-Glucosylation in a-Glucosylated Steviol Glycosides' 9 11 12

Table IV.C.1-1 Summary of the Proposed Food-Uses for a-Glucosylated Steviol Glycosides as a General Purpose Sweetener 15 Table IV.C.2-1 Intakes of Intense Sweeteners and Predicted Intakes of Rebaudioside A (Renwick, 2008) Table IV.C.2-2 Intakes of Intense Sweeteners and Predicted Intakes of ct-Glucosylated Steviol Glycosides 19 18

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GRAS Exemption Claim for a-Glucosylated Steviol Glycosides

I.GRAS Exemption Claim A. Claim of Exemption from the Requirement for Premarket Approval Pursuant to Proposed 21 CFR §170.36(c) (1) [62 FR 18938 (17 April 1997)] (U.S. FDA, 1997)

a-Glucosylated steviol glycosides have been determined by Toyo Sugar Refining Co., Ltd. (hereafter Toyo Sugar) and Nippon Paper Chemicals Co., Ltd. (hereafter Nippon Paper) to be Generally Recognized as Safe (GRAS) for use in a variety of traditional food products, consistent with Section 201(s) of the Federal

Food, Drug, and Cosmetic Act. This determination is based on scientific procedures as described in the

following sections, under the conditions of its intended use in food. Therefore, the use of aglucosylated steviol glycosides in food as described below is exempt from the requirement of premarket approval. Signed,

(b) (6)

Fe)7 2301/

NAME Yoshihisa lida General Manager POSITION

DateFebruary 23, 2011

B. Name and Address of Notifier

NAME: Yoshihisa lida POSITION : General Manager Toyo Sugar Refining Co., Ltd. 18-20, Nihonbashi ,Koami-cho, Chuo-ku, Tokyo 103-0016 Japan Telephone : +8140)3-3668-7871 Facsimile : +8140)3-3668-7879 Email: [email protected] NAME:Yasuyuki Nakamura POSITION : General Manager Nippon Paper Chemicals Co., Ltd. 1-2-2, Hitotsubashi, Chiyoda-ku, Tokyo 100-0003 Japan Telephone : +8140)3-6665-5900 Facsimile : +8140)3-3217-3280 Email: [email protected]

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C.

Common Name of the Notified Substance

a-Glucosylated Steviol Glycosides

D.

Conditions of Intended Use in Food

Toyo Sugar and Nippon Paper intend to market a-glucosylated steviol glycosides (trade names SK Sweet Z3, aG Sweet PX) as a general purpose sweetening agent, in accordance with current good manufacturing practices (cGMP), in the United States (U.S.) in a variety of traditional food products. Most other high intensity sweeteners (HIS), including aspartame, have been approved by the United States Food and Drug Administration (FDA) as general purpose sweeteners with no restriction on their use in specific foods or use-levels. Hence, the foods to which aspartame are added and the use level is controlled by technological properties (e.g., sweetness potency). Although a-glucosylated steviol glycosides are slightly less sweet than aspartame (137 vs. 200 times sweeter than 6% sucrose, respectively), the uses for a-glucosylated steviol glycosides primarily reflect those currently permitted for aspartame in the United States.

E.

Basis for the GRAS Determination

Pursuant to Title 21, Section 170.30 of the Code of Federal Regulations (CFR) § 170.30, a-glucosylated steviol glycosides have been determined by Toyo Sugar and Nippon Paper to be GRAS on the basis of scientific procedures (U.S. FDA, 2010a). This GRAS determination is based on data generally available in the public domain pertaining to the safety of a-glucosylated steviol glycosides and unchanged steviol glycosides, as discussed herein, and on consensus among a panel of experts who are qualified by scientific training and experience to evaluate the safety of a-glucosylated steviol glycosides as a component of food [see Appendix A, entitled, "EXPERT PANEL CONSENSUS STATEMENT CONCERNING

THE GENERALLY RECOGNIZED AS SAFE (GRAS) STATUS OF a-GLUCOSYLATED STEVIOL GLYCOSIDES FOR USE AS A GENERAL PURPOSE SWEETENING AGENT"].

At the request of Toyo Sugar and Nippon Paper, an Expert Panel ("the Expert Panel") of independent scientists, qualified by their relevant national and international experience and scientific training to evaluate the safety of food ingredients, was specially convened on June 30, 2010 to conduct a critical and comprehensive evaluation of the available pertinent data and information, and to determine whether the intended uses of a-glucosylated steviol glycosides as a general purpose sweetening agent are safe and suitable and would be GRAS based on scientific procedures. The Panel consisted of the following qualified scientific experts: Dr. Samuel Cohen, Ph.D., M.D., D.A.B.P., F.A.T.S (University of Nebraska Medical Center), Dr. Ian Munro, Ph.D., F.A.T.S., FRCPath (Cantox Health Sciences International), and Dr. William Waddell, M.D., F.A.T.S (University of Louisville). The Expert Panel convened on behalf of Toyo Sugar and Nippon Paper, independently and collectively, critically evaluated the data and information summarized herein and concluded that the intended uses in traditional foods described herein for a-glucosylated steviol glycosides, meeting appropriate foodToyo Sugar Refining Co., Ltd. Nippon Paper Chemicals Co., Ltd. February 23, 2011

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grade specifications and manufactured according to cGMP, are safe and suitable and GRAS based on scientific procedures. It is also the Expert Panel's opinion that other qualified and competent scientists reviewing the same publicly available toxicological and safety information would reach the same conclusion. a-Glucosylated steviol glycosides are GRAS based on scientific procedures for its intended use as a general purpose sweetening agent; therefore, it is excluded from the definition of a food additive and thus may be marketed and sold for its intended purpose in the U.S. without the promulgation of a food additive regulation under Title 21 of the CFR.

F.Availability of Information

The data and information that serve as the basis for this GRAS Notification will be sent to the U.S. Food and Drug Administration (FDA) upon request, or will be available for review and copying at reasonable times at the offices of: Toyo Sugar Refining Co., Ltd. 18-20, Nihonbashi-Koamicho, Chuo-ku, Tokyo 103-0016 Japan or Nippon Paper Chemicals Co., Ltd. 1-2-2, Hitotsubashi, Chiyoda-ku, Tokyo 100-0003 Japan Should the FDA have any questions or additional information requests regarding this notification, Toyo Sugar and Nippon Paper will supply these data and information.

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II.Detailed Information about the Identity of the Substance

A.Identity

Steviol glycosides (steviol conjugated with glucose, xylose, and/or rhamnose via13-glucosidic bonds) are natural constituents of the Stevia rebaudiana plant. At least 10 different steviol glycosides have been identified in the leaves of S. rebaudiana (stevioside, rebaudioside A, B, C, D, E, and F, dulcoside A, rubusoside, and steviolbioside) with rebaudioside A and stevioside typically identified as the principal sweetening constituents. a-Glucosylated steviol glycosides are enzymatically modified steviol glycosides, such that additional glucose moieties are bonded to the original steviol glycoside structure via a(1--4) linkages. a-Glucosylated steviol glycosides are a mixture of a-glucosylated stevioside, rebaudioside A, rebaudioside C, dulcoside A, steviolbioside, rubusoside, rebaudioside B. The resulting product is produced as a white to slightly yellowish powder with a slight to characteristic odor. As demonstrated in Figure ll.A-1, the structural formulae of a-glucosylated steviol glycosides share the same hydrocarbon backbone, steviol, and have multiple sites where the a(1-4)-glucose linkage may occur. Analytical data determining the degree of a-glycosylation of the steviol glycosides, as discussed in greater detail in Section ll.C.2, have demonstrated that a-glucosylated steviol glycosides are primarily mono-, di-, and triglucosylated.

HOH2C 910 HOH2C 0 H0H2C 0

\

HOH2C

}-K±) 0

--0

OH CH3 --CH2

OH HOH2C

_-- 0

HOH2C

H3CH c_o O OH OH

Molecular weight = 967 g/mol* a-Glucosylstevioside

Molecular weight = 1129 g/mol* a-Glucosylrebaudioside A

R = H or a-glucopyranosyl * Molecular weights provided are for monoglucosylated steviol glycosides

Figure II.A-1 Structural Formulae of a-Glucosylated Steviol Glycosides

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HOH2C RO HO RO OH RO / HO

/

HOH2C

HO

OH

CH3

---- CH2

H3

HO

OH

CH2

HOH2C

H 3C t Ho 0 OH HOH2C

H3C

OH

Molecular weight = 951 g/mol* a-Glucosyldulcoside A

Molecular weight = 1113 g/mol* a-Glucosylrebaudioside C

Molecular weight = 805 g/mol* a-Glucosylsteviolbioside

R = H or a-glucopyranosyl

Molecular weight = 805 g/mol* ct-Glucosylrubusoside

* Molecular weights provided are for monoglucosylated steviol glycosides Figure IIA-1 Structural Formulae of a-Glucosylated Steviol Glycosides

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000 010

HOH2C

HOH2C

OH

CH3

c -

CH2

H3C OH

Molecular weight = 976 g/mol* a-Glucosylrebaudioside B

R = H or a-glucopyranosyl * Molecular weights provided are for monoglucosylated steviol glycosides

Figure ILA-1 Structural Formulae of a-Glucosylated Steviol Glycosides

B.Method of Manufacture

A schematic diagram of the general manufacturing process employed to produce a-glucosylated steviol glycosides is illustrated in Figure II.B-1. a-Glucosylated steviol glycosides are produced via an enzymatic reaction of dextrin with a high-purity S. rebaudiana extract meeting the current specification for steviol glycoside preparations as determined by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) (i.e., >95% steviol glycosides). The starting S. rebaudiana extract powder and dextrin are dissolved in hot water, along with a solution of cyclodextrin glucanotransferase (CGTase) derived from Bacillus stearothermophilus. The mixture is allowed to react for 20 to 40 hours and the CGTase subsequently is neutralized by boiling the mixture for 30 minutes. After allowing the mixture to cool, a- and 13-amylase solutions (derived from Bacillus subtilis and barley, respectively) are added and the mixture is incubated for 20 to 40 hours. a- and I3-Amylase are then neutralized by boiling the mixture for 30 minutes. Impurities are removed from the a-glucosylated steviol glycoside mixture by eluting the mixture through an adsorption resin and ion-exchange resins (strong acidic and strong basic resins) with ethanol or methanol. The resulting a-glucosylated steviol glycosides then undergo standard concentration, sterilization, and spray-drying procedures to yield a final product that is designated as aG Sweet PX or SK Sweet Z3. All raw materials and processing aids used in the manufacture of GOS are suitable food-grade materials and are used in accordance with applicable U.S. federal regulations as described in Table II.B-1, below.

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TableRaw Materials and Processing Aids Used in the Manufacturing of Glucosylated Steviol Glycosides

Material Stevia rebaudiana extract powder Dextrin Water Cyclodextrin glucanotransferase (CGTase) derived from Bacillus stearothermophilus a-Amylase 13-Amylase Adsorption and ion-exchange resins (strong acidic and strong basic resins) Ethanol Methanol Use Reaction substrate Reaction substrate Solvent Processing aid

Regulatory Status

a-

GRAS (GRN1252, 253, 275, 278, 282, 287, 303, 304, 318, 323, and 329) (U.S. FDA, 2008a,b, 2009a-d, 2010b-f) 21 CFR §184.1277 (U.S. FDA, 2010g) 21 CFR §184.1012 (U.S. FDA, 2010g);EFSA (2007); U.S. FDA (2000, 2001, 2004); Commission of the European Communities (2003) 21 CFR §184.1148 (U.S. FDA, 2010g) 21 CFR §184.1443a (U.S. FDA, 2010g) 21 CFR §173.25 (U.S. FDA, 2010g) 21 CFR §184.1293 & 21 CFR §172.340 (U.S. FDA, 2010g) 21 CFR §172.859, 21 CFR §172.869, 21 CFR §173.250 & 21 CFR §172.867 (U.S. FDA, 2010g)

Processing aid Processing aid Processing aid Processing aid Processing aid

CFR, Code of Federal Regulations; EFSA, European Food Safety Authority; GRAS, Generally Recognized As Safe; U.S FDA, United States Food and Drug Administration

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9

Stevia Extract

4--

Dissolution Enzyrne(CGTase) Ft ea c dor' I

4

Enzyme( $-Amylase) Enzyme( -Amylase)

Reaction 2

N eutraliza tion

Purification Adsorption Resin !on-exchange Resin

4-- Mfl OH or Et-CH

Ftrat on

y Drir

alpha-Glucosylated Steviol Glycosides Figure II.B-1 Schematic Overview of the Manufacturing Process for a-Glucosylated Steviol Glycosides

C.Specifications and Analytical Data for Food Grade Material

C.1Chemical and Microbiological Specifications

a-Glucosylated steviol glycosides are produced in accordance with cGMP, and in order to ensure a consistent and safe product, Toyo Sugar and Nippon Paper have established food-grade specification pa ra m eters for the final ingredient. These specifications conform to those established by JECFA at their 69th meeting for steviol glycosides in terms of purity, but not in terms of steviol glycoside components (JECFA, 2008). A summary of the chemical and microbial specifications for a-glucosylated steviol Toyo Sugar Refining Co., Ltd. Nippon Paper Chemicals Co., Ltd. February 23, 2011

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glycosides is provided in Table ll.C.1-1. Analyses of representative, non-consecutive lots demonstrated compliance with final product chemical and microbiological specifications.

Table H.C.1-1 Chemical Specifications for a-Glucosylated Steviol Glycosides

Specification Parameter Description Specification White to light yellow powder Odorless or having a slight characteristic odor Solubility pH Assay2 Total Ash Loss on Drying Residual Solvent Freely soluble in water and ethanol Between 4.5 and 7.0 (1% aqueous solution) Not less than 95% Not more than 1% Not more than 6% (105°C, 2h) Not more than 200 mg/kg methanol Not more than 5,000 mg/kg ethanol Arsenic Lead Viable Count Yeasts Molds Coliforms Salmonella Not more than 1 mg/kg Not more than 1 mg/kg Not more than 1,000 CFU/g Less than 10 CFU/g Less than 10 CFU/g Negative/1 g Negative/25 g FAO/JECFA (2006) (Vol 4) 1 (p. 41) FAO/JECFA (2006) (Vol 4) (pp. 36-38) MHLW (2009) (8th Ed.) (pp. 257-258, 379);3JECFA, 2008; 4 FAO/JECFA, 2006 (Vol 4) (pp. 20-24) FAO/JECFA (2006) (Vol 4; Method I) (pp. 53-54) FAO/JECFA (2006) (Vol 4) (p. 61) FAO/JECFA (2006) (Vol 4) (pp. 87-90) FAO/JECFA (2006) (Vol 4) (pp. 87-90) FAO/JECFA (2006) (Vol 4) (p. 68) FAO/JECFA (2006) (Vol 4) (p. 66) MHLW (2009) (8th Ed.)3 FDA BAM (2001) Chapter 18 FDA BAM (2001) Chapter 18 MHLW (2009) (8th Ed.) FDA BAM (2007) Chapter 5 Visual/Organoleptic Method

CFU, colony forming units; FDA BAM, Food and Drug Administration Bacteriological Analytical Manual 1 FAO/JECFA. Combined Compendium of Food Additive Specifications, Volume 4 [Online Edition]. Rome, Italy: Food and Agriculture Organization of the United Nations (FAO) /Joint FAO/WHO Expert Committee on Food Additives (JECFA), 2006. Available from: http://www.fao.oredocrep/O09/a0691e/a0691e00.htm. 2 Total of 7 steviol glycosides (reacted and unreacted): stevioside, rebaudioside A, rebaudioside C, dulcoside A, steviolbioside, rubusoside, rebaudioside B 3 MHLW. Japan's Specifications and Standards for Food Additives, 8 th edition. 2009. 4 JECFA (2008). Steviol glycosides. In: Compendium of Food Additive Specifications. Joint FAO/WHO Expert Committee on Food Additives (JECFA), 69th Meeting, June 17-26, 2008, Rome, Italy. (FAO/JECFA Monographs no. 5). Rome, Italy: Food and Agriculture Organization of the United Nations (FAO) / World Health Organization (WHO), pp. 75-78. Available at:

itp://ftp.fao.org/docrepfiao/011/10345e/iO345e.pdf

C.2Additional Analyses

Additional product analyses were conducted using high performance liquid chromatography (HPLC) on the starting S. rebaudiana extract and the final product to determine content of reacted and unreacted steviol glycosides in the final products. From these results, it was determined that approximately 90% of the steviol glycosides in the final products are a-glucosylated, with approximately 5% of the steviol glycosides unreacted. Of the glycosides present in a-glucosylated steviol glycosides, approximately 75% are stevioside, 15% are rebaudioside A, 3% are rebaudioside C, and <1% is dulcoside A, with the remaining 5% representing steviolbioside, rubusoside, and rebaudioside B. Furthermore, as Toyo Sugar Refining Co., Ltd. Nippon Paper Chemicals Co., Ltd. February 23, 2011

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demonstrated in Table ll.C.2-1, below, a-glucosylated steviol glycosides are primarily mono-, di-, and triglucosylated, with smaller amounts of tetra- and penta-glucosylated steviol glycosides. Consistent with the distribution of the glycosides and degree of a-glucosylation, Figure ll.C.2-1 demonstrates that the majority of a-glucosylated steviol glycosides are mono-, di-, and tri-glucosylated stevioside and mono-, di-, and tri-glucosylated rebaudioside A.

Table II.C.2-1 Degree of a-Glucosylation in a-Glucosylated Steviol Glycosidesi

Unreacted (%) Mono-glucosylated (%) Di-glucosylated (%) Tri-glucosylated (%) Tetra-glucosylated (%) Penta-glucosylated (%) steviolbioside, and rebaudioside B) 5 20 34 28 11 1

1 Based on the presence of 7 steviol glycosides (stevioside, rebaudioside A, rebaudioside C, dulcoside A, rubusoside,

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4stevioside 10reb-A 21mono-glucosylated

25rnono-64ucosyletact retr-A 27di-glucosylitted Erteviosids 29dingluoosylated reb-A 30 . 31 tri-glucosylated stevioside 32tri-glucosylated reb-A

stavioside

33etre-giucosylated steviosido 34tetra-gluoosylated reb-A Brite- ucos I tevi

Figure

Degree of a-Glucosylation of Stevioside and Rebaudioside A in a-Glucosylated Steviol Glycosides

From the above information, the weighted average molecular weight of the reacted and unreacted steviol glycosides present in a-glucosylated steviol glycosides was calculated to be 1,084 g/mol. Therefore, in order to convert a-glucosylated steviol glycosides to steviol equivalents, the ratio of the molecular weight of a-glucosylated steviol glycosides to that of steviol was determined to be approximately 3.4.

III. Self-Limiting Levels of Use

The use of a-glucosylated steviol glycosides in food is largely limited by the desired sweetness intended for a particular food or beverage product; therefore, the use of a-glucosylated steviol glycosides as a general purpose sweetener in foods is self-limiting based on its organoleptic properties.

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IV. Basis for GRAS Determination A.Introduction

The determination that a-glucosylated steviol glycosides are GRAS is on the basis of scientific procedures and the information supporting the general recognition of the safe use of a-glucosylated steviol glycosides includes: · · · · The recognized history of safe consumption of unreacted steviol glycosides in humans, data pertaining to the identity, intended use, and estimated intake of a-glucosylated steviol glycosides, the expected metabolic fate of a-glucosylated steviol glycosides and unreacted steviol glycosides, and preclinical and human studies assessing the safety of a-glucosylated steviol glycosides and unreacted steviol glycosides.

Moreover, these data were reviewed by a panel of experts, qualified by scientific training and experience to evaluate the safety of ingredients as components of food, who concluded that the intended uses of a-glucosylated steviol glycosides are safe and suitable and would be GRAS based on scientific procedures [see Appendix A, entitled, "EXPERT PANEL CONSENSUS STATEMENT CONCERNING

THE GENERALLY RECOGNIZED AS SAFE (GRAS) STATUS OF a-GLUCOSYLATED STEVIOL GLYCOSIDES FOR USE AS A GENERAL PURPOSE SWEETENING AGENT1 A summary of these data is presented herein.

B.Conditions of Intended Use in Food and Consumer Exposure

B.1History of Use of Steviol Glycosides

S. rebaudiana

and its isolated glycosides (most commonly stevioside) have been consumed for centuries became a popular herbal tea ingredient in the United States (Blumenthal, 1995; Ferlow,

by humans, in various countries, as sweeteners in foods and beverages (Geuns, 2003). In the 1980s,

S. rebaudiana

2005). There have been no reports of adverse effects following the use of these natural sweeteners (Lee, 1979; Ferlow, 2005). In 1995, the use of stevioside in Asia was reported to be approximately 160,000 tones, calculated on a sucrose equivalency basis; however, by 1999, such use reportedly increased to approximately 200,000 tones SE (Anonymous, 2001). Stevioside has been used as a sweetener in Japan for more than 30 years, and its use has been reported to be safe, without the occurrence of adverse effects (Ferlow, 2005). a-Glucosylated steviol glycosides also have been consumed in Japan for more than 20 years.

B.2Current Regulatory Status of Steviol Glycosides

a-Glucosylated steviol glycosides are approved for use in Japan as a sweetening agent in various foods. Although a-glucosylated steviol glycosides are not currently permitted for addition to food and beverages in the U.S., several GRAS Notifications for purified steviol glycosides 05% purity) have been submitted to the FDA. Of the 15 GRAS Notifications submitted, the FDA has responded with "no Toyo Sugar Refining Co., Ltd. Nippon Paper Chemicals Co., Ltd. February 23, 2011

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questions" to 11 (GRN 252, 253, 275, 278, 282, 287, 303, 304, 318, 323, and 329) (U.S. FDA, 2008a,b, 2009a-d, 2010b-f). The remaining 4 Notifications are currently under review. Additionally, 1 Notification for enzyme modified steviol glycosides has been submitted to the FDA and is currently under review. Steviol glycosides also are approved for use in foods in a number of other jurisdictions and countries including Australia and New Zealand, Japan, Korea, Argentina, Paraguay, Brazil, Russia, Switzerland, Turkey, and Ukraine. Although steviol glycosides are not permitted yet for use as a food additive throughout the European Union, high-purity steviol glycoside preparations are approved for use in Switzerland and France. In April 2010, the European Food Safety Agency (EFSA) established an acceptable daily intake (ADI) for steviol glycosides, expressed as steviol equivalents, of 4 mg/kg body weight/day, based on a no-observed-effect level (NOEL) of 970 mg/kg body weight/day from a 2-year study in rats (Toyoda et 1997) and a safety factor of 100 (EFSA, 2010). This ADI is the same as the one established by JECFA established at their 69 th meeting (JECFA, 2009).

al.,

B.3Sweetening Potency

Although there is no industry-wide agreement on a common sucrose equivalency at which to quote sweetness potencies, realistic use-levels of HIS are generally in the range of 4 to 8% SE. Therefore, 6% SE has been determined to represent reasonable average value to compare HIS. Following the organoleptic evaluation of ct-glucosylated steviol glycosides by qualified panelists, the potency was determined to be 137 times that of sucrose at 6% SE.

C.Estimated Intake of the a-Glucosylated Steviol Glycosides

C.1Intended Use of a-Glucosylated Steviol Glycosides and Levels of Use in Foods

a-Glucosylated steviol glycosides are intended for use as a general purpose sweetening agent, in accordance with cGMP. Although a-glucosylated steviol glycosides are slightly less sweet than aspartame, the uses for a-glucosylated steviol glycosides primarily reflect those currently permitted for aspartame in the United States. A summary of the proposed uses of a-glucosylated steviol glycosides as a general purpose sweetener is provided in Table IV.C.1-1.

Table IV.C.1-1Summary of the Proposed Food-Uses for a-Glucosylated Steviol Glycosides

as a General Purpose Sweetener

Food Category

Proposed Food Uses

Alcoholic Beverages Baked Goods and Baking Mixes

Aromatized Alcoholic Beverages (Excluding Beer) Cakes Cookies Crackers French Toast, Pancakes, and Waffles Muffins, Scones and Doughnuts Pastries and Pie Crust Sweet Breads and Rolls

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Table IV.C.1-1Summary of the Proposed Food-Uses for a-Glucosylated Ste yiol Glycosides as a General Purpose Sweetener

Food Category Proposed Food Uses

Cones (ice cream, soft ice cream) Beverages and Beverage Bases Carbonated Beverages Coffee and Tea Drinks Energy, Sport, and Electrolyte Drinks Fruit Flavored Drinks Meal Replacements (non-milk based), Not for Weight Reduction Meal Replacements (non-milk based) for Weight Reduction Vitamin and Enhanced Waters Breakfast Cereals Ready to Eat Breakfast Cereals Instant and Regular Hot Breakfast Cereals Chewing Gum Condiments and Relishes Chewing Gum Ketchup Mustard Olives and Pickles Relish Confections and Frostings Cocoa Mixes Cocoa-Based Spreads and Fillings Frostings, Icings, and Coatings Dairy-Products Analogs Fats and Oils Soy-Based Beverages Emulsified Sauces Fat-Based Desserts Mayonnaise and Mayonnaise-Type Dressings Salad Dressings Frozen Dairy Desserts and Mixes Frozen Yogurt Ice Cream, Novelties, and Frozen Milk Desserts Fruit and Water Ices Gelatins, Puddings, and Fillings Edible Ices, Sherbet, and Sorbet Flans, Custards, and Other Egg-Based Desserts Puddings and Other Milk-Based Desserts Grain Products and Pastas Cereal and Granola Bars Energy, Meal Replacement, and Fortified Bars Gravies and Sauces Clear Sauces Tomato-Based Sauces Soy sauce Tartar sauce Water and Milk-Based Sauces, Gravies, and Dressings, Including Mixes

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Table IV.C.1-1Summary of the Proposed Food-Uses for a-Glucosylated Steviol Glycosides as a General Purpose Sweetener

Food Category Hard Candy Proposed Food Uses Breath-Freshening Micro Mints with No Added Sugar Freshening Throat Pastilles with No Added Sugar Hard Candy Jams and Jellies Milk Products Jams, Jellies, Preserves, and Marmalades Fermented Milks, Plain Flavored Milk, Milk Drinks, and Mixes (not cocoa) Milk-Based Meal Replacements, Not for Weight Reduction Milk-Based Meal Replacements, For Weight Reduction Yogurt Yogurt Drinks Nut and Nut Products Nut Spreads Processed Whole Nuts, Coated Nuts, and Mixtures Processed Fruits and Fruit Juices Canned or Bottled Fruit Coconut Milk and Coconut Cream Fruit Fillings For Pastries Fruit Juices Fruit Puree Fruit-Based Desserts Processed Vegetables and Vegetable Juices Cooked Vegetables Mixtures Vegetable Juices Vegetable Purees Salty Snacks Corn-Based Snacks Potato -- Based Chips Soft Candy Cocoa and Chocolate Products Soft Candy, Nougats, and Marzipans Sugar Substitutes Sweet Sauces, Toppings, and Syrups Table Top Sugar Substitutes Cocoa Syrups Fruit Sauces, Syrups, and Toppings Sweet Sauces and Toppings (not fruit, not syrups) Dietary Supplements

C.2Estimated Consumption of a-Glucosylated Steviol Glycosides from Proposed Food Uses

Numerous studies in the U.S., Canada, Australia/New Zealand, and countries in the EU have identified the intakes of aspartame and other HIS through post-market surveillance data. Although intake modeling is commonly used to estimate the consumption of a particular ingredient, a more realistic, but conservative approach is to estimate the intake of a-glucosylated steviol glycosides based on the intake Toyo Sugar Refining Co., Ltd. Nippon Paper Chemicals Co., Ltd. February 23, 2011

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figures reported in these published studies. Using published data on dietary exposures to approved intense sweeteners, such as aspartame from post-market surveillance studies, with adjustment for their relative sweetness intensities, Renwick (2008) estimated the intake of rebaudioside A, assuming a relative sweetness for rebaudioside A of 200 times that of sucrose. The data used in these analyses were primarily from studies that used specifically designed food diaries combined with actual use levels or approved levels in different foods and beverages. These data were pooled in order to provide a realistic, but conservative estimate of potential consumption of rebaudioside A. The estimated consumption of rebaudioside A, as calculated by Renwick (2008), is presented in Table IV.C.2-1.

Table IV.C.2-1Intakes of Intense Sweeteners and Predicted Intakes of Rebaudioside A (Renwick, 2008)

Intakes of Intense Sweeteners (as sucrose equivalents) Population Group Mean Intake (mg/kg bw/d) 255 280 425 672 Heavy Consumer (mg/kg bw/d) 675 897 990 908 Predicted Intakes of Rebaudioside A Mean Intake (mg/kg bw/d) 1.3 1.4 2.1 3.4 Heavy Consumer (mg/kg bw/d) 3.4 4.5 5.0 4.5 Predicted Intakes of Rebaudioside A (as steviol equivalents) Mean Intake (mg/kg bw/d) 0.43 0.46 0.69 1.12 Heavy Consumer (mg/kg bw/d) 1.12 1.48 1.64 1.48

Non-diabetic Adults Diabetic Adults Non-diabetic Children Diabetic Children

Using the same principles as Renwick (2008), the predicted intake of a-glucosylated steviol glycosides were calculated based on the intakes of intense sweeteners (as sucrose equivalents) based on published data and relative a-glucosylated steviol glycosides sweetness of 137 times that of sucrose as discussed in Section IV.B.3. As presented in Table IV.C.2-2, the predicted intakes of a-glucosylated steviol glycosides are all below the current ADI defined by the JECFA for steviol glycosides (JECFA, 2009) of 0 to 4 mg/kg body weight/day as steviol.

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Table IV.C.2-2Intakes of Intense Sweeteners and Predicted Intakes of a-Glucosylated Steviol Glycosides Intakes of Intense Sweeteners (as sucrose equivalents) Population Group Mean Intake (mg/kg bw/d)

255 280 425 672

Predicted Intakes of a-Glucosylated Steviol Glycosides Heavy Consumer (mg/kg bw/d)

4.9 6.5 7.2 6.6

Predicted Intakes of a-Glucosylated Steviol Glycosides (as steviol equivalents)1 Mean Intake (mg/kg bw/d)

0.56 0.59 0.91 1.44

I HeavyI Mean Intake Consumer(mg/kg (mg/kgbw/d) bw/d)

6751.9 8972.0 990 908 3.1 4.9

Heavy Consumer (mg/kg bw/d)

1.44 1.91 2.12 1.94

Non-diabetic Adults Diabetic Adults Non-diabetic Children Diabetic Children

Calculated based on the ratio of the molecular weight of a-glucosylated steviol glycosides(weighted average) to that of steviol (3.4)

D.Metabolic Fate of a-Glucosylated Steviol Glycosides

The available data indicate that steviol glycosides escape digestion in the upper gastrointestinal tract as rodent and human digestive enzymes are not capable of hydrolyzing p-glycosidic bonds between the aglycone steviol and the 13-glucose moieties (Hutapea et al., 1997; Geuns et al., 2007). Steviol glycosides, therefore, pass undigested through the upper portion of the gastrointestinal tract and enter the colon intact where they are subjected to microbial degradation. The a-glucopyranosyl moiety of a-glucosylated steviol glycosides, however, may be hydrolyzed by salivary and/or pancreatic a-amylase to the non-modified steviol glycoside form due to the presence of an a(1-->4) glycosidic bond; although, this has not been confirmed experimentally. It would be expected that both modified steviol glycosides that escape cleavage in the oral cavity and upper intestine and non-modified steviol glycosides would follow the same metabolic pathway as other steviol glycosides, whereas the cleaved glucose moiety would be absorbed in the intestine and follow normal carbohydrate metabolism pathways. Studies investigating the hydrolysis of non-modified steviol glycosides by the gut microflora have demonstrated that stevioside and rebaudioside A are completely hydrolyzed to steviol following in vitro incubation with the cecal microflora of mice, rats, and hamsters and human fecal microflora (Wingard et

al., 1980; Hutapea et al., 1997; Gardana et al., 2003; Geuns et al., 2003; Koyama et al., 2003a; Renwick

and Tarka, 2008). Similarly, in vitro data have confirmed that a-glucosylated steviol glycosides are degraded to the non-modified glycoside form, which is then hydrolyzed to the aglycone steviol through the action of the intestinal microflora (Koyama et al., 2003a). The metabolic pathway of a-glucosylated steviol glycosides was determined to be similar to non-modified steviol glycosides, with the a-glucosylated steviol glycosides being a-deglucosylated to the "parent" steviol glycoside (i.e., a-monoglucosylsteviosidesteviosidesteviol). Therefore, due to the similarities in metabolic

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pathways, the metabolic fate and safety of ct-glucosylated steviol glycosides can be established based on the studies conducted with non-modified steviol glycosides. By measuring radioactivity or concentrations of steviol in plasma, a number of authors have investigated the in vivo absorption characteristics following administration of steviol or steviol glycosides. These studies have demonstrated that steviol glycosides are hydrolyzed slowly to steviol, which is then absorbed from the gastrointestinal tract and distributed to a number of organs and tissues, including liver, spleen, adrenal glands, fat, and blood (Nakayama et al., 1986; Sung, 2002; Koyama et al., 2003b; Wang et al., 2004; Roberts and Renwick, 2008). In studies conducted with rats or humans, steviol has been shown to primarily undergo conjugation with glucuronic acid to form steviol glucuronide in the liver (Wingard et al., 1980; Nakayama et al., 1986; Kraemer and Maurer, 1994; Sung, 2002; Geuns and Pietta, 2004; Simonetti et al., 2004; Geuns et al., 2006, 2007; Roberts and Renwick, 2008; Wheeler et al., 2008). Although the metabolism of steviol glycosides has been demonstrated to be similar in rats and humans, the excretion of steviol glucuronide differs slightly in that the primary route of excretion in rats is in the feces via the bile, and in humans is in the urine. The inter-species difference in the route of elimination of systemically absorbed steviol as steviol glucuronide (via the bile in rats and in the urine in humans) occurs as a result of the lower molecular weight threshold for biliary excretion in rats (325 Da) as compared to humans (500 to 600 Da; molecular weight of steviol glucuronide is 495 Da) (Renwick, 2007). Overall, the data demonstrate that steviol glycosides show similar pharmacokinetics in the rat; they are all metabolized in the gut to steviol prior to absorption followed by glucuronidation in the liver and excretion in the feces via the bile. Therefore, the results of toxicology studies on stevioside or rebaudioside A can be used to support the safety assessment of other steviol glycosides. Moreover, although the primary routes of elimination of steviol glucuronide differs between rats and humans, the metabolism of steviol glycosides and pharmacokinetics are quite similar, which confirms the rat as an acceptable model for risk assessment in humans. The difference in the route of elimination is considered to be of no toxicological significance due to the fact that the water soluble phase II metabolites are rapidly cleared in both species.

E.Potential Cleavage of a-Glucopyranosyl Moieties

As mentioned previously, the a-glucopyranosyl moiety of a-glucosylated steviol glycosides, may be hydrolyzed by salivary and/or pancreatic a-amylase to the non-modified steviol glycoside form due to the presence of an a(1--44) glycosidic bond. The cleaved glucose moiety, therefore, may be absorbed in the intestine and follow normal carbohydrate metabolism pathways, thereby potentially increasing blood glucose levels. To establish the maximum potential levels of glucose cleaved from a-glucosylated steviol glycosides following consumption, it was assumed that all a-glucopyranosyl moieties would be hydrolyzed and available for absorption in the intestine. Therefore, based on the estimated consumption of a-glucosylated steviol glycosides from their proposed use as a general purpose sweetener (see Section Toyo Sugar Refining Co., Ltd. Nippon Paper Chemicals Co., Ltd. February 23, 2011

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IV.C.2), the mean and heavy-consumer intakes of glucose potentially available from a-glucosylated steviol glycosides following hydrolysis by digestive enzymes was calculated' for four population groups, including non-diabetic adults, diabetic adults, non-diabetic children, and diabetic children. On a per kilogram basis, non-diabetic adults have the lowest estimated intake of glucose from aglucosylated steviol glycosides, while diabetic children have the highest estimated intake of glucose from a-glucosylated steviol glycosides; thus, for both the mean and heavy-consumer estimated intakes of glucose from a-glucosylated steviol glycosides, the lower end of the range is represented by estimated intakes for non-diabetic adults, while the upper end of the range is represented by estimated intakes for diabetic children. The mean intake of glucose from a-glucosylated steviol glycosides is estimated to range from 0.67 to 1.70 mg/kg body weight/day. The heavy consumer intake of glucose from a-glucosylated steviol glycosides is estimated to range from 1.70 to 2.49 mg/kg body weight/day. Assuming the most conservative scenario -- that a diabetic child or a diabetic adult is exposed to 2.49 mg/kg body weight/day of glucose from the consumption of a-glucosylated steviol glycosides, the total amount of glucose that the diabetic child or the diabetic adult would consume, assuming child and adult body weights of 20 kg and 70 kg, respectively, is 49.8 or 174.3 mg/day, respectively. These amounts of glucose are equivalent to 0.20 kcal/day or 0.70 kcal/day, respectively, which are negligible.

F.Toxicological Studies

An extensive database of literature on steviol glycosides, as well as crude S. rebaudiana extracts, exists. Due to the long history of uses of S. rebaudiana and interest in the economic development of S. rebaudiana-based products, many early safety studies were conducted with crude Stevia extracts or were carried out using non-standardized protocols. The outcomes from these studies have led to questions concerning the effects of steviol glycosides on the reproductive and renal systems, as well as on glucose homeostasis and blood pressure. However, the relevance of these studies is limited given the lack of appropriate characterization of the test materials and deficiencies in study design. Similarly, studies conducted with steviol are of little relevance to the safety of steviol glycosides given the fact that the plasma levels of steviol following oral administration are expected to be much higher that those achieved following oral administration of steviol glycosides due to the slow hydrolysis of the glycosides to steviol in the gut. Instead, Good Laboratory Practices (GLP)-compliant studies conducted with wellcharacterized and high-purity material 05% steviol glycosides) were deemed to be pivotal to the safety of a-glucosylated steviol glycosides. These studies served as the basis for JECFA's evaluation of steviol glycosides and establishment of an ADI of 4 mg/kg body weight, expressed as steviol equivalents. In addition, unpublished GLP-compliant studies conducted on a-glucosylated steviol glycosides provide additional support for the safety of Nippon's and Toyo's products.

1

Calculated using ratio of the weighted average molecular weight of a-glucosylated steviol glycosides (1,084 g/mol) to average molecular weight of available glucose (375.5 g/mol). Ratio was determined to be -2.9:1.

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The majority of the toxicological data available to support the safety of a-glucosylated steviol glycosides has previously been reviewed by the FDA through the numerous Notifications for steviol glycoside preparations (see Section IV.B.2, above). Therefore, these studies are briefly summarized in Section IV.F.1 for completeness. Additionally, any data further corroborating the safety of a-glucosylated steviol glycosides are discussed in Section IV.F.2.

F.1Pivotal Data to the Safety of a-Glucosylated Steviol Glycosides

Acute toxicity studies demonstrate that high-purity steviol glycosides are of low acute oral toxicity >15 g/kg body weight) to mice, rats, and hamsters (Toskulkao

et al., (LD50

1997). With respect to longer-term

exposure, the potential toxicity of high-purity steviol glycosides was evaluated in one 4-week study and three 13-week studies. In these studies, no evidence of systemic toxicity was reported following dietary administration of up to 100,000 or 50,000 ppm of rebaudioside A (97% purity) or stevioside (96.5% purity) to male and female Han Wistar or Sprague-Dawley rats for 4 and 13 weeks, respectively. These dietary concentrations are equivalent to up to 4,136 and 1,332 mg steviol equivalents/kg body weight/day, respectively. Body weight gains were reported to be significantly decreased in the 13-week studies conducted by Curry and Roberts (2008) and Nikiforov and Eapen (2008) with rebaudioside A; however, this effect was suggested to be due to the inclusion of a high concentration of a non-nutritive substance in the diet and acclimation to the intensely sweet diets and thus was not considered to be an adverse effect. Other reported changes in hematological or clinical chemistry parameters or organ weights following dietary administration of rebaudioside A or stevioside were sporadic, limited to one sex, were within historical control ranges, or were determined to be related to physiological adaptations. Histopathological examinations of the organs and tissues also revealed no significant effects. Based on the lack of any biologically or toxicologically significant findings, the no-observedadverse-effect level (NOAEL) was determined to be the highest dietary concentration tested in each study, the highest of which among the 13-week studies was 50,000 ppm, equivalent to 1,332 and 1,487 mg steviol equivalents/kg body weight/day in male and female rats, respectively. Single-and multi-generational studies designed to assess the potential reproductive and developmental toxicity of high-purity steviol glycosides in rats have revealed no significant effects on fertility, mating performance, estrous cycle duration, gestation length, gestation index, litter size, offspring survival, or incidence of fetal abnormalities (Akashi and Yokoyama, 1975; Mori et

et al.,

1981; Usami et al., 1995; Curry 2008). Similar to the 13-week subchronic studies, decreases in body weight and weight gains

al.,

were observed early in the study periods and following weaning, and were not accompanied by significant decreases in food consumption of food utilization. Consistent with the subchronic studies, these changes were considered to be due to acclimation to the diet and were not considered to represent an adverse effect. The NOAELs were determined to be the highest dose tested in each study, which was 25,000 ppm in Han Wistar rats fed rebaudioside A in the diet over 2 generations (equivalent to 656 and 728 mg steviol equivalents/kg body weight/day in males and females, respectively). In single generation studies conducted in rats with high-purity stevioside, the NOAELs following dietary or gavage administration were reported to range from 34 to 952 mg steviol equivalents/kg body weight/day. In addition, a 2-generation study conducted in hamsters demonstrated no significant effects on

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reproduction or development and no evidence of toxicity following gavage administration of up to 2,500 mg/kg body weight/day of stevioside (90% purity), equivalent to 900 mg steviol equivalents/kg body weight/day (Yodyingyuad and Bunyawong, 1991). High-purity stevioside (95.6% purity) was demonstrated to possess no carcinogenic potential or evidence of toxicity in a 2-year study in which F344 rats were fed diets containing up to 5% stevioside (equivalent to 799 and 955 mg steviol equivalents/kg body weight/day in males and females, respectively) (Toyoda et al., 1997). Data pertaining to the genotoxicity of high-purity steviol glycosides also indicate a lack of genotoxic activity in vitro or in vivo (JECFA, 2009). The database of literature pertinent to the safety of high-purity steviol glycosides also includes published studies conducted in humans to determine the tolerability and potential pharmacological effects with respect to glucose homeostasis and blood pressure. These studies demonstrate that steviol glycosides are well-tolerated in normotensive, hypotensive, and type-2 diabetic subjects following consumption of doses of up to 1,500 mg/day (21.4 mg/kg body weight/day for a 70 kg individual) for up to 2 years (Oviedo et al., 1970; Alvarez et al., 1981; Boeckh and Humboldt, 1981; Curi et al., 1986; Boeckh-Haebisch, 1992; Chan et al., 2000; Hsieh et al., 2003; Anonymous, 2004a,b; Gregersen et al., 2004; Temme et al., 2004; Cavalcante da Silva et al., 2006; Ferri et al., 2006; Jeppesen et al., 2006; Geuns et al., 2007; Barriocanal et al., 2008; Maki et al., 2008a,b). Studies specifically designed to assess the effects of rebaudioside A on glucose homeostasis and blood pressure also demonstrate a lack of pharmacological effects at doses of up to 1,000 mg/day (14.3 mg/kg body weight/day for a 70 kg individual) for up to 16 weeks (Maki et al., 2008a,b).

F.2Corroborative Data to the Safety of a-Glucosylated Steviol Glycosides

Similar to non-modified steviol glycosides, a-glucosylated steviol glycosides are of low acute oral toxicity, as no deaths or signs of toxicity following single gavage administration of up to 60 g/kg body weight to mice (Toyo Sugar, unpublished[a]). Data pertaining to the genotoxicity of a-glucosylated steviol glycosides also indicate a lack of genotoxic activity in vitro or in vivo (Toyo Sugar, unpublished[b]). In an unpublished 13-week toxicity study conducted in accordance with Good Laboratory Practices (GLP), male and female Charles River rats (10/sex/group) were randomized to receive a diet supplemented with either 0 (control), 1.25, 2.5, or 5.0% a-glucosylated steviol glycosides (0, 253, 519, or 1,059 mg steviol equivalents/kg body weight/day for males and 0, 289, 601, or 1,153 mg steviol equivalents/kg body weight/day for females, respectively) (Hooks et al., 1988). No deaths, clinical signs of toxicity, or abnormalities in ophthalmoscopic examinations were observed among the study groups throughout the treatment period. Although male and female rats in the 2.5 and 5.0% groups had slight decreases in body weight gain when compared to the control group, these decreases did not reach statistical significance and were deemed by the authors to be of no toxicological importance. Likewise, food consumption was marginally decreased among males and females in the 1.25% group, but was attributed to represent natural variation and was not related to the test-article.

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Food conversion efficiency and water consumption were unaffected by the a-glucosylated steviol glycoside-containing diets. Consistent with the findings of the subchronic toxicity studies conducted with non-modified steviol glycosides, various statistically significant increases and decreases in hematology, clinical chemistry, and urinalysis measurements, as well as in relative and absolute organ weights, were observed following the administration of a-glucosylated steviol glycosides. The absence of dose-dependency, observations in both sexes, and test article-related macroscopic or microscopic changes in all organs examined led the authors to conclude that these findings were of no toxicological significance. Therefore, the authors concluded that under the conditions of the study, a-glucosylated steviol glycosides did not cause any signs of toxicity in rats after 13 weeks of administration at a level of 5.0% in the diet (1,059 and 1,153 mg/kg body weight/day for males and females, respectively), the highest dose tested.

G.Summary and Basis for GRAS Conclusion

The GRAS determination for the use of a-glucosylated steviol glycosides as a general purpose sweetener is based on scientific procedures. a-Glucosylated steviol glycosides are produced in accordance with cGMP as a result of an enzymatic reaction of dextrin with a high-purity S. rebaudiana extract meeting the current specification for steviol glycoside preparations as determined by JECFA (i.e., >95% steviol glycosides) to yield a product containing >95% of total reacted and unreacted steviol glycosides. Analytical data using HPLC have demonstrated that a-glucosylated steviol glycosides are primarily mono-, di-, and tri-glucosylated, with smaller amounts of tetra- and penta-glucosylated steviol glycosides. Toyo Sugar and Nippon Paper have established chemical and microbiological specifications that are consistent with other food-grade materials and meet the minimum specifications for steviol glycosides established by JECFA. Batch analysis data confirm that that the products are in compliance with the specifications. ct-Glucosylated steviol glycosides are intended for use as a general purpose sweetening agent, in accordance with good manufacturing practice. Based on published dietary exposure data for other approved sweeteners in the U.S. market (e.g., aspartame) and adjusting for relative sweetness intensity, the intake of a-glucosylated steviol glycosides was estimated for non-diabetic children and adults, and diabetic children and adults. The mean intake for these 4 population groups is estimated to range from 1.9 to 4.9 mg/kg body weight/day (0.56 to 1.44 mg/kg body weight/day as steviol equivalents), for nondiabetic adults and diabetic children, respectively. For the heavy consumer, the intake is estimated to range from 4.9 to 7.2 mg/kg body weight/day (1.44 to 2.12 mg/kg body weight/day as steviol equivalents), in non-diabetic adults and non-diabetic children, respectively. The estimated intakes of aglucosylated steviol glycosides are all below the ADI of 4 mg/kg body weight, expressed as steviol equivalents, established by JECFA. An extensive database of literature on steviol glycosides, as well as crude S. rebaudiana extracts, exists. Early safety studies conducted with crude S. rebaudiana extracts or carried out using non-standardized Toyo Sugar Refining Co., Ltd. Nippon Paper Chemicals Co., Ltd. February 23, 2011

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protocols led to questions concerning the effects of steviol glycosides on the reproductive and renal systems, as well as on glucose homeostasis and blood pressure. However, the relevance of these studies is limited given the lack of appropriate characterization of the test materials and deficiencies in study design. Instead, GLP-compliant studies conducted with well-characterized and high-purity material 05% steviol glycosides) were deemed to be pivotal to the safety of steviol glycosides. These studies served as the basis for JECFA's evaluation of steviol glycosides and establishment of an ADI of 4 mg/kg body weight, expressed as steviol equivalents (JECFA, 2009). This ADI also was supported recently following EFSA's review of the available safety data (EFSA, 2010). Moreover, the majority of the toxicological data available to support the safety of steviol glycosides has previously been reviewed by the FDA through the numerous Notifications for steviol glycoside preparations, to which the FDA responded with no questions. Following oral administration, steviol glycosides are not readily absorbed from the upper small intestine. Human digestive enzymes are not capable of hydrolyzing B-glycosidic bonds and thus steviol glycosides escape digestion in the upper gastrointestinal tract. Although not confirmed experimentally, the aglucopyranosyl moiety of a-glucosylated steviol glycosides, however, may be hydrolyzed by digestive enzymes to the non-modified steviol glycoside form due to the presence of an a(1-->4) glycosidic bond. It would be expected that the resulting non-modified steviol glycoside would follow the same metabolic pathway as other steviol glycosides, whereas the cleaved glucose moiety would be absorbed in the intestine and follow normal carbohydrate metabolism pathways. However, the amount of glucose released during hydrolysis in the upper gastrointestinal tract is negligible (0.2 or 0.7 kcal/day for children and adults, respectively). In the colon, non-modified steviol glycosides are completely hydrolyzed to steviol by microbes of the Bacteroidaceae species (predominantly Bacteroides). Similar to non-modified steviol glycosides, in vitro data have confirmed that a-glucosylated steviol glycosides are degraded to the non-modified glycoside form, which is then hydrolyzed to the aglycone steviol (i.e., a-monoglucosylstevioside stevioside --> steviol) through the action of the intestinal microflora. Therefore, due to the similarities in metabolic fate, the safety of a-glucosylated steviol glycosides can be established based on studies conducted with non-modified steviol glycosides. Furthermore, as individual steviol glycosides show similar pharmacokinetics in the rat and humans, the results of toxicology studies on individual steviol glycosides are applicable to the safety of steviol glycosides in general. In the liver, steviol primarily is conjugated with glucuronic acid to form steviol glucuronide in both rats and humans, and subsequently excreted via the bile in rats and the urine in humans. The differences in the route of elimination are due to the lower molecular weight threshold for biliary excretion in rats (325 Da) as compared to humans (500 to 600 Da). Although the primary routes of elimination of steviol glucuronide differs between rats and humans, the metabolism of modified and non-modified steviol glycosides and pharmacokinetics are quite similar, which confirms the rat as an acceptable model for risk assessment in humans.

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The available toxicological data have demonstrated that steviol glycosides are of low acute oral toxicity. Additionally, outcomes from subchronic, reproductive, and developmental toxicity studies conducted with high-purity steviol glycosides were similar and did not indicate any toxicologically or biologically relevant effects. The NOAELs from these studies ranged between 25,000 and 50,000 ppm (equivalent to 656 and 1,487 mg steviol equivalents/kg body weight/day in rats, respectively). High-purity stevioside (95.6% purity) was demonstrated to possess no carcinogenic potential or evidence of toxicity in the pivotal 2-year study in which F344 rats were fed diets containing up to 5% stevioside (equivalent to 799 and 955 mg steviol equivalents/kg body weight/day in males and females, respectively). Data pertaining to the genotoxicity of high-purity steviol glycosides also indicate a lack of genotoxic activity in vitro or in

vivo.

Published studies conducted in humans to determine the tolerability and potential pharmacological effects with respect to glucose homeostasis and blood pressure demonstrate that steviol glycosides are well-tolerated in normotensive, hypotensive, and type-2 diabetic subjects following consumption of doses of up to 1,500 mg/day (21.4 mg/kg body weight/day for a 70 kg individual) for up to 2 years. Studies specifically designed to assess the effects of steviol glycosides on glucose homeostasis and blood pressure also demonstrate a lack of pharmacological effects at doses of up to 1,000 mg/day (14.3 mg/kg body weight/day for a 70 kg individual) for up to 16 weeks. The safety of a-glucosylated steviol glycosides is further corroborated through the lack of toxicologically or biologically significant effects observed in a subchronic toxicity in which rats were administered diets containing up to 5% a-glucosylated steviol glycosides (equivalent to 1,059 and 1,153 mg steviol equivalents/kg body weight/day for males and females, respectively), as well as a lack of in vitro and in

vivo genotoxic activity in 2 unpublished studies. Together, the above data provided support the

conclusion that the consumption of a-glucosylated steviol glycosides under the intended conditions of use would not be expected to produce adverse effect in consumers. Finally, the Expert Panel convened on behalf of Toyo Sugar and Nippon Paper, independently and collectively, critically evaluated the data and information summarized above, and concluded that the intended uses of a-glucosylated steviol glycosides, trade named SK Sweet Z3 or aG Sweet PX, produced consistently with cGMP and meeting appropriate food grade specifications described herein, are safe and suitable. Furthermore, the Expert Panel unanimously concluded that the intended uses of aglucosylated steviol glycosides, meeting appropriate food-grade specifications and produced consistent with cGMP, are GRAS based on scientific procedures. It is also Toyo Sugar and Nippon Paper's opinion that other qualified scientists reviewing the same publicly available toxicological and safety information would reach the same conclusion. Therefore, Toyo Sugar and Nippon Paper have concluded that a-glucosylated steviol glycosides are GRAS under the intended conditions of use on the basis of scientific procedures. Because a-glucosylated steviol glycosides are GRAS based on scientific procedures for its intended uses in food, they are excluded from the definition of a food additive and thus may be marketed and sold for the uses designated above in the U.S. without the promulgation of a food additive regulation under Title 21 of the CFR.

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V.References

Akashi H, Yokoyama Y (1975). [Security of dried-leaves extracts of stevia--report of toxicological test]. Shokuhin Kogyo [Food Ind] 18(20):34-43 [Japanese]. Alvarez M, Bazzote RB, Godoy GL, Cury R, Botion LM (1981). Efeito do extrato aquoso de Stevia rebaundiana Bertoni sobre parametros bioquimicos de pesoas adultas normais. [Hypoglycemic effect of Stevia rebaudiana Bertoni]. Arq Biol Tecnol 24(1):178 [abstract H-35] [Portuguese]. Anonymous (2001). Developments in the high-intensity sweeteners markets. Zuckerindustrie 126(12):970. Anonymous (2004a). Evaluation of the Ingestion of Stevioside, Orally, In Humans Through A Randomised 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. Cited In: JECFA, 2006. Anonymous (2004b). Evaluation of the Ingestion of Stevioside, Orally, In Humans Through A Randomised 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 MaringS. Submitted to WHO by State University of Campinas, Brazil. Cited In: JECFA, 2006. Barriocanal LA, Palacios M, Benitez G, Benitez S, Jirnenez JT, Jimenez N et al. (2008). 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(1):37-41. Blumenthal M (1995). FDA lifts import alert on stevia. Herb can be imported only as dietary supplement; future use as a sweetener is still unclear. HerbalGram (35 - Fall):17-18. Boeckh EMA, Humboldt G (1981). Avaliacao clinica do efeito crô nico do edulcorante natural Stevia rebaudiana Bertoni sobre o teste de tolerância a glicose, parâmetros clinicos e electrocardiográficos em individus normais, Cienc Cult 32(Suple.): 208-210 [Portuguese]. Boeckh-Haebisch EMA (1992). Pharmacological trial of a concentrated crude extract of Stevia rebaudiana (Bert.) Bertoni in healthy volunteers. Arq Biol Tecnol 35(2):299-314. Cavalcante da Silva GE, Assef AH, Cordeiro Albino C, de Araujo Funari Ferri L, Tasin G, Takahashi MH et al. (2006). Investigation of the tolerability of oral stevioside in Brazilian hyperlipidemic patients. Braz Arch Biol Technol 49(4):583-587.

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Chan P, Tomlinson B, Chen Y, Liu 1, Hsieh M, Cheng J (2000). A double-blind placebo-controlled study of the effectiveness and tolerability of oral stevioside in human hypertension. Br J Clin Pharmacol 50(3):215-220. Commission of the European Communities (2003). Commission Directive 2003/95/EC of 27 October 2003 amending Directive 96/77/EC laying down specific purity criteria on food additives other than colours and sweeteners. OffJ Eur Union 46(L283):71-77. Available at: httpVeurlex.europa.eu/LexUriSery/LexUriServ.dohri=01 ±:2003:283:0071:0077:EN:PDF Curi R, Alvarez M, Bazotte RB, Botion LM, Godoy JL, Bracht A (1986). Effect of Stevia rebaudiana on glucose tolerance in normal adult humans. Braz J Med Biol Res 19(6):771-774. Curry LL, Roberts A (2008). Subchronic toxicity of rebaudioside A. Food Chem Toxico146(Suppl. 7):S11S20. Curry LL, Roberts A, Brown N (2008). Rebaudioside A: two-generation reproductive toxicity study in rats. Food Chem Toxicol 46(Suppl. 7):S21-S30. EFSA (2007). Opinion of the Scientific Committee on a request from EFSA on the introduction of a Qualified Presumption of Safety (QPS) approach for assessment of selected microorganisms referred to EFSA. (Question number: EFSA-Q-2005-293, Adopted: 19 November 2007 by European Food Safety Authority). EFSA J 587:1-16. Available at: http://www.efsa.europa.eu/en/scdocs/doc/587.pdf. EFSA (2010). EFSA Panel on Food Additives and Nutrient Sources (ANS); Scientific Opinion on safety of steviol glycosides for the proposed uses as a food additive (Question number: EFSA-Q-2007-071, EFSA-Q-2008-387, EFSA-Q-2008-401, Adopted: 10 March 2010 by European Food Safety Authority). EFSA J 2010;8(4):1537. [85 pp.]. doi:10.2903/j.efsa.2010.1537. Available at: http://www.efsa.europa.eu/en/scdocs/scdoc/1537.htm. Ferlow K (2005). Stevia - The sweetest substance on Earth. NutraCos 4(2, Suppl.):10-11. Ferri LAF, Alves-Do-Prado W, Yamada SS, Gazola S. Batista MR, Bazotte RB (2006). Investigation of the antihypertensive effect of oral crude stevioside in patients with mild essential hypertension. Phytother Res 20(9):732-736. Gardana C, Simonetti P, Canzi E, Zanchi R, Pietta P (2003). Metabolism of stevioside and rebaudioside A from Stevia rebaudiana extracts by human microflora. J Agric Food Chem 51(22):6618-6622. Geuns JMC (2003). Stevioside. Phytochemistry 64(5):913-921. Geuns JMC, Pietta P (2004). 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: JECFA, 2006. Geuns JM, Malheiros RD, Moraes VM, Decuypere EM, Compernolle F, Buyse JG (2003a). Metabolism of stevioside by chickens. J Agric Food Chem 51(4):1095-1101.

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Geuns JMC, Buyse J, Vankeirsbilck A, Temme EHM, Compernolle F, Toppet S (2006). Identification of steviol glucuronide in human urine. J Agric Food Chem 54(7):2794-2798. Geuns JMC, Buyse J, Vankeirsbilck A, Temme EHM (2007). Metabolism of stevioside by healthy subjects. Exp Biol Med 232(1):164-173. Gregersen S, Jeppesen PB, Hoist JJ, Hermansen K (2004). Antihyperglycemic effects of stevioside in type 2 diabetic subjects. Metabolism 53(1):73-76. Hooks WN, Hopkins MN, Gibson WA, Offer 1M, Gopinath C (1988). a-Glucosyl Steviol Glycoside Toxicity to Rats by Repeated Dietary Administration for 13 Weeks. (SKP 36/871363). Prepared by Cambridgeshire, Engl.: Huntingdon Research Centre Ltd. For Tokyo, Japan: Sanyo Kokusaka Pulp Co. Ltd. / Toyo Seito K.K. [Unpublished]. Hsieh MH, Chan P, Sue YM, Liu JC, Liang TH, Huang TY et al. (2003). Efficacy and tolerability of oral stevioside in patients with mild essential hypertension: a two-year, randomized, placebocontrolled study. Clin Ther 25(11):2797-2808. Hutapea AM, Toskulkao C, Buddhasukh D, Wilairat P. Glinsukon T (1997). Digestion of stevioside, a natural sweetener, by various digestive enzymes.1 Clin Biochem Nutr 23:177-186. JECFA (2006). Steviol glycosides. In: Safety Evaluation of Certain Food Additives. Sixty-third Meeting of the Joint FAO/WHO Expert Committee on Food Additives, June 8-17, 2004, Geneva, Switz. (WHO Food Additives Series, no 54). Geneva, Switz.: World Health Organization (WHO), International Programme on Chemical Safety (IPCS), pp. 117-144, 638. Available at:

http://whdlibdoc.who.int/publications/2006/9241660546 eng.pdf.

JECFA (2008). Steviol glycosides. In: Compendium of Food Additive Specifications. Joint FAO/WHO Expert Committee on Food Additives (JECFA), 69th Meeting, June 17-26, 2008, Rome, Italy. (FAO/JECFA Monographs no. 5). Rome, Italy: Food and Agriculture Organiazation of the United Nations (FAO) / World Health Organization (WHO), pp. 75-78. Available at:

ftp://ftp ,fao,org/docrep/fao/011/iO345e/10345e.pdf.

JECFA (2009). Steviol glycosides (addendum). In: Safety Evaluation of Certain Food Additives. Sixty-ninth Meeting of the Joint FAO/WHO Expert Committee on Food Additives (WHO Food Additives Series, no 60). Geneva, Switz.: World Health Organization (WHO), Food and Agriculture Organization of the United Nations, pp. 183-220. Available at:

http://whdlibdoc.who.int/pubhcations/2009/9789241660600 eng.pdf.

Jeppesen PB, Barriocanal L, Meyer MT, Palacios M, Canete F, Benitez S et al. (2006). Efficacy and tolerability of oral stevioside in patients with type 2 diabetes: a long-term, randomized, doubleblinded, placebo- controlled study. Diabetologia 49(Suppl. 1):511-512 [Abstract No. 08431. Koyama E, Kitazawa K, Ohori Y, Izawa 0, Kakegawa K, Fujino A et al. (2003a). In vitro metabolism of the glycosidic sweeteners, stevia mixture and enzymatically modified stevia in human intestinal microflora. Food Chem Toxicol 41(3):359-374.

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Koyama E, Sakai N, Ohori Y, Kitazawa K, Izawa 0, Kakegawa K et al. (2003b). Absorption and metabolism of glycosidic sweeteners of stevia mixture and their aglycone, steviol, in rats and humans. Food Chem Toxicol 41(6):875-883. Kraemer T, Maurer HH (1994). On the metabolism of the sweetener stevioside in humans. Eur J Pharm Sci 2(1&2):103 [abstract FC12]. Lee CK (1979). Carbohydrate sweeteners: structural requirements for taste. In: Bourne GH, editor. Some Special Aspects of Nutrition. (World Review of Nutrition and Dietetics, vol. 33). Basel, Switz.: Karger AG, pp. 142-197. Maki KC, Curry LL, Reeves MS, Toth PD, McKenney JM, Farmer MV et al. (2008a). Chronic consumption of rebaudioside A, a steviol glycoside, in men and women with type 2 diabetes mellitus. Food Chem Toxicol 46(Suppl. 7):S47-S53. Maki KC, Curry LL, Carakostas MC, Tarka SM, ReevesMS, Farmer MV et al. (2008b). The hemodynamic effects of rebaudioside A in healthy adults with normal and low-normal blood pressure. Food Chem Toxicol 46(Suppl. 7):S40-S46. Mori N, Sakanoue M, Takeuchi M, Shimpo K, Tanabe T (1981). Effect of Stevioside on fertility in rats. Shokuhin Eiseigaku Zasshi 22(5):409-414 [Japanese]. Nakayama K, Kasahara D, Yamamoto F (1986). Absorption, distribution, metabolism and excretion of stevioside in rats. Shokuhin Eiseigaku Zasshi 27(1):1-8. Nikiforov Al, Eapen AK (2008). A 90-day oral (dietary) toxicity study of rebaudioside A in Sprague-Dawley rats. Int J Toxicol 27(1):65-80. Oviedo CA, Franciani G, Moreno R, Maas LC (1970). Hypoglycaemic action of Stevia Rebaudiana Bertoni (Kaa-he-6). In: Rodriguez RR, Ebling FJG, Henderson I, editors. Seventh Congress of the International Diabetes Federation, Aug. 23-28, 1970, Buenos Aires, Argentina. (International Congress Series, no 209). Amsterdam, Netherl.: Excerpta Medica Foundation / International Diabetes Foundation (IDF), pp. 92-93 [abstract 208]. Renwick AG (2007). Toxicokinetics [section on elimination: excretion via the gut]. In: Hayes W, editor. Principles and Methods of Toxicology, 5th edition. Philadelphia (PA): Taylor and Francis/CRC Press, p. 188. Renwick AG (2008). The use of a sweetener substitution method to predict dietary exposures for the intense sweetener rebaudioside A. Food Chem Toxicol 46(Suppl. 7):S61-569. Renwick AG, Tarka SM (2008). Microbial hydrolysis of steviol glycosides. Food Chem Toxicol 46(Suppl. 7):570-S74. Roberts A, Renwick AG (2008). Comparative toxicokinetics and metabolism of rebaudioside A, stevioside, and steviol in rats. Food Chem Toxicol 46(Suppl. 7):S31-S39.

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Simonetti P, Gardana C, Bramati L, Pietta PG (2004). Bioavailability of stevioside from Stevia rebaudiana in humans: preliminary report. In: Geuns JMC, Buyse J, editors. Safety of Stevioside: Proceedings of the First Symposium Sponsored by KULeuven, April 16, 2004, Leuven, Belgium. Heverlee, Belgium: Euprint ed., pp. 51-62. Sung LH (2002). 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. Cited In: JECFA, 2006. Temme EH, Vankeirsblick A, Buyse J (2004). A short term study of stevioside in healthy subjects. In: Geuns JMC, Buyse J, editors. Safety of Stevioside: Proceedings of the First Symposium Sponsored by KULeuven, April 16, 2004, Leuven, Belgium. Heverlee, Belgium: Euprint ed., pp. 63-74. Toskulkao C, Chaturat L, Temcharoen P, Glinsukon T (1997). Acute toxicity of stevioside, a natural sweetener, and its metabolite, steviol, in several animal species. Drug Chem Toxicol 20(1&2):3144. Toyo Sugar (unpublished[a]). Safety of alpha-Glucosyltransferase treated Stevia (Acute toxicity). Tokyo, Japan: Toyo Sugar Refining Co., Ltd. Toyo Sugar (unpublished[b]). Mutagenesis Experiments about a-Glucosyltransferase treated Stevia. Tokyo, Japan: Toyo Sugar Refining Co., Ltd. Toyoda K, Matsui H, Shoda T, Uneyama C, Takada K, Takahashi M (1997). Assessment of the carcinogenicity of stevioside in F344 rats. Food Chem Toxicol 35(6):597-603. U.S. FDA (1997). Substances generally recognized as safe; Proposed rule (21 CFR Parts 170, 184, 186, and 570) [Docket No. 97N-0103]. Fed Regist (US) 62(74):18937-18964. U.S. FDA (2000). [GRAS Notice No. 46 -- Substance: Gamma Cyclodextrin]. Submitted to: Washington (DC): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Premarket Approval (HFS-200) by Adrian (MI): Wacker Biochem Corp. Available at: http://www.accessdata.fda.gov/scriptsficn/fcnDetailNavigation.cfm?rpt=grasListing&id=46. U.S. FDA (2001). [GRAS Notice No. 74 -- Substance: Beta-Cyclodextrin]. Submitted to: Washington (DC): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Premarket Approval (HFS-200) by Adrian (MI): Wacker Biochem Corp. Available at: http://www.accessdatalda.gov/scripts/fcnjfcnDetailNavigation.cfm?rpt=grasListing&id=74. U.S. FDA (2004). [GRAS Notice No. 155 -- Substance: a-Cyclodextrin]. Submitted to: Washington (DC): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety by Adrian (MI): Wacker Biochem Corp. Available at: httplZwww.accessdata.tda.,,gpv6.criptsjfcn/fcnDetailNavti3tion.cfm?v,ErasListinOtid=1.55.

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U.S. FDA (2008a). Agency Response Letter GRAS Notice No. GRN 000253 [Rebaudioside A purified from Stevia Rebaudiana (Bertoni) Bertoni]. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN); College Park, Maryland. Available at: http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSateGRAS/GRASLis tings/ucm154989.htrn. U.S. FDA (2008b). Agency Response Letter GRAS Notice No. GRN 000252 [Rebaudioside A purified from Stevia Rebaudiana (Bertoni) Bertoni]. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN); College Park, Maryland. Available at: http://www.fda.gov/FoodgoodIngredientspackaging/GenerallyRecognizedasSafeGRAS/GRASLis tings/ucm154988.htm. U.S. FDA (2009a). Agency Response Letter GRAS Notice No. GRN 000275 [Purified Steviol Glycosides With Rebaudioside A as the Principal Component]. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at: http://www.fda.gov/Food/FoodIngredientsPackaging/GenerailyRecognizedasSafeGRAS/GRASLis tings/ucm171539.htm. U.S. FDA (2009b). Agency Response Letter GRAS Notice No. GRN 000278 [Rebaudioside A Purified From Stevia Rebaudiana (Bertoni) Bertoni]. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at: http://www.ida.gov/Food/FoodIngredientsPackaging/GenerailyRecognizedasSafeGRAVGRASLis tings/ucm174946.htm. U.S. FDA (2009c). Agency Response Letter GRAS Notice No. GRN 000282 [Rebaudioside A Purified From the Leaves of Stevia Rebaudiana (Bertoni) Bertoni]. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at: http://www.fda.gov/Food/FoodIngredientsPackaging/GenerailvRecognizedasSafeGRAS/GRASLis tings/ucni185772.htrn. U.S. FDA (2009d). Agency Response Letter GRAS Notice No. GRN 000287 [Purified Steviol Glycosides With Rebaudioside A and Stevioside As the Principal Components]. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at: http://www.fda.gov/Food/FoodlngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASLis tings/ucm181937,htm. U.S. FDA (2010a). Part 170--Food additives. §170.30--Eligibility for classification as generally recognized as safe (GRAS). In: U.S. Code of Federal Regulations (CFR). Title 21: Food and Drugs (Food and Drug Administration). Washington (DC): U.S. Government Printing Office (GPO), pp. 13-15. Available at: Available at: http://www.access.gpogovyinarakfr/cfr-table-search.htrni#page1.

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U.S. FDA (2010b). Agency Response Letter GRAS Notice No. GRN 000303 [Rebaudioside A purified from the leaves of Stevia rebaudiana (Bertoni) Bertoni]. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at: http://www.fcta.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRASJGRASLis tings/ucm207985.htrn U.S. FDA (2010c). Agency Response Letter GRAS Notice No. GRN 000304 [Purified steviol glycosides with rebaudioside A and stevioside as the principal components]. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at: http://www.fc,a.gov/FoodgoodIngredientsPackaging/GenerailyRecognizedasSafeGRAS/GRASLIS tings/uon207c03.htm U.S. FDA (2010d). Agency Response Letter GRAS Notice No. GRN 000318 [Rebaudioside A purified from the leaves of Stevia rebaudiana (Bertoni) Bertoni]. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at: http://www.foa.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASLis tings/ucm213311.htm U.S. FDA (2010e). Agency Response Letter GRAS Notice No. GRN 000323 [Purified steviol glycosides with rebaudioside A and stevioside as the principal components]. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at: http://www.fd:a.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASLis tingshicrn219341.htrn U.S. FDA (2010f). Agency Response Letter GRAS Notice No. GRN 000329 [Rebaudioside A purified from the leaves of Stevia rebaudiana (Bertoni) Bertoni]. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at: http://ww`ALfda.gov/Food/FoodlngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASLis tings/uon228941.htm U.S. FDA (2010g). U.S. Code of Federal Regulations (CFR). Title 21: Food and Drugs (Food and Drug Administration). Washington (DC): U.S. Government Printing Office (GPO). Available at: Available at: http://www.access.gpo.govhara/cfr/cfr-table-search.html#pagel [See Table for CFR sections].

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Table of CFR Sections Referenced (Title 21--Food and Drugs) Section § Section Title Part

172--Food additives permitted for direct addition to food for human consumption 172.340 172.859 172.867 172.869 173--Secondary direct food additives permitted in food for human consumption 184--Direct food substances affirmed as generally recognized as safe 173.25 173.250 184.1148 184.1277 184.1293 184.1443a Fish protein isolate Sucrose fatty acid esters Olestra Sucrose oligoesters lon-exchange resins Methyl alcohol residues Bacterially-derived carbohydrase enzyme preparation Dextrin Ethyl alcohol Malt

Usami M, Sakemo K, Kawashima K, Tsuda M, Ohno Y (1995). Teratogenicity study of stevioside in rats. Eisei Shikenjo Hokoku [Bull Natl Inst Hyg Sci] 113:31-35 [Japanese]. Wang LZ, Goh BC, Fan L, Lee HS (2004). Sensitive high- performance liquid chromatography/mass spectrometry method for determination of steviol in rat plasma. Rapid Commun Mass Spectrom 18(1):83-86. Wheeler A, Boileau AC, Winkler PC, Compton JC, Prakash I, Jiang X et al. (2008). Pharmacokinetics of rebaudioside A and stevioside after single oral doses in healthy men. Food Chem Toxicol 46(Suppl. 7):S54-S60. Wingard RE Jr, Brown JP, Enderlin FE, Dale JA, Hale RL, Seitz CT (1980). Intestinal degradation and absorption of the glycosidic sweeteners stevioside and rebaudioside A. Experientia 36(5):519520. Yodyingyuad V. Bunyawong S (1991). Effect of stevioside on growth and reproduction. Hum Reprod 6(1):158-165.

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Appendix A Expert Panel Consensus Statement Concerning the Generally Recognized as Safe (GRAS) Status of a-Glucosylated Steviol Glycosides for use as a General Purpose Sweetening Agent

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Expert Panel Consensus Statement Concerning the Generally Recognized as Safe (GRAS) Status of a-Glucosylated Steviol Glycosides for Use as a General Purpose Sweetening Agent

June 30, 2010

INTRODUCTION

At the request of Toyo Sugar Refining Co., Ltd. (herein "Toyo Sugar") and Nippon Paper Chemicals Co., Ltd. (herein "Nippon Paper"), an Expert Panel (the "Panel") of independent scientists, qualified by their relevant national and international experience and scientific training to evaluate the safety of food ingredients, was specially convened to conduct a critical and comprehensive evaluation of the available pertinent data and information, and determine whether the intended uses of a-glucosylated steviol glycosides as a food ingredient are safe and suitable and would be Generally Recognized as Safe (GRAS) based on scientific procedures. The Panel consisted of: Dr. Samuel Cohen, Ph.D., M.D., D.A.B.P., F.A.T.S (University of Nebraska Medical Center), Dr. Ian Munro, Ph.D., F.A.T.S., FRCPath (Cantox Health Sciences International), and Dr. William Waddell, M.D., F.A.T.S (University of Louisville). The Panel, independently and collectively, critically examined a comprehensive package of scientific information and data on a-glucosylated steviol glycosides from the literature and other published sources through June 2010. This information was presented in a dossier [Documentation Supporting the Evaluation of a-Glucosylated Steviol Glycosides as Generally Recognized as Safe (GRAS) for Use as a General Purpose Sweetening Agent] that was submitted by Toyo Sugar and Nippon Paper to the Panel. In addition, the Panel evaluated other information deemed appropriate or necessary. The information evaluated by the Panel included details pertaining to the method of manufacture and product specifications, supporting analytical data, intended use-levels in specified food products, consumption estimates for all intended uses, and a comprehensive assessment of the available scientific literature pertaining to the safety of a-glucosylated steviol glycosides. Following independent, critical evaluation of such data and information, the Panel convened on June 30, 2010 and unanimously concluded that the intended uses in traditional foods described herein for a-glucosylated steviol glycosides, meeting appropriate food-grade specifications as described in the supporting dossier and manufactured according to current Good Manufacturing Practice (cGMP), are safe and suitable and GRAS based on scientific procedures. A summary of the basis for the Panel's conclusion is provided below.

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SUMMARY

a-Glucosylated steviol glycosides are produced in accordance with cGMP as a result of an enzymatic reaction of dextrin with a high-purity Stevie rebaudiana extract (>95% steviol glycosides) to yield a product containing >95% of total reacted and unreacted steviol glycosides. Analytical data using High Performance Liquid Chromatography (HPLC) have demonstrated that a-glucosylated steviol glycosides are primarily mono-, di-, and tri-glucosylated, with smaller amounts of tetra- and penta-glucosylated steviol glycosides. Toyo Sugar and Nippon Paper have established chemical and microbiological specifications that are consistent with other foodgrade materials and meet the minimum specifications for steviol glycosides established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Batch analysis data confirm that that the products are in compliance with the specifications. a-Glucosylated steviol glycosides are intended for use as a general purpose sweetening agent, in accordance with good manufacturing practice. Based on published dietary exposure data for other approved sweeteners in the U.S. market (e.g., aspartame) and adjusting for relative sweetness intensity, the intake of a-glucosylated steviol glycosides was estimated for nondiabetic children and adults, and diabetic children and adults. The mean intake for these 4 population groups is estimated to range from 1.9 to 4.9 mg/kg body weight/day (0.53 to 1.37 mg/kg body weight/day as steviol equivalents), whereas for heavy consumers, the intake is estimated to range from 4.9 to 7.2 mg/kg body weight/day (1.37 to 2.02 mg/kg body weight/day as steviol equivalents. The estimated intakes of a-glucosylated steviol glycosides are all below the acceptable daily intake (ADI) of 4 mg/kg body weight, expressed as steviol equivalents, established by JECFA (2009). Several GRAS Notifications for purified steviol glycosides (95% purity) have been submitted to the United States Food and Drug Administration (FDA). Of the 11 GRAS Notifications submitted, the FDA has responded with "no questions" to 9 (GRN 000252, 000253, 000275, 000278, 000282, 000287, 000303, 000304, and 000318) (U.S. FDA, 2008a,b, 2009a-d, 2010a-c). The remaining 2 Notifications are currently under review. Moreover, the European Food Safety Authority (EFSA) released their conclusions regarding the safety of purified steviol glycosides in April 2010. Similar to JECFA's conclusions, EFSA established an acceptable daily intake (ADI) for steviol glycosides, expressed as steviol equivalents, of 4 mg/kg body weight/day (EFSA, 2010). The safety of a-glucosylated steviol glycosides is supported by a number of published and unpublished studies on a-glucosylated steviol glycosides and non-enzymatically modified steviol glycosides. A published in vitro study in which a-glucosylated steviol glycosides were degraded by fecal microflora to steviol glycosides, which were then hydrolyzed to the aglycone steviol, demonstrates that the metabolic fate of a-glucosylated steviol glycosides follows that of nonmodified steviol glycosides (Koyama et al., 2003). Thus, data that are pivotal to the safety of

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steviol glycosides also are pivotal to the safety of a-glucosylated steviol glycosides under the conditions of intended use. Studies conducted in rats and humans indicate that steviol glycosides, including rebaudioside A and stevioside, are metabolized by lower gut microflora to steviol by successive removal of glucose moieties. Steviol is subsequently absorbed from the colon, subjected to glucuronidation in the liver, and excreted primarily in the feces (via bile) of rats or the urine of humans (as steviol glucuronide). Although the primary routes of elimination of steviol glucuronide differ between rats and humans, the metabolism of steviol glycosides and pharmacokinetics are quite similar, which confirms the rat as an acceptable model for risk assessment in humans (Roberts and Renwick, 2008; Wheeler et al., 2008). Therefore, the results of toxicology studies on stevioside or rebaudioside A can be used to support the safety assessment of other steviol glycosides. An extensive database of literature on steviol glycosides, as well as crude S. rebaudiana extracts, exists. Due to the long history of uses of S. rebaudiana and interest in the economic development of S. rebaudiana-based products, many early safety studies were conducted with crude Stevie extracts or were carried out using non-standardized protocols. The outcomes from these studies have led to questions concerning the effects of steviol glycosides on the reproductive and renal systems, as well as on glucose homeostasis and blood pressure. However, the relevance of these studies is limited given the lack of appropriate characterization of the test materials and deficiencies in study design. Similarly, studies conducted with steviol are of little relevance to the safety of steviol glycosides given the fact that the plasma levels of steviol following oral administration are expected to be much higher than those achieved following oral administration of steviol glycosides due to the slow hydrolysis of the glycosides to steviol in the gut. Instead, good laboratory practices (GLP)-compliant studies conducted with well-characterized and high-purity material (95% steviol glycosides) were deemed to be pivotal to the safety of a-glucosylated steviol glycosides. These studies served as the basis for JECFA's evaluation of steviol glycosides and establishment of an ADI of 4 mg/kg body weight, expressed as steviol equivalents. In addition, unpublished GLP-compliant studies conducted on a-glucosylated steviol glycosides provide additional support for the safety of Nippon Paper's and Toyo Sugar's products. Acute toxicity studies demonstrate that high-purity steviol glycosides are of low acute oral toxicity (LD50 >15 g/kg body weight) to mice, rats, and hamsters (Toskulkao et al., 1997), while a study of a-glucosylated steviol glycosides in mice resulted in no deaths or signs of toxicity following single gavage administration of up to 60 g/kg body weight (Anonymous). With respect to longer-term exposure, the potential toxicity of high-purity steviol glycosides was evaluated in one 4-week study and three 13-week studies (Aze et al., 1991; Curry and Roberts, 2008; Nikiforov and Eapen, 2008). In these studies, no evidence of systemic toxicity was reported following dietary administration of up to 100,000 or 50,000 ppm of rebaudioside A (97% purity) or stevioside (96.5% purity), respectively, to male and female Han Wistar or Sprague-Dawley

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rats for 4 and 13 weeks, respectively. These dietary concentrations are equivalent to up to 4,136 and 1,332 mg steviol equivalents/kg body weight/day, respectively. Body weight gains were reported to be significantly decreased in the 13-week studies conducted by Curry and Roberts (2008) and Nikiforov and Eapen (2008) with rebaudioside A; however, this effect was suggested to be due to the inclusion of a high concentration of a non-nutritive substance in the diet and acclimation to the intensely sweet diets and thus was not considered to be an adverse effect. Other reported changes in hematological or clinical chemistry parameters or organ weights following dietary administration of rebaudioside A or stevioside were sporadic, limited to one sex, were within historical control ranges, or were determined to be related to physiological adaptations. Histopathological examinations of the organs and tissues also revealed no significant effects. Based on the lack of any biologically or toxicologically significant findings, the no-observed-adverse-effect level (NOAEL) was determined to be the highest dietary concentration tested in each study, the highest of which among the 13-week studies was 50,000 ppm, equivalent to 1,332 and 1,487 mg steviol equivalents/kg body weight/day in male and female rats, respectively. The results of an unpublished 13-week toxicity study on a-glucosylated steviol glycosides in male and female Charles River CD Sprague-Dawley rats are consistent with those of the published studies on steviol glycosides (Hooks et al., 1988). No statistically significant changes in body weight gain were reported and changes in hematology, clinical chemistry, and urinalysis parameters and organ weights were deemed not to be toxicologically significant given the absence of dose-dependency, observations in only one sex, and a lack of microscopic changes in the organs. The NOAEL was determined to be 5% in the diet, the highest concentration tested, which is equivalent to 1,008 and 1,098 mg steviol equivalents/kg body weight/day. Single- and multi-generational studies designed to assess the potential reproductive and developmental toxicity of high-purity steviol glycosides in rats have revealed no significant effects on fertility, mating performance, estrous cycle duration, gestation length, gestation index, litter size, offspring survival, or incidence of fetal abnormalities (Akashi and Yokoyama, 1975; Mod et al., 1981; Usami et al., 1995; Curry et al., 2008). Similar to the 13-week subchronic studies, decreases in body weight and weight gains were observed early in the study periods and following weaning, and were not accompanied by significant decreases in food consumption of food utilization. Consistent with the subchronic studies, these changes were considered to be due to acclimation to the diet and were not considered to represent an adverse effect. The NOAELs were determined to be the highest dose tested in each study, which was 25,000 ppm in Han Wistar rats fed rebaudioside A in the diet over 2 generations (equivalent to 656 and 728 mg steviol equivalents/kg body weight/day in males and females, respectively) (Curry et al., 2008). In single generation studies conducted in rats with high-purity stevioside, the NOAELs following dietary or gavage administration were reported to range from 34 to 952 mg steviol equivalents/kg body weight/day (Akashi and Yokoyama, 1975; Mori et al., 1981; Usami et al., 1995). In addition, a 2-generation study conducted in hamsters demonstrated no

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significant effects on reproduction or development and no evidence of toxicity following gavage administration of up to 2,500 mg/kg body weight/day of stevioside (90% purity), equivalent to 900 mg steviol equivalents/kg body weight/day (Wasuntarawat et al., 1998). High-purity stevioside (95.6% purity) was demonstrated to possess no carcinogenic potential or evidence of toxicity in a 2-year study in which F344 rats were fed diets containing up to 5% stevioside (equivalent to 799 and 955 mg steviol equivalents/kg body weight/day in males and females, respectively) (Toyoda et al., 1997). Data pertaining to the genotoxicity of high-purity steviol glycosides and a-glucosylated steviol glycosides also indicate a lack of genotoxic activity in vitro or in vivo. The database of literature pertinent to the safety of high-purity steviol glycosides also includes published studies conducted in humans to determine the tolerability and potential pharmacological effects with respect to glucose homeostasis and blood pressure. These studies demonstrate that steviol glycosides are well-tolerated in normotensive, hypotensive, and type-2 diabetic subjects following consumption of doses of up to 1,500 mg/day (21.4 mg/kg body weight/day for a 70 kg individual) for up to 2 years (Oviedo et al., 1970; Alvarez et al., 1981; Boeckh and Humboldt, 1981; Curi et al., 1986; Boeckh-Haebisch, 1992; Chan et al., 2000; Hsieh et al., 2003; Anonymous, 2004a,b; Gregersen et al., 2004; Temme et al., 2004; Cavalcante da Silva et al., 2006; Ferri et al., 2006; Jeppesen et al., 2006; Geuns et al., 2007; Barriocanal et al., 2008; Maki et al., 2008a,b). Studies specifically designed to assess the effects of rebaudioside A on glucose homeostasis and blood pressure also demonstrate a lack of pharmacological effects at doses of up to 1,000 mg/day (14.3 mg/kg body weight/day for a 70 kg individual) for up to 16 weeks (Maki et al., 2008a,b). The scientific evidence examined by the Panel demonstrates that under the conditions of intended use, a-glucosylated steviol glycosides would not produce any adverse health effects.

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CONCLUSIONS

We, the Expert Panel, have, independently and collectively, critically evaluated the data and information summarized above and conclude that the intended uses of a-glucosylated steviol glycosides, meeting appropriate food-grade specifications presented in the supporting dossier [Documentation Supporting the Evaluation of a-Glucosylated Steviol Glycosides as Generally Recognized as Safe (GRAS) for Use as a General Purpose Sweetening Agent] and produced consistent with current Good Manufacturing Practices (cGMP), are safe and suitable. We further conclude that the intended uses of a-glucosylated steviol glycosides, meeting appropriate food-grade specifications presented in the supporting dossier and produced consistent with cGMP, are Generally Recognized as Safe (GRAS) based on scientific procedures. It is our opinion that other qualified experts would concur with these conclusions.

(b) (6)

Dr. Samuel Cohen, Ph.D., M.D., D.A.B.P., F.A.T.S. University of Nebraska Medical Center

Date

(b) (6)

D/ an Mun , Cantox Health Sciences International

(b) (6)

Dr. William Waddell, M. ., F.A.T.S. University of Louisville Date

7No

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REFERENCES

Akashi H, Yokoyama Y (1975). [Security of dried-leaves extracts of stevia--report of toxicological test]. Shokuhin Kogyo [Food Ind] 18(20):34-43 [Japanese]. Alvarez M, Bazzote RB, Godoy GL, Cury R, Botion LM (1981). Efeito do extrato aquoso de Stevia rebaundiana Bertoni sobre parametros bioquimicos de pesoas adultas normais. [Hypoglycemic effect of Stevia rebaudiana Bertoni]. Arq Biol Tecnol 24(1):178 [abstract H-35] [Portuguese]. Anonymous (2004a). Evaluation of the Ingestion of Stevioside, Orally, In Humans Through A Randomised 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 Maringa and the Academical Hospital of Maringa. Submitted to WHO by State University of Campinas, Brazil. Cited In: JECFA, 2006. Anonymous (2004b). Evaluation of the Ingestion of Stevioside, Orally, In Humans Through A Randomised 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. Cited In: JECFA, 2006. Anonymous (Undated). Safety of alpha-Glucosyltransferase treated Stevia (Acute Toxicity) [Unpublished]. Aze Y, Toyoda K, Imaida K, Hayashi S, Imazawa T, Hayashi Y et al. (1991). Subchronic oral toxicity study of stevioside in F344 rats. Eisei Shikenjo Hokoku [Bull Natl Inst Hyg Sci] (109):48-54 [Japanese]. Barriocanal LA, Palacios M, Benitez G, Benitez S, Jimenez JT, Jimenez N et al. (2008). 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(1):37-41. Boeckh EMA, Humboldt G (1981). Avaliagao clinica do efeito cre· nico do edulcorante natural Stevia rebaudiana Bertoni sobre o teste de tolerância a glicose, parâmetros clinicos e electrocardiográficos em individus normais. Cienc Cult 32(Suple.): 208-210 [Portuguese]. Boeckh-Haebisch EMA (1992). Pharmacological trial of a concentrated crude extract of Stevia rebaudiana (Bert.) Bertoni in healthy volunteers. Arq Biol Tecnol 35(2):299-314. Cavalcante da Silva GE, Assef AH, Cordeiro Albino C, de Araujo Funari Ferri L, Tasin G, Takahashi MH et al. (2006). Investigation of the tolerability of oral stevioside in Brazilian hyperlipidemic patients. Braz Arch Biol Technol 49(4):583-587.

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Chan P, Tomlinson B, Chen Y, Liu J, Hsieh M, Cheng J (2000). A double-blind placebocontrolled study of the effectiveness and tolerability of oral stevioside in human hypertension. Br J Clin Pharmacol 50(3):215-220. Curi R, Alvarez M, Bazotte RB, Botion LM, Godoy JL, Bracht A (1986). Effect of Stevia rebaudiana on glucose tolerance in normal adult humans. Braz J Med Biol Res 19(6):771-774. Curry LL, Roberts A (2008). Subchronic toxicity of rebaudioside A. Food Chem Toxicol 46(Suppl. 7):S11-S20. Curry LL, Roberts A, Brown N (2008). Rebaudioside A: two-generation reproductive toxicity study in rats. Food Chem Toxicol 46(Suppl. 7):S21-S30. EFSA (2010). EFSA Panel on Food Additives and Nutrient Sources (ANS); Scientific Opinion on safety of steviol glycosides for the proposed uses as a food additive (Question number: EFSA-Q-2007-071, EFSA-Q-2008-387, EFSA-Q-2008-401, Adopted: 10 March 2010 by European Food Safety Authority). EFSA J 2010;8(4):1537. [85 pp.]. doi:10.2903/j.efsa.2010.1537. Available at: http://www.efsa.europa.eu/en/scdocs/scdoc/1537.htm. Ferri LAF, Alves-Do-Prado W, Yamada SS, Gazola S, Batista MR, Bazotte RB (2006). Investigation of the antihypertensive effect of oral crude stevioside in patients with mild essential hypertension. Phytother Res 20(9):732-736. Geuns JMC, Buyse J, Vankeirsbilck A, Temme EHM (2007). Metabolism of stevioside by healthy subjects. Exp Biol Med 232(1):164-173. Gregersen S, Jeppesen PB, Ho1st JJ, Hermansen K (2004). Antihyperglycemic effects of stevioside in type 2 diabetic subjects. Metabolism 53(1):73-76. Hooks WN, Hopkins MN, Gibson WA, Offer JM, Gopinath C (1988). a-Glucosyl Steviol Glycoside Toxicity to Rats by Repeated Dietary Administration for 13 Weeks. (SKP 36/871363). Prepared by Cambridgeshire, Engl.: Huntingdon Research Centre Ltd. For Tokyo, Japan: Sanyo Kokusaka Pulp Co. Ltd. / Toyo Seito K.K. [Unpublished]. Hsieh MH, Chan P, Sue YM, Liu JC, Liang TH, Huang TY et al. (2003). Efficacy and tolerability of oral stevioside in patients with mild essential hypertension: a two-year, randomized, placebo- controlled study. Clin Ther 25(11):2797-2808. JECFA (2006). Steviol glycosides. In: Safety Evaluation of Certain Food Additives. Sixty-third Meeting of the Joint FAO/VVHO Expert Committee on Food Additives, June 8-17, 2004, Geneva, Switz. (WHO Food Additives Series, no 54). Geneva, Switz.: World Health Organization (WHO), International Programme on Chemical Safety (IPCS), pp. 117-144, 638. Available at: httpl/whqiibdoc.whojnt/publications/2006/9241660546 eng.pdt. JECFA (2009). Steviol glycosides (addendum). In: Safety Evaluation of Certain Food Additives. Sixty-ninth Meeting of the Joint FAO/WHO Expert Committee on Food Additives (WHO Food Additives Series, no 60). Geneva, Switz.: World Health Organization (WHO), Food and Agriculture Organization of the United Nations, pp. 183-220. Available at: 'nttp://\Nhqhbdoc.who.int/publicationsi2009/9789241660600 e,nci.pdf.

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Jeppesen PB, Barriocanal L, Meyer MT, Palacios M, Canete F, Benitez S et al. (2006). Efficacy and tolerability of oral stevioside in patients with type 2 diabetes: a long-term, randomized, double-blinded, placebo- controlled study. Diabetologia 49(Suppl. 1):511512 [Abstract No. 0843]. Koyama E, Kitazawa K, Ohori Y, Izawa 0, Kakegawa K, Fujino A et al. (2003). In vitro metabolism of the glycosidic sweeteners, stevia mixture and enzymatically modified stevia in human intestinal microflora. Food Chem Toxicol 41(3):359-374. Maki KC, Curry LL, Reeves MS, Toth PD, McKenney JM, Farmer MV et al. (2008a). Chronic consumption of rebaudioside A, a steviol glycoside, in men and women with type 2 diabetes mellitus. Food Chem Toxicol 46(Suppl. 7):S47-S53. Maki KC, Curry LL, Carakostas MC, Tarka SM, ReevesMS, Farmer MV et al. (2008b). The hemodynamic effects of rebaudioside A in healthy adults with normal and low-normal blood pressure. Food Chem Toxicol 46(Suppl. 7):S40-S46. Mori N, Sakanoue M, Takeuchi M, Shimpo K, Tanabe T (1981). Effect of Stevioside on fertility in rats. Shokuhin Eiseigaku Zasshi 22(5):409-414 [Japanese]. Nikiforov AI, Eapen AK (2008). A 90-day oral (dietary) toxicity study of rebaudioside A in Sprague-Dawley rats. Int J Toxicol 27(1):65-80. Oviedo CA, Franciani G, Moreno R, Maas LC (1970). Hypoglycaemic action of Stevia Rebaudiana Bertoni (Ka6-he-6). In: Rodriguez RR, Ebling FJG, Henderson I, editors. Seventh Congress of the International Diabetes Federation, Aug. 23-28, 1970, Buenos Aires, Argentina. (International Congress Series, no 209). Amsterdam, Netherl.: Excerpta Medica Foundation / International Diabetes Foundation (IDF), pp. 92-93 [abstract 208]. Roberts A, Renwick AG (2008). Comparative toxicokinetics and metabolism of rebaudioside A, stevioside, and steviol in rats. Food Chem Toxicol 46(Suppl. 7):S31-S39. Temme EH, Vankeirsblick A, Buyse J (2004). A short term study of stevioside in healthy subjects. In: Geuns JMC, Buyse J, editors. Safety of Stevioside: Proceedings of the First Symposium Sponsored by KULeuven, April 16, 2004, Leuven, Belgium. Heverlee, Belgium: Euprint ed., pp. 63-74. Toskulkao C, Chaturat L, Temcharoen P, Glinsukon T (1997). Acute toxicity of stevioside, a natural sweetener, and its metabolite, steviol, in several animal species. Drug Chem Toxicol 20(1&2):31-44. Toyoda K, Matsui H, Shoda T, Uneyama C, Takada K, Takahashi M (1997). Assessment of the carcinogenicity of stevioside in F344 rats. Food Chem Toxicol 35(6):597-603. U.S. FDA (2008a). Agency Response Letter GRAS Notice No. GRN 000253 [Rebaudioside A purified from Stevia Rebaudiana (Bertoni) Bertond. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN); College Park, Maryland. Available at: http://www.fda.gov/Food/FoodIngredientsPackaqing/GenerallyRecognizedassafeGRAS/ (3,RASListinqs/ucm154989,htm.

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U.S. FDA (2008b). Agency Response Letter GRAS Notice No. GRN 000252 [Rebaudioside A purified from Stevia Rebaudiana (Bertoni) Bertond. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN); College Park, Maryland. Available at: http://www.fda.gov/Food/FoodingredientsPackaging/GenerallyRecocinizedasSafeGRAS/ GRASListings/ucm154988.htm. U.S. FDA (2009a). Agency Response Letter GRAS Notice No. GRN 000275 [Purified Steviol Glycosides With Rebaudioside A as the Principal Component]. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at: http://www.fda.gov/Food/FoodingredientsPackaging/GenerallyRecognizedasSafeGRAS/ GRASUstings/ucm171539.htm. U.S. FDA (2009b). Agency Response Letter GRAS Notice No. GRN 000278 [Rebaudioside A Purified From Stevia Rebaudiana (Berton° Berton°. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at: http://www.fdaclowfood/FoodIncredientsPackaging/GenerallyRecognizedasSafeGRAS/ GRASListings/ucm174946.htm. U.S. FDA (2009c). Agency Response Letter GRAS Notice No. GRN 000282 [Rebaudioside A Purified From the Leaves of Stevia Rebaudiana (Berton° Berton°. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at: http://www.fda.gov/Foold/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/ GRASListinqs/ucm185772.htm. U.S. FDA (2009d). Agency Response Letter GRAS Notice No. GRN 000287 [Purified Steviol Glycosides With Rebaudioside A and Stevioside As the Principal Components]. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at: http://wwwida.gov/Food/FoodIncredientsPackaging/GenerallyRecognizedasSafeGRAS/ GRASListings/ucm181937.htm. U.S. FDA (2010a). Agency Response Letter GRAS Notice No. GRN 000303 [Rebaudioside A purified from the leaves of Stevia rebaudiana (Berton° Berton°. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at: http://www.fdagov/Food/FoodInqredientsPackaqinq/GenerallyRecoqnizedasSafeGRAS/ GRASListings/ucm207985,htm. U.S. FDA (2010b). Agency Response Letter GRAS Notice No. GRN 000304 [Purified steviol glycosides with rebaudioside A and stevioside as the principal components]. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at: http://www.fdagoviFood/FoodingredientsPackaqing/GeneraHyRecoqnizedasSafeGRAS/ GRASListings/ucrn207983.htm.

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U.S. FDA (2010c). Agency Response Letter GRAS Notice No. GRN 000318 IRebaudioside A purified from the leaves of Stevia rebaudiana (Bertoni) Bertonil. College Park (MD): U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Food Additive Safety. Available at: http://www.fdagov/Food/FoodlncredientsPackaging/GenerallyRecognizedasSafeGRAS/ GRASListinqs/ucm213311.htm. Usami M, Sakemo K, Kawashirna K, Tsuda M, Ohno Y (1995). Teratogenicity study of stevioside in rats. Eisei Shikenjo Hokoku [Bull Natl Inst Hyg Sci] 113:31-35 [Japanese]. Wasuntarawat C, Terncharoen P, Toskulkao C, Mungkornkarn P, Suttajit M, Glinsukon T (1998). Developmental toxicity of steviol, a metabolite of stevioside, in the hamster. Drug Chem Toxicol 21(2):207-222. Wheeler A, Boileau AC, Winkler PC, Cornpton JC, Prakash I, Jiang X et al. (2008). Pharmacokinetics of rebaudioside A and stevioside after single oral doses in healthy men. Food Chern Toxicol 46(Suppl. 7):S54-S60.

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From: iida [mailto:[email protected]] Sent: Saturday, July 30, 2011 2:35 AM To: Carlson, Susan Cc: Erives, Gladys* Subject: GRAS Notice No.GRN 000375 Dear Dr. Susan Carlson (CC: Dear Dr. Gladys Erives) Thank you for your FAX. And I'm sorry ,I didn't reply to your e-mail for GRAS Notice No.GRN 00375. I have little chance to English e-mail. Therefore, I think that I couldn't check your two e-mail. Dr. Ashley Roberts from Cantox would be acting as the Nippon and Toyo agent regarding FDA scientific and regulatory matters. And that Dr. Roberts will follow-up with the FDA next week to respond to the questions outlined in the fax sent to them on July 28th. Best regards, Yoshihisa Iida Toyo Sugar Refining Co., Ltd.

From: [email protected] [mailto:[email protected]] Sent: Sunday, July 31, 2011 8:16 PM To: Carlson, Susan Cc: Erives, Gladys*; [email protected]; [email protected] Subject: RE: GRAS Notice No.GRN 000375 Dear Dr. Susan Carlson (CC: Dear Dr. Gladys Erives) Thank you for your FAX on July 28th. And sorry, for your inconvenient due to my slow responce regarding for your e-mail for GRAS Notice No.GRN 00375. But sorry, I did not find out your two e-mail before getting your urgent FAX on last week. We (Nipponn Paper Chemicals & Toyo Sugar & Cantox) will responde to your three questions through Dr. Ashley Roberts soon. Best Regards, Yasuyuki Nakamura

************************************* 1-2-2 TEL:080-1266-8494 FAX:03-3217-3280 NIPPON PAPER CHEMICALS CO.,LTD. SALES DIV. Yasuyuki Nakamura TEL:81-80-1266-8494 FAX:81-3-3217-3280 *****************************************

From: Ashley Roberts Intertek [mailto:[email protected]] Sent: Wednesday, August 03, 2011 2:29 PM To: Carlson, Susan Subject: RE: GRAS Notice No.GRN 000375 Dear Dr. Carlson, Re GRAS Notice number GRN 000375 In response to your fax dated July 28th 2011 to Nippon Paper Chemicals Co Ltd and Toyo Sugar Refining Co Ltd please find following our response to your list of clarifications. Firstly, the companies involved intend to market enzyme modified steviol glycosides as a general purpose sweetener in accordance with cGMP. For clarity and if required, the table outlining the potential food categories can be omitted from the notification. Based upon the fact that it is the intention to market as a general purpose sweetener and to remove the list of food categories thereby addresses the potential concern regarding the inclusion of the dietary supplement category. In addition, both Nippon Paper and Toyo Sugar confirm that enzyme modified steviol glycosides are not intended for use in meat or poultry products thereby removing the need for USDA review. I hope this brief response clarifies the issues outlined in your fax to both parties, if not please do not hesitate to contact me for further information Kind Regards ================================ Ashley Roberts, Ph.D. Senior Vice President Food & Nutrition Group Cantox Health Sciences International An Intertek Company 2233 Argentia Road, Suite 308 Mississauga, Ontario L5N 2X7 CANADA Tel: +1 905-542-2900 Fax: +1 905-542-1011 www.cantox.com www.intertek.com/green

From: Ashley Roberts Intertek [mailto:[email protected]] Sent: Friday, August 05, 2011 10:16 AM To: Carlson, Susan Cc: Erives, Gladys* Subject: RE: GRN 375, two technical questions Dear Dr. Carlson, In response to your e-mail below Please find following the answers to your technical questions regarding enzyme modified steviol glycosides (GRN 375). 1) The concentration of dextrin (maltodextrin) is approximately 30-45% of the reaction mixture. The excess dextrin is then removed via purification through ion-exchange chromatography. 2) The HPLC conditions for the chromatogram provided on page 13 of the notification are as follows, Column : Shodex RSpak DC-613(6mm×150mm) Column temp. : 40 A) CH3CNH2O = 82 18 v/v % B) CH3CNH2O = 50 50 v/v % Linear gradient : A) B) , 60min Flow rate : 1 ml/min Detector : UV210nm I hope our response satisfactorily addresses the remaining technical questions? Please do not hesitate to contact me further if I can be of further assistance Kind Regards ================================ Ashley Roberts, Ph.D. Senior Vice President Food & Nutrition Group Cantox Health Sciences International An Intertek Company 2233 Argentia Road, Suite 308 Mississauga, Ontario L5N 2X7 CANADA Tel: +1 905-542-2900 Fax: +1 905-542-1011 www.cantox.com www.intertek.com/green Mobile phase :

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GRAS Notice 000375: Enzyme modified steviol glycosides