GRAS Notice (GRN) No. 910
https://www.fda.gov/food/generally-recognized-safe-gras/gras-notice-inventory

Center for Regulatory Services, Inc.
5200 Wolf Run Shoals Road
Woodbridge, VA 22192-5755
703 590 7337 (Fax 703 580 8637)
smcdley@cfr-sclviccs.com.,._
consultants to the regulated industry

February 3, 2020

Dr. Susan Carlson
Director, Division of Biotechnology and GRAS Notice Review (HFS-255)
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition
Food and Drug Administration
5001 Campus Drive
College Park, MD 20740
Dear Dr. Carlson:
SUBJECT:

Transmittal of the NOMAD BIOSCIENCE GmbH GRAS Notice for THAUMATIN II
For use as Sweetener in Foods and Beverages

Enclosed you will find the GRAS notice for THAUMATIN II as produced by Nicotiana
benthamiana as a food and beverage sweetener as submitted by NOMAD BIOSCIENCE GmbH.
I have provided a CD of the GRAS notice and all the cited references.
Should you have any questions on this filing, please contact me, at your convenience.

Sincerely,/
Kristi 0. Smedley, Ph.D.
Consultant to NOMAD BIOf CIENCE GmbH
Attachments
FDA Form 3667 (Hard Copy and CD-Copy)
THAUMATIN II GRN NARRATIVE ofNotice (Hard Copy and CD-Copy)
Appendices (as appended to Narrative) (CD-copy)
Full Complement of References (CD-copy)-~: Yuri Gleba, Nomad

FEB O4 2020
OFFICE OF
FOOD ADDITIVE SAFETYForm Approved: OMB No. 0910-0342; Expiration Date: 02/29/2016(See last page for OMB Statement)

FDA USE ONLYDATE OF RECEIPTGRN NUMBER

000910
DEPARTMENT OF HEALTH AND HUMAN SERVICES
Food and Drug Administration

GENERALLY RECOGNIZED AS SAFE
(GRAS) NOTICEII Feb 4, 2020

ESTIMATED DAILY INTAKEINTENDED USE FOR INTERNETNAME FOR INTERNETKEYWORDS

Transmit completed form and attachments electronically via the Electronic Submission Gateway (see Instructions); OR Transmit
completed form and attachments in paper format or on physical media to: Office of Food Additive Safety (HFS-200), Center for
Food Safety and Applied Nutrition, Food and Drug Administration, 5100 Paint Branch Pkwy., College Park, MD 20740-3835.

PART I – INTRODUCTORY INFORMATION ABOUT THE SUBMISSION
1. Type of Submission (Check one)



New



Amendment to GRN No.

Supplement to GRN No.

2.

All electronic files included in this submission have been checked and found to be virus free. (Check box to verify)
Most recent presubmission meeting (if any) with
3a. For New Submissions Only:
FDA on the subject substance (yyyy/mm/dd):
3b. For Amendments or Supplements: Is youramendment or supplement submitted in
response to a communication from FDA?

(Check one)Yes If yes, enter the date ofcommunication (yyyy/mm/dd):NoPART II – INFORMATION ABOUT THE NOTIFIER
Name of Contact Person

Position

Yuri Gleba, Ph.D.

Chief Executive Officer

Company (if applicable)
1a. Notifier

Nomad Bioscience GmbH
Mailing Address (number and street)

Biozentrum Halle, Weinbergweg 22
City

State or Province

Zip Code/Postal CodeCountry

Halle/Saale

Saxony-Anhalt

D-06120

Germany

Telephone Number

Fax Number

E-Mail Address

49 345 1314 2606

49 345 1314 2601

gleba@nomadbioscience.com

1b. Agentor Attorney(if applicable)

Name of Contact Person

Position

Kristi O. Smedley, Ph.D.

Sponsor's US Regulatory Representative

Company (if applicable)

Center for Regulatory Services, Inc.
Mailing Address (number and street)

5200 Wolf Run Shoals Rd.
City

Woodbridge

State or Province

Zip Code/Postal CodeCountry

Virginia

22192

United States of America

Telephone Number

Fax Number

E-Mail Address703-590-7337

703-580-8637

smedley@cfr-services.com

FORM FDA 3667 (2/13)

Page 1 of 4

PART III – GENERAL ADMINISTRATIVE INFORMATION
1. Name of Substance

THAUMATIN II
3. For paper submissions only:

2. Submission Format: (Check appropriate box(es))
Electronic Submission GatewayElectronic files on physical media
with paper signature pagePaper
If applicable give number and type of physical media

Submission consists of one (1) CD containing electronic files of GRAS Notice

Number of volumes
Total number of pages

4. Does this submission incorporate any information in FDA’s files by reference? (Check one)
No (Proceed to Item 6)[8l Yes (Proceed to Item 5)

5. The submission incorporates by reference information from a previous submission to FDA as indicated below (Check all that apply)[8l a) GRAS Notice No. GRN



[8l

738

b) GRAS Affirmation Petition No. GRPc) Food Additive Petition No. FAPd) Food Master File No. FMFe) Other or Additional (describe or enter information as above) GRN 775

6. Statutory basis for determination of GRAS status (Check one)

[8l Scientific Procedures (21 CFR 170.30(b))

Experience based on common use in food (21 CFR 170.30(c))

7. Does the submission (including information that you are incorporating by reference) contain information that you view as trade secretor as confidential commercial or financial information?
Yes (Proceed to Item 8)[8l No (Proceed to Part IV)

8. Have you designated information in your submission that you view as trade secret or as confidential commercial or financial information(Check all that apply)
Yes, see attached Designation of Confidential Information
Yes, information is designated at the place where it occurs in the submission
No







9. Have you attached a redacted copy of some or all of the submission? (Check one)
Yes, a redacted copy of the complete submission
Yes, a redacted copy of part(s) of the submission
No

PART IV – INTENDED USE
1. Describe the intended use of the notified substance including the foods in which the substance will be used, the levels of use in such
foods, the purpose for which the substance will be used, and any special population that will consume the substance (e.g., when a substance would be an ingredient in infant formula, identify infants as a special population).

THAUMATIN II is a plant-made non-caloric sweetening product comprised of the protein thaumatin II, intended for application to foods
or beverages by itself or blended with other sweeteners to achieve the desired flavoring effect. THAUMATIN II is produced by a newplant-based process, but the component protein is (a) identical in amino acid sequence to commercially available thaumatin II (a.k.a.
thaumatin 2) extracted from the fruit of the Katemfe bush, or made recombinantly by fermentation; (b) a simple polypeptide with noglycosylation or other non-natural post-translational modifications; and (c) produced in high purity. Extracted and recombinantthaumatins are allowed for use in multiple foods and beverages at application rates up to 150 ppm (mg/kg or mg/L) in the USA (FEMA
GRASa 3732 and FEMA GRASa 3814), and up to 400 ppm in the EU (E 957) and in several other countries in Asia, Australasia, the Middle
East and South America. As a sweetener, thaumatins (E 957) are allowed without limitation (Quantum satis) because the proteins are
considered non-toxic. The same application rates are envisioned for THAUMATIN II as those for commercially available products.
THAUMATIN II is not intended for use in infant formulas.

2. Does the intended use of the notified substance include any use in meat, meat food product, poultry product, or egg product?
(Check one)

Yes

FORM FDA 3667 (2/13)

No

Page 2 of 4

PART V – IDENTITY
1. Information about the Identity of the SubstanceName of Substance 1

Thaumatin II

CAS no. and UniProt entry no.

Registry
Used(CAS, EC)

Registry No.2

53850 34 3 P02884

Biological Source(if applicable)

Substance Category(FOR FDA USE ONLY)

Plant, recombinant12

Note: Extracted "thaumatin" consisting of single
proteins or mixtures is CAS No. 53850-34-3.
Recombinant THM-B is CAS No. 553850-34-3.

3Include chemical name or common name. Put synonyms (whether chemical name, other scientific name, or common name) for each respective
item (1 - 3) in Item 3 of Part V (synonyms)
2
Registry used e.g., CAS (Chemical Abstracts Service) and EC (Refers to Enzyme Commission of the International Union of Biochemistry (IUB), now
carried out by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB))
1

2. Description
Provide additional information to identify the notified substance(s), which may include chemical formula(s), empirical formula(s), structural
formula(s), quantitative composition, characteristic properties (such as molecular weight(s)), and general composition of the substance. For
substances from biological sources, you should include scientific information sufficient to identify the source (e.g., genus, species, variety,strain, part of a plant source (such as roots or leaves), and organ or tissue of an animal source), and include any known toxicants that
could be in the source.

Detailed information regarding the identity, manufacturing process, functionality, uses and safety of the notified substance isincorporated in the Notice documents provided.

3. Synonyms
Provide as available or relevant:1

Thaumatin-2

2

3

FORM FDA 3667 (2/13)

Page 3 of 4

PART VI – OTHER ELEMENTS IN YOUR GRAS NOTICE(check list to help ensure your submission is complete – check all that apply)
Any additional information about identity not covered in Part V of this form
Method of Manufacture
Specifications for food-grade material
Information about dietary exposure
Information about any self-limiting levels of use (which may include a statement that the intended use of the notified substance is
not-self-limiting)Use in food before 1958 (which may include a statement that there is no information about use of the notified substance in foodprior to 1958)
Comprehensive discussion of the basis for the determination of GRAS status
Bibliography
Other Information
Did you include any other information that you want FDA to consider in evaluating your GRAS notice?




Yes

~ No

Did you include this other information in the list of attachments?
Yes



No

PART VII – SIGNATURE
1. The undersigned is informing FDA that

NOMAD BIOSCIENCE GMBH
(name of notifier)has concluded that the intended use(s) of THAUMATIN II(name of notified substance)

described on this form, as discussed in the attached notice, is (are) exempt from the premarket approval requirements of section 409 of theFederal Food, Drug, and Cosmetic Act because the intended use(s) is (are) generally recognized as safe.

2.

---------(name of notifier)(name of notifier)agrees to make the data and information that are the basis for thedetermination of GRAS status available to FDA if FDA asks to see them.
agrees to allow FDA to review and copy these data and information during
customary business hours at the following location if FDA asks to do so.

(address of notifier or other location)(name of notifier)agrees to send these data and information to FDA if FDA asks to do so.

OR
The complete record that supports the determination of GRAS status is available to FDA in the submitted notice and in GRP No.

(GRAS Affirmation Petition No.)

3. Signature of Responsible Official,
Agent, or Attorney

Kristi Smedley
FORM FDA 3667 (2/13)

Digitally signed by Kristi Smedley
DN: cn=Kristi Smedley, o=Center for Regulatory Services, Inc., ou,
email=smedley@cfr-services.com, c=US
Date: 2020.02.03 08:08:52 -05'00'

Printed Name and Title

Date (mm/dd/yyyy)

Kristi O. Smedley, Ph.D.

02/03/2020

Page 4 of 4

PART VIII – LIST OF ATTACHMENTS
List your attached files or documents containing your submission, forms, amendments or supplements, and other pertinent information.
Clearly identify the attachment with appropriate descriptive file names (or titles for paper documents), preferably as suggested in theguidance associated with this form. Number your attachments consecutively. When submitting paper documents, enter the inclusive page
numbers of each portion of the document below.
Attachment
NumberFolder Location (select from menu)

Attachment Name

(Page Number(s) for paper Copy Only)

GRN for THAUMATIN II as a non-caloric sweetener for use infoods and beverages(PDF of Notification)

Submission

THAUMATIN II GRN References - For FDA Internal Review Only
(PDFs of All Cited References - Not for Republication)

Submission

OMB Statement: Public reporting burden for this collection of information is estimated to average 150 hours per response, including
the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing andreviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information,
including suggestions for reducing this burden to: Department of Health and Human Services,Food and Drug Administration, Office of Chief
Information Officer, 1350 Piccard Drive, Room 400, Rockville, MD 20850. (Please do NOT return the form to this address.). An agency may
not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB
control number.
FORM FDA 3667 (2/13)

Page 5 of 4

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANANOMAD BIOSCIENCE GmbH
Biozentrum Halle
Weinbergweg 22
D-06120 Halle/Saale
Germany
Tel. 49 345 1314 2606
Fax. 49 345 1314 2601
3 February 2020
Antonia Mattia, Ph.D.
Director, Division of Biotechnology and GRAS Notice Review (HFS-255)
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition
Food and Drug Administration
5100 Paint Branch Parkway
College Park, MD 20740
Re: GRAS Notice for THAUMATIN II Sweetener from Nicotiana benthamiana
Dear Dr. Mattia,
Nomad Bioscience GmbH ("Nomad"; “Notifier”) is submitting this GRAS Notice for its THAUMATIN II
sweetener from Nicotiana benthamiana. Nomad has concluded, under FDA’s Final Rule pertaining to 21 CFR
170 (August 17, 2016), that the naturally occurring protein Thaumatin II when produced recombinantly in theplant species Nicotiana benthamiana is Generally Recognized as Safe (GRAS) for use as a food and beverage
sweetener. The product could be used singly and in blends with sugar or other sweetening agents.
This Notice follows Notifier’s GRN 738 for its THAUMATIN product consisting of thaumatin I and/or thaumatin
II proteins produced in edible plant species (e.g., spinach, leafy beet and lettuce), for which Nomad received
from CFSAN a "No Questions" letter on 18 April 2018. GRN 738 established THAUMATIN as a GRAS sweetenerin the USA, whereas before then the GRAS status pertained to thaumatins used only as flavor modifiers, as
specifically described in FEMA GRASa No. 3732 (Oser 1984) for natural (extracted) thaumatins, and in FEMA
GRASa 3814 (Smith 1996) for recombinant versions of these proteins.
Differences between this Notice and our prior GRN 738 include: (1) modification of the manufacturing process
to expand the list of production host plants to include Nicotiana benthamiana; (2) selection of Thaumatin II
as the preferred sweetening active ingredient; and (3) adjustment of product application rates and dietaryintake estimates due to projected replacement of up to 50% (w/w) of sugar in food and beverages withThaumatin II. Data and justifications for these changes are included in this submission.
In this Notice we describe production of Thaumatin II in the non-food plant species Nicotiana benthamiana(N. benthamiana). This host enables higher expression of recombinant proteins including thaumatins, is moreamenable to controlled indoor cultivation, and yields better process economics. This host has been used to
produce human biopharmaceuticals that have been shown safe in multiple clinical studies under FDA INDs.
The same species has also been accepted by FDA for producing GRAS food antimicrobials such as Nomad’s
COLICIN (GRN 775; 26 October 2018), ENDOLYSIN (GRN 802; 10 May 2019) and SALMOCIN (21 October 2019).
We describe here a process using the same host to produce Thaumatin II.
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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
Regardless of production host plant, the amino acid sequence of Nomad's Thaumatin II is identical to thesequence of the same proteins extracted and purified from the fruit of the Katemfe tree (Thaumatococcus
daniellii); the original source of thaumatins and the main source of commercial thaumatin-containing
products. In the USA, EU, Japan and elsewhere, isolates of the Katemfe fruit have been marketedcommercially since the mid-1970s under various trade names (e.g. Talin; San Sweet T-100), as low-calorie
sweeteners and flavor modifiers. Results of extensive preclinical safety studies and human clinical exposure
allowed adoption by the EU of a working maximum consumption level of 1.1 mg thaumatin/kg body mass per
day as a highly safe level for all subpopulations of consumers (EFSA 2015). Based on that body of work, andthe physicochemical equivalency of Thaumatococcus-derived and Notifier’s recombinantly produced
thaumatins, Nomad claimed the same maximum safe level of 1.1 mg/kg-day in GRN 738.
Since submission of GRN 738, Nomad has conducted blinded sensory evaluation studies in-house and at two
independent sensory evaluation organizations. These studies confirmed that the threshold of sweetness
detection of Nomad’s Thaumatin II is equivalent to that of native proteins, and further, that Nomad’s
Thaumatin II can replace up to 50 wt% of the sucrose in test solutions while retaining the same sensation ofsweetness. Therefore, the projected application rates and estimates for average moderate and average
maximum levels of intake provided in this Notice take this new information into account.
In the current Notice, Nomad documents the identity, manufacturing process, product quality, safety, dietary
exposure and potential risks from consumption of its THAUMATIN II product containing Thaumatin II as thehigh-intensity sweetener, when produced in the plant host N. benthamiana. While describing the
manufacturing process and organoleptic properties, this Notice frequently references GRN 738 and GRN 775
for brevity and to rely on FDA’s original review memoranda for our prior GRAS notices.
Our submission complies with the 7-part format prescribed by FDA in its Final Rule for the GRAS Noticeprocess (August 17, 2016), and includes a CD containing PDFs of the following documents:
1. FDA Form 3667 Nomad Bioscience GRN for THAUMATIN II sweetener from Nicotiana benthamiana
2. GRN for THAUMATIN II sweetener from Nicotiana benthamiana (Parts 1-7), which includes:
APPENDIX A: Nomad's Safety Data Sheet for THAUMATIN II from Nicotiana benthamiana
APPENDIX B: THAUMATIN II Nicotiana benthamiana Manufacturing Process
APPENDIX C: THAUMATIN II Characterization
3. Copies of references cited in the GRN
If the Agency has any questions or requires additional information to aid their review of Nomad’s findings
and GRAS conclusion, please contact us at the address listed above. For convenience, you may also contact
our regulatory and product development representatives in the USA, Dr. Kristi Smedley at Center for
Regulatory Services Inc., Woodbridge, VA (Tel 703-590-7337; Email smedley@cfr-services.com), and Dr.
Daniel Tusé at DT/Consulting Group, Sacramento, CA (Tel 707-290-9528; Email daniel@dt-cg.com).
Sincerely,

Yuri Gleba, Ph.D.
Chief Executive Officer
Nomad Bioscience GmbH

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA

Table of Contents
Table of Contents ................................................................................................................................................ 3
Table of Figures ................................................................................................................................................... 5
Table of Tables .................................................................................................................................................... 6
1. General Introduction and Claim of Exemption from Premarket Approval Requirements ..........................7
1.1. Submission of Notice .............................................................................................................................. 7
1.2. Name and Address of Notifier ................................................................................................................ 7
1.3. Common or Usual Name of the Notified Substance ............................................................................... 8
1.4. Conditions of Use .................................................................................................................................... 8
1.5. Statutory Basis for Notifier’s GRAS Conclusion ...................................................................................... 8
1.6. Not Subject to Preclearance ................................................................................................................... 8
1.7. Availability of Information for FDA Review ............................................................................................ 9
1.8. Public Disclosure ..................................................................................................................................... 9
1.9. Certification ............................................................................................................................................ 9
2. Identity, Method of Manufacture, Specification, Technical Effect ............................................................ 10
2.1. Identity, Structural and Functional Information ................................................................................... 10
2.2. Method of Manufacture ....................................................................................................................... 122.3. Composition and Specification ............................................................................................................. 13
2.4. Technical Effect and Suitability of Use .................................................................................................. 15
2.5. Overall Conclusion ................................................................................................................................ 15
3. Dietary Exposure ........................................................................................................................................ 15
3.1. Application rates of THAUMATIN II in various food categories ............................................................ 15
3.2. Estimated dietary intake of THAUMATIN II in foods and beverages .................................................... 18
3.3. Additional exposure to thaumatins (intake not related to THAUMATIN product) ..............................19
3.4. Dietary Exposure to Host- and Process-Derived Impurities ................................................................. 19
4. Information on Any Self-Limiting Levels of Use ......................................................................................... 21
5. Experience Based on Common Use in Food Before 1958 .......................................................................... 21
6. Basis for Conclusion of THAUMATIN II’s GRAS Status ................................................................................ 21
6.1. THAUMATIN II Overall Safety ............................................................................................................... 22
6.1.1

Allergenic potential of THAUMATIN II ........................................................................................ 23

6.1.2

Host impurities ............................................................................................................................ 24

6.1.3

Process modification to lower host alkaloid content in THAUMATIN II product .......................31

6.1.4

Process impurities in N. benthamiana-based THAUMATIN II manufacture ...............................33

6.2. Safety in Relation to Dietary Intake of THAUMATIN II ......................................................................... 35
6.3. Occupational safety .............................................................................................................................. 39
6.4. GRAS conclusion for N. benthamiana-produced THAUMATIN II sweetener ........................................39
7. Supporting Data and Information .............................................................................................................. 41
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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
References ........................................................................................................................................................ 44
APPENDIX A.

THAUMATIN II Safety Data Sheet ........................................................................................... 55

APPENDIX B.

THAUMATIN II Manufacturing Process ................................................................................... 61

B.1. Introduction and Rationale ................................................................................................................... 61
B.2. Organism Used and Gene Expression Cassette .................................................................................... 61
B.3. Procedure.............................................................................................................................................. 62
B.4. Manufacturing Facilities ....................................................................................................................... 66
B.5. Waste Handling and Disposal ............................................................................................................... 66
B.6. Justification of Specification ................................................................................................................. 66
APPENDIX C.

THAUMATIN II Characterization ............................................................................................. 68

C.1. Purification of Thaumatin II from Plant Biomass .................................................................................. 68
C.2. Confirmation of Identity ....................................................................................................................... 69
C.3. Stability of Purified Thaumatin II .......................................................................................................... 70
C.4. Manufacturing Yield and Consistency .................................................................................................. 71
C.5. Analysis of Residual Alkaloid Content ................................................................................................... 72
C.6. Analysis of Elemental Impurities........................................................................................................... 72
C.7. Determination of Sweetness Intensity ................................................................................................. 74
C.8. Stability of Thaumatin II in the Presence of Sugar ................................................................................ 81
C.9. Bioburden and Residual Vector ............................................................................................................ 82
C.10. Summary .............................................................................................................................................. 82

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA

Table of Figures
Figure 2-1 Structural model of thaumatin II .................................................................................................... 10
Figure 2-2. Amino acid sequence of mature Thaumatin II .............................................................................. 11
Figure 3-1. Thaumatin II sweetness and aftertaste ......................................................................................... 16
Figure 6-1. Biosynthetic pathway of major alkaloids in Nicotiana species ......................................................28
Figure 6-2. Bioavailability of nicotine in humans as a function of route of administration ............................30
Figure B-1. Agrobacterium expression vector for thaumatin proteins ............................................................61
Figure B-2. Schematic of vector for thaumatin expression in plants ...............................................................62
Figure B-3. Diagram of modified process for producing THAUMATIN II in Nicotiana benthamiana ..............63
Figure C-1. One-step purification of cold-extracted Thaumatin II by Capto S CEX chromatography ..............68
Figure C-2. Molecular stability of purified Thaumatin II as determined by ISD ...............................................69
Figure C-3. Determination of Thaumatin II mass by MALDI-TOF .....................................................................69
Figure C-4. Stability of purified Thaumatin II ................................................................................................... 70
Figure C-5. Thaumatin II sweetness intensity and duration of aftertaste .......................................................75
Figure C-6. Sweetness intensity of sucrose and sucrose + Thaumatin II solutions..........................................76
Figure C-7. Sensory profile of sucrose and sucrose + Thaumatin II solutions .................................................77
Figure C-8. Sweetness intensity of Thaumatin II and sugar-replacing potential .............................................80
Figure C-9. Stability of Thaumatin II in the presence of sugar ......................................................................... 81

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA

Table of Tables
Table 2-1. Relative sweetness of natural and artificial sweeteners compared to sucrose ..............................10
Table 2-2. Specification of THAUMATIN II Product.......................................................................................... 14
Table 3-1. Estimated application rates of THAUMATIN II (Thaumatin II) sweetener ......................................17
Table 3-2. Estimated exposure to host alkaloids from Thaumatin II produced in N. benthamiana................20
Table 6-1. Bioinformatic amino acid scan for allergenic sequences in Thaumatin II ......................................24
Table 6-2. Estimated pharmacological thresholds of nicotine ........................................................................ 29
Table 6-3. Levels of nicotine and anabasine in N. benthamiana .....................................................................31
Table 6-4. Residual levels of N. benthamiana alkaloids in purified Thaumatin II ............................................32
Table 6-5. Average nicotine content in common vegetables .......................................................................... 32
Table 6-6. Consumption, nicotine content and dietary nicotine exposure from common vegetables ...........33
Table 6-7. Estimated exposure to elemental impurities in Thaumatin II ........................................................34
Table 6-8. EFSA Currently authorized and extended use levels (2015) for thaumatin (E 957) .......................36
Table 6-9. FEMA Updated thaumatin use levels (2018) .................................................................................. 37
Table 7-1. Information supporting THAUMATIN II sweetener GRAS conclusion ............................................41
Table B-1. Justification of Specification Release Limits for THAUMATIN II Product ........................................67
Table C-1. Stability of Thaumatin II during storage ......................................................................................... 71
Table C-2. Consistency of production yield and purity of Thaumatin II ..........................................................71
Table C-3. Residual alkaloid impurities in N. benthamiana-produced Thaumatin II .......................................72
Table C-4. Multi-batch analysis of elemental impurities in N. benthamiana-produced Thaumatin II ............73
Table C-5. Thaumatin II and sucrose sweetness intensity scores .....................................................................75
Table C-6. Thaumatin II sensory detection threshold ....................................................................................... 78
Table C-7. Thaumatin II sweetness detection threshold .................................................................................. 79
Table C-8. Multi-batch analyses of bioburden and residual Agrobacterium inoculum ....................................82

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA

1. General Introduction and Claim of Exemption from Premarket Approval
Requirements
Nomad Bioscience GmbH (“Nomad”; Notifier) THAUMATIN II product containing Thaumatin II protein
sweetener is produced using a plant-based manufacturing process to match the amino acid sequence of
naturally occurring thaumatin II protein. Thaumatins (both extracted and recombinant) have been extensively
studied scientifically for many decades pre and post commercialization, and a large public database of
information exists regarding manufacturing methods, functionality, applications and safety. This extensive
public record enables comparison of Nomad's new product to the currently available thaumatin-containing
products. Results of a literature search and analysis of publicly available information on which Nomad based
its conclusions of the safety of its THAUMATIN product were included in GRN 738 and are updated in thisNotice.
Native thaumatins found in the arils of the fruit of Thaumatococcus danielli have been used for centuries inWest Africa as sweeteners and flavor modifiers. There is no ethnobotanical record of ill effects associatedwith consumption of impure thaumatins. Products containing industrially produced thaumatins have enjoyedan excellent safety record since their first use in human foods in the mid-1970s with expanded approvals in
the 1990s. Nomad's plant-made thaumatins, including those produced in edible species as described in
GRN 738 and those produced in N. benthamiana as described in the present submission, are identical inamino acid composition, sequence and structure to the native thaumatins.
This Notice provides exposure estimates from consumption of foods that would be treated with Nomad's
N. benthamiana-produced THAUMATIN II sweetener and includes a corresponding risk assessment. Under
any use scenario, we project that THAUMATIN II can be used safely at the levels needed for functionality.
Notifier therefore concludes that under the conditions of use described herein, N. benthamiana-produced
THAUMATIN II is generally recognized as safe and therefore should be exempt from premarket approval
procedures under 21 CFR 170.36(a)(I). THAUMATIN II is not intended for use in infant formulas.

1.1.

Submission of NoticeThis Notice is submitted in compliance with Subpart E of FDA’s Final Rule of the GRAS Notification process(August 17, 2016) 21 CFR 170.203-170.285.

1.2.

Name and Address of Notifier
NOMAD BIOSCIENCE GmbH
Biozentrum Halle
Weinbergweg 22
D-06120 Halle/Saale, Germany
Office: 49 345 1314 2606
Fax:
49 345 1314 2601
Notifier’s US Representative
Kristi O. Smedley, Ph.D.
Center for Regulatory Services, Inc.
5200 Wolf Run Shoals Rd.
Woodbridge, VA 22192
Office: 703-590-7337; Mobile: 703-786-7674
Fax: 703-580-8637
eMail: smedley@cfr-services.com

Notifier's US Representative
Daniel Tusé, Ph.D.
DT/Consulting Group2695 13th Street
Sacramento, CA 95818
Telephone: 707-290-9528
Fax: 916-822-4124
Email: daniel@dt-cg.com
Page | 7

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA

1.3.

Common or Usual Name of the Notified Substance
THAUMATIN II

1.4.

Conditions of Use

What is claimed as GRAS in this Notice is:
A THAUMATIN II composition consisting of recombinant, Nicotiana benthamiana-produced
Thaumatin II protein meeting the target Specification, for application to food and beverages as asweetener singly and/or in combination with other caloric and/or non/low caloric sweeteners.
Native “thaumatin” is comprised of several related proteins, but the major sweetening properties reside in
two very similar proteins, thaumatin I and thaumatin II. Thaumatins are naturally occurring proteins that canmodify taste perception contextually, depending on their concentration and the properties of the food matrix
into/onto which the thaumatins are applied. Nomad's product can be comprised of thaumatin I and/or
thaumatin II, as described in GRN 738.
However, new manufacturing process and sensory information generated by Nomad since submission of GRN
738 suggests that thaumatin II can satisfy production and functionality requirements as a single active
ingredient; therefore, this Notice focuses on a low-calorie, high-intensity sweetener comprised only ofthaumatin II. Hence, Notifier's product "THAUMATIN II" is formulated to contain recombinant, N.
benthamiana plant-produced, purified Thaumatin II (capitalized in this Notice to differentiate it from thesame protein obtained from other sources). Thaumatin II can be applied to foods up to the safety limits
allowed for thaumatins from native sources. The sources of information used to document and support
Thaumatin II’s properties, functionality, safety attributes and our GRAS conclusion are listed in Table 7-1.
This Notice pertains to Thaumatin II’s use as a sweetener. In independent sensory evaluations, the thresholdof detected sweetness of Notifier’s Thaumatin II was in the same range as published values for highly purethaumatins from other sources. Further, Thaumatin II was able to replace up to 50 wt% of the sugar in test
solutions consisting of different Thaumatin II/sucrose ratios. Therefore, we anticipate consumption ofThaumatin II at levels considerably lower, but in any event not higher, than those specified for the currently
available thaumatins (i.e., safe consumption up to 1.1 mg/kg-day body weight basis for all age groups, EFSA
E957 October 28, 2015).
Additional intake and safety information for subpopulations potentially consuming THAUMATIN II, includingchildren, adults and the elderly, is provided in Section 3 (Dietary Exposure) of this Notice.

1.5.

Statutory Basis for Notifier’s GRAS Conclusion

The statutory basis of the GRAS status in through scientific procedures in accordance with 21 CFR 170.30(b):GRAS Conclusion.
In accordance with the information provided in this Notice, it is Nomad Bioscience’s conclusion that Nicotiana
benthamiana-produced THAUMATIN II is generally recognized as safe when used as a sweetener on foods
and beverages at application rates that would collectively amount to not more than 1.1 mg/kg-day (bodyweight basis) additive exposure in a typical human diet.

1.6.

Not Subject to Preclearance

Notifier has concluded that THAUMATIN II as manufactured via its plant-based process using the hostN. benthamiana is generally recognized as safe, and as such the substance is not subject to pre-market
approval requirements of the Federal Food Drug and Cosmetic Act.
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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA

1.7.

Availability of Information for FDA Review

All data and information that serve as a basis for the GRAS conclusions are included in this Notice.

1.8.

Public Disclosure

The information provided in this Notice is publicly available and not subject to exception under 170.225(c)(8).
All information contained in this Notice can be shared without restriction.

1.9.

Certification

On behalf of Nomad Bioscience GmbH (Notifier), I certify that to the best of my knowledge, this GRAS Notice
is complete, representative, and balanced with respect to the information provided, favorable orunfavorable, known to me and pertinent to the evaluation of the safety and GRAS status of our Nicotiana
benthamiana-produced THAUMATIN II sweetener product.

Yuri Gleba, Ph.D.
Chief Executive Officer
NOMAD BIOSCIENCE GmbH
Biozentrum Halle
Weinbergweg 22
D-06120 Halle/Saale
Germany

Page | 9

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA

2. Identity, Method of Manufacture, Specification, Technical Effect
2.1.

Identity, Structural and Functional Information

Identity
In GRN 738, Notifier claimed substitutable use of thaumatin I or thaumatin II and/or their mixtures to achieve
the desired sweetening effect. Recent studies summarized in this submission show that Thaumatin II can beproduced more efficiently than Thaumatin I and also to possess a superior flavor profile relative toThaumatin I, including fewer off-flavor notes. While development studies characterized both proteins,
Notifier’s commercial product will likely contain Thaumatin II as the preferred high-intensity sweetener.
The history of thaumatin development and commercialization was included in GRN 738 (Section 2.1, pg 9;
Section 6.1, pp 15-16) and is only summarized here for brevity. The product Talin® was commercialized
beginning in the 1970s by Tate & Lyle (UK) with a claimed sweetness of 1,600-2,700 times that of a 7-10%
solution of sucrose (van der Wel 1972). A similar, mixture was sold in Japan under the brand San Sweet
T-100®. Thaumatins are currently available from several vendors, including Naturex, Beneo Palatinit, Natex,
KF Specialty Ingredients and several others. The chemistry and applications of thaumatins have been
extensively described in several original and review articles (e.g. Lee 1987; Kim 1988; Hough 1993;
Shallenberger 1993; Witty 1998; Zemanek 1995; Suami 1997; Greenly 2003). Table 2-1 lists the sweetness
ranking of thaumatins relative to sucrose (from Greenly 2003).
Thaumatins I and II have similar properties, amino acid composition, sweetness, molecular weight (both are~22 kDa) and highly similar amino acid sequences, differing by only 5 amino acid residues. Each protein is asingle polypeptide chain of 207 amino acids with 8 intramolecular disulphide linkages (Suami 1997). X-ray
crystallography of thaumatin I revealed the features of the protein's backbone (de Vos 1985; Kim 1988; Ogata
1992). Circular dichroism studies (van der Wel 1984) showed few α-helices, but many β-pleated sheet strands
and bends. It is thought that the constrained structure of thaumatins is responsible for inducing taste
sensation. Either heat denaturation or cleavage of the disulphide bridges results in loss of sweetness,suggesting that the tertiary structure of the protein triggers taste modulation through a highly stereoselective
process (Kaneko 2001; Hough 1993). A molecular diagram of thaumatin II determined from crystallography
at 0.99-Å resolution is shown in Figure 2-1 (from Masuda 2014).
Table 2-1. Relative sweetness of natural andartificial sweeteners compared to sucrose
N ER

N EOTAME
T l t /\ U M/\TI N
ALITAM Cl) H CS U CR.!.. LOS E
SACC HARINS T E V IA
ACE:SUL F AME:- K
A S PARTAME
G I 'l' CYh'WI-I I ./ IN

CYC L AMAT E:

13,0003 , 000
2,000
2,000

600
300
300
200
180
100

30

HFCS

1. 5

1-I O N E Y

1. 5
1.0
1.0
1.0

SUCROSE.:T AGAT 05E

X YL ITO LE RYT HR I TO L

0 .D
0 .9
0. 7

I S.OMA.LT

0 . 65HSH
M A L T I TO L

Figure 2-1 Structural model of thaumatin II

5\','EETN E55/5 U CR05 E

MA

N ITO L

0 .5

S0110 1T O L

0.5
0 .6

l., Ac;: r 11· n L

0. 4

T R E l l/,, LOSE:

Page | 10

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA

Structural Information for Plant-Made Thaumatin II
The amino acid sequences of thaumatins I and II, including Notifier's plant-expressed thaumatins, were
included in GRN 738 (Section 2.1, pg 10). The amino acid sequence of Thaumatin II is shown in Figure 2-2(Faus 2008). Notifier's plant-expressed, mature Thaumatin II has an identical amino acid sequence to thecorresponding protein from Thaumatococcus (APPENDIX C, Section C.2).
The sequence shown is for the mature protein; thaumatins including Thaumatin II are natively expressed as
pre-proproteins containing N-terminal signal and C-terminal peptide additions. These precursors areprocessed in planta to yield the intermediate proprotein devoid of the N-terminal peptide but still containing
the C-terminal addition. Subsequent processing in planta yields the nature-identical sequence of amino acidsin the mature, isolated thaumatin. Thaumatin II does not contain glycosylation sites on its 207-aa backbone;hence, like the protein derived from Thaumatococcus, Notifier's Thaumatin II is a non-glycosylated
polypeptide.
60
SGRGICRTGDCGGLLQCKRFGRPPTTLAEFSLNQYGKDYIDISNIKGFNVPMDFSPTTRG120

CRGVRCAADIVGQCPAKLKAPGGGCNDACTVFQTSEYCCTTGKCGPTEYSRFFKRLCPDA180

FSYVLDKPTTVTCPGSSNYRVTFCPTA

207

Figure 2-2. Amino acid sequence of mature Thaumatin II
A Safety Data Sheet (SDS) for THAUMATIN II (Thaumatin II produced in N. benthamiana) is found in
APPENDIX A. The method applied to manufacture Notifier's Thaumatin II protein in the plant hostN. benthamiana is described in APPENDIX B. Analytical methods used to define the physicochemical andfunctional properties of Notifier's recombinant Thaumatin II are summarized in APPENDIX C. Notifier's
Nicotiana-expressed plant-made Thaumatin II conforms to its predicted composition and shares the aminoacid sequence of the native Thaumatococcus protein.

Quantitative Composition
THAUMATIN II is prepared in bulk as a dry powder as described in APPENDIX B. The Specification for the bulk
product is found in Table 2-2. Typically, THAUMATIN II is supplied as a solid of >95% purity. Thaumatin II ishighly water soluble (>20% w/v). The bulk product is dissolved/diluted in water or other suitable foodcompatible vehicle or directly mixed into foods or beverages to achieve the desired effect.
THAUMATIN can be used singly or in combination with other sweeteners and food additives to enhance thesweet flavor profile of foods and beverages. The amount of THAUMATIN II used is food- and applicationspecific and depends on the end result desired in each flavor formulation.

Mode of Action
The mode of action of thaumatins was reviewed in GRN 738 (Section 2.1, pg 11) and results of more recent
studies are included in this submission. Thaumatins and other sweet-tasting proteins interact with taste
receptors in the tongue to impart the neurophysiological sensation of sweetness. Sweet-tasting proteins aswell as aspartame can be perceived by humans, apes, and Old World monkeys but not New World monkeys
and rodents (Masuda 2013). Natural sweetness perception occurs when a sugar such as sucrose or othersweetener dissolves in saliva and binds to the heterodimeric T1R2–T1R3 receptors, which belong to the
G-protein-coupled receptors (GPCRs) family (Li 2002; Margolskee 2001; Nelson 2001). These receptors have
Page | 11

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
multiple binding sites (Vigues 2008) that are activated upon interaction with compounds that elicit sweet
taste. Each sweetener exhibits different binding properties on the same receptors (Fernstrom 2012), leading
to the varying perceptions of sweetness for the different proteins.
In addition to their history of use by African, Western and Eastern cultures, thaumatins have been studied
systematically for their flavor modifying properties in vitro and ex vivo as well as in animal studies in vivo. As
defined in whole animal studies using various species and in isolated gustatory cells in vitro, thaumatins havea demonstrable effect on various groups of taste receptors and these interactions affect perception of taste(Bartoszewski 2003; Faus 2008; Glaser 2000; Greenly 2003; Hellekant 1996; Kaneko 2001; Kinghorn 1986; Lim
2012; Nagarajan 2017; Ohta 2011; Sardesai 1991; Schiffman 1995; Suami 1997; Tinti 2000; van der Wel 1972;
Witty 1990). Thaumatins find multiple uses in food technology because these proteins can impart a sweet
flavor, enhance desirable flavor notes, or mask or ameliorate unpleasant flavors in food components.
At the molecular level, the electrical charge distribution on the thaumatin molecules appears to mediate theirinteraction with the taste receptors. In addition to sweetness, thaumatins' effect in the presence of more
complex flavor compounds suggests that flavor modification depends on the nature of the individual
molecules present (Natex 2017). The strength of the interaction between the thaumatin molecules and thetaste receptors may account for thaumatins' intensity and the duration of sweetness perception.
Natural thaumatin was originally found to consist of six closely related proteins (I, II, III, a, b and c), all with a
MW of ~22 kDa (207 amino acids) (van der Wel 1972; Ledeboer 1984). Thaumatin I and II are robust becauseof their multiple disulfide bridges, but can lose their sweetness on heating at alkaline pH; the proteins arestable at neutral to acidic pH even at elevated temperatures (Lim 2012). The 3-D structure of thaumatin I (deVos 1985; Ogata 1992) revealed that the protein consists of three domains: (a) an 11 strand, flattened βsandwich (aa 1–53, 85–127 and 178–207, domain I); (b) a small disulfide-rich region (54–84, domain III); and(c) a large disulfide-rich region (128–177, domain II). Subsequently, the crystal structure of plant-extracted
thaumatin II was determined at 1.27-Å resolution (Masuda 2011) and the structure of a recombinant versionof the same molecule was determined at 0.99-Å resolution (Masuda 2014). These studies revealed very
similar structures for thaumatin I and II and enabled subsequent in-depth structure-activity studies.
Modification of lysine residues in the structure of thaumatins was found to reduce their sweetness (Kaneko
2001). Phosphopyridoxylation of lysine residues Lys78, Lys97, Lys106, Lys137 and Lys187 markedly reduced
sweetness. The intensity of sweetness was returned to that of native thaumatins by dephosphorylation ofthese phosphopyridoxylated lysine residues except Lys106. These lysine residues occurred in thaumatins, but
not in non-sweet thaumatin-like proteins, suggesting that these lysine residues were required for sweetness.
The sweetness threshold of natural (plant extracted) thaumatins was reported by Masuda et. al. (2016; 2018)as ~50 nM and sweeter versions have been generated by amino acid substitutions. Masuda (2016) found thata D21N mutation enhanced sweetness and reduced the sensory detection threshold from 50 nM to 31 nM.
Nomad determined a sweetness threshold of 37 nM for its native-sequence, purified Thaumatin II.

2.2.

Method of Manufacture

The THAUMATIN plant-based manufacturing process was described in APPENDIX B of GRN 738 and included
expression and extraction of thaumatin I and/or II proteins in food species of plants. Plant hosts included
Spinacia oleracea (spinach), Beta vulgaris (red beet) or Lactuca sativa (lettuce). These same hosts were foundacceptable for the manufacture of antimicrobial proteins as food safety processing aids, as described in
Notifier's GRN 593 and GRN 676. The safety features of these host species were detailed in GRN 593.

Page | 12

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
In the present submission, Notifier asserts that other plant hosts can be used to produce thaumatin proteins
without impacting the safety or functionality of the final product. Such species include members of the genus
Nicotiana, specifically the species N. benthamiana. Reduction in the levels of N. benthamiana-associatedimpurities such as pyridine alkaloids that are not found in edible species hosts is achieved through the processmodification described in APPENDIX B of this Notice. The modification enables inclusion of N. benthamiana
to the list of candidate plant species for producing thaumatin proteins.
Nomad includes N. benthamiana as an additional host for its manufacturing process because the species isamenable to large-scale indoor cultivation and yields better process economics relative to its food-species
counterparts. Nomad has described use of N. benthamiana in the manufacture of its food safety processing
aids and has used scientific procedures to conclude GRAS status for the products COLICIN (GRN 775),
ENDOLYSIN (GRN 802) and SALMOCIN (GRN 824). In the present Notice, Nomad asserts its GRAS conclusion
for N. benthamiana-produced Thaumatin II.
Regardless of host used, the plant-derived biomass remaining after thaumatin protein extraction is treated
and discarded (disposed) and is not used as a human food or animal feed product, additive or supplement.

2.3.

Composition and Specification

Characteristic Properties
The characteristic properties of plant-made THAUMATIN II are summarized in the Specification. In recent
studies Notifier has determined that the manufacture of Thaumatin II is more efficient than the manufactureof Thaumatin I. Additionally, Notifier has determined in sensory studies conducted in house and at
independent sensory evaluation organizations, that Thaumatin II has superior flavor characteristics overThaumatin I, including comparable sweetness with fewer/shorter-lasting off-flavor effects. Hence, a current
target Specification for a product consisting of Thaumatin II as the preferred active ingredient is provided inTable 2-2.
As reported in GRN 738 (Section 6.2, pp 17 -21), Notifier studied at the molecular level the allergenic potentialof plant-expressed thaumatins for use in food and determined, from published information, that Notifier's
thaumatins should pose no higher allergenic risk than the Thaumatococcus-derived commercial thaumatinscurrently on the market (see current Notice’s Section 6.1.1 Allergenic potential of THAUMATIN II).

Formulation
THAUMATIN II is provided in bulk as a dry powder, which the end-user can dissolve and/or dilute in water orother food-acceptable vehicle according to instructions and mixed in foods and beverages at levels sufficientto achieve the desired level of sweetness.

Content of Potential Human Toxicants in THAUMATIN II
Thaumatin proteins comprise an approved product and are non-toxic when used as intended. Processimpurities derived from the manufacture of thaumatins in edible plants are also non-toxic as described in
GRN 738. There are two potential human toxicants that may remain in low levels in the THAUMATIN II
product when it is manufactured using the host plant N. benthamiana; namely, the alkaloids nicotine andanabasine. As discussed in GRN 775, the host plant N. benthamiana shares metabolic pathways with othermembers of the plant family Solanaceae, which includes tomato, pepper, potato, eggplant and others. All
these plants contain low residual yet measurable levels of pyridine alkaloids.

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
The purpose of including a chromatographic purification step during downstream processing is to produce
Thaumatin II with levels of residual alkaloids that are in the same range as the levels consumed in common
vegetables in a typical diet.
In addition to residual alkaloids, some heavy metals may be present in low amounts in the final product. The
residual levels of all impurities in the final product are low, and at those levels the impurities are not expected
to pose a risk to any subpopulation of consumers when the product THAUMATIN II is used as specified.
Formulation excipients included in the final product are approved for food applications. There are no otherknown sources of potential human toxicants in THAUMATIN II.

Specification
The current Specification for THAUMATIN II is shown in Table 2-2. The process used to manufacture theproduct in N. benthamiana with this specification, including the current experimentally determined values
used to justify this specification, are shown in APPENDIX B (THAUMATIN II Manufacturing Process).
Table 2-2. Specification of THAUMATIN II Product
THAUMATIN II (Thaumatin II) Bulk ProductParameter

Specification limit

MethodAppearance

Powder, yellowish to light tan

VisualMinimum total thaumatin content(as percent of total protein)> 95%

CGE (capillary gelelectrophoresis)

Solubility (DI water)> 200 mg/mL

VisualpH of a 1% solution (excipient-dependent)

2.7 – 6.0

Potentiometric

Heavy metals (sum of Class 1 metals Pb,
Cd, Hg, As)< 5 ng/mg thaumatin

USP38<233>

Lead (Pb)< 1 ng/mg thaumatin

USP38<233>

Host alkaloid: Nicotine< 20 ng/mg thaumatin

HPLC/MS

Host alkaloid: Anabasine< 5 ng/mg thaumatin

HPLC/MS

Bioburden< 5,000 CFU total per g

USP32<61>

Agrobacterium (vector) per 10 g sample

0 (absent)

Selective plate-based assay

Undesirable microorganisms, including
Escherichia coli, Pseudomonas aeruginosa,
Salmonella spp. or coagulase-positive
Staphylococcus spp., per 25 g sample

0 (absent)

USP32<1111>

Stability (dry powder; 0-20°C) as <3% lossof thaumatin protein> 6 months

CGE; thaumatin peak in 0.1%(w/v) solution, Tm relative to T0

Page | 14

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA

2.4.

Technical Effect and Suitability of Use

Notifier has determined that the intended use of THAUMATIN II does not raise safety concerns; as such,physical or technical data are not required. Nevertheless, technical effect information was developed by
Notifier in its efforts to characterize its product, including residual impurities, and to assess Thaumatin II’s
sugar replacement potential. This information is therefore included in this Notice to enable additional
estimates of application rates and dietary exposure (see Section 3 Dietary Exposure).

2.5.

Overall Conclusion

The results of studies supporting this Notice show that Thaumatin II can be produced consistently using
recombinant expression in the plant host Nicotiana benthamiana. The recombinant thaumatin can be readily
purified from plant biomass and exhibits the characteristic properties of extracted thaumatin II. The
molecular composition of Notifier's Thaumatin II is the same as that of their native counterpart. The genesequence used in the expression vector encodes protein precursors that are correctly processed in planta toyield the same mature thaumatin protein that is isolated from Thaumatococcus, including identical aminoacid sequence. Regardless of source, including the new host N. benthamiana, the mature Thaumatin II protein
lacks glycosylation because the polypeptide backbone does not contain glycan addition sites.
The safety, physicochemical and taste-modifying properties of fermentation-derived recombinantthaumatins match those of native (extracted) thaumatins (Smith 1996) and both are available commercially.
Ample evidence of safety of thaumatins exists in the published literature. Thaumatin's approval for use infood and beverages in the USA, EU countries, Japan, Australia, Israel and elsewhere underscore thaumatin's
excellent record of safety obtained during more than 40-years of pre- and post-market surveillance.
In sum, the main difference between Notifier's N. benthamiana-produced Thaumatin II protein andcommercially available extracted or fermentation-derived thaumatin II is the method of manufacture.

3. Dietary Exposure
3.1.

Application rates of THAUMATIN II in various food categories

Average maximum application rates in all allowed uses
Thaumatin is listed in the Codex General Standard for Food Additives and permitted for general use in food(Codex Alimentarius 2016). In Europe, it is approved as a Sweetener or as a Flavour Enhancer (EFSA E 957) for
use in a number of different categories. In the USA, natural (extracted) thaumatin has GRAS classification(FEMA GRASa No. 3732; Oser 1984) as a flavor modifier, as does a recombinant version of the proteins("thaumatin B-recombinant"; FEMA GRASa 3814; Smith 1996).
Notifier’s GRN 738 established plant-made thaumatin as a GRAS sweetener in the USA. In the EU and Japan,
the acceptable daily intake (ADI) limit for thaumatin table-top sweeteners is "not specified" (Quantum satis)
because the proteins are considered to be non-toxic. Based on their sweetening and flavor-modifying profile,thaumatins are applied to foods in the 1-400 ppm range, or 1-400 mg thaumatin/kg or /L food or beverage.
When used at these maximum allowed safe levels and on the same foods, the use rates and dietary exposureof Notifier's product described in GRN 738 were expected to be the same as those for currently available
thaumatin products. In the USA, FEMA's suggested application rates range from 1-150 ppm, because theyexclude uses of thaumatin as a sweetener (Cohen 2016; Cohen 2018).

Page | 15

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANAIn GRN 738, Notifier used the more inclusive EU database for estimating the maximum safe dietary intake ofthaumatins from THAUMATIN because it represented a higher potential intake scenario (1-400 ppm) bycovering all food categories and use rates encompassed by EU statutes (EFSA 2015). Based on the use rates
in those statutes, a safe maximum intake from a typical diet of thaumatin-treated products was defined as
1.1 mg/kg-day; for an adult person of 70 kg body mass, maximum safe intake would be 77 mg/person-day.

Average moderate application rate as a sweetener
Although the 1.1 mg/kg body mass/day upper limit of consumption of thaumatin in all uses provides a safety
reference that is indicative of thaumatin’s very low toxicity, it is unlikely that anyone would consume thatamount, especially as a sweetener, as most uses of thaumatin are in flavor modification (Green 1999).
Recently, Nomad conducted blinded sensory evaluation studies to determine the sweetening potency(intensity) of its Thaumatin II when mixed in various ratios with sucrose in highly pure water. A representative
panel of results is shown in Figure 3-1. The perceived sweetness (intensity units relative to 10% sucrose w/v
in water) is shown in the ordinate and the duration of the effect is shown in the abscissa (time in seconds).
Linear regression analyses of these data were then used to compare sweetness at various levels and to
estimate the sugar replacement ratios. For example, it was calculated that 4.6 ppm Thaumatin II produced
an equivalent sweetness sensation as 5% (w/v) sucrose, as it could replace 1/2 of the sugar in the positive
control (10% sugar) with minimal sensory detection of the substitution by test subjects.
Stated another way, 4.6 mg Thaumatin II was found to be as sweet at 50 g of sucrose; meaning that at thatconcentration Thaumatin II is >10,000-times sweeter than sugar on a weight basis. Such results suggest that
THAUMATIN II could be applied to some foods as a sweetener at very low rates, enabling potential
replacement of up to one-half of the caloric sweetener typically added to food and beverages (Section 3.2).
15
14

13
12

~

10% sugar {control)

11~5%Sugar, 3.SppmThaumatin II10. -s% Sugar, Sppm Thaumatin II

98- - s% Sugar, 7ppm Thaumatin II7......... 6% Sugar, 3.Sppm Thaumatin II6-+- 6%Sugar, SppmThaumatin II5- - 6% Sugar, 7ppm Thaumatin II

4

-

3

7%Sugar, 3.SppmThaumatin II.....,_ 7% Sugar, Sppm Thaumatin II2
10
Sweetness

20s

40s

60 s

80s

100s

120s

Figure 3-1. Thaumatin II sweetness and aftertaste
Sweetness intensity was evaluated by a panel of tasters in blinded fashion, after holding samples in their mouth for 5 seconds
before expectorating. Sweet aftertaste was evaluated every 20 seconds for 2 minutes. 10% sucrose (w/v; sugar) was used as acontrol. Solutions with the same sugar content are grouped using the same color on the graph but using different patterns.
Different levels of Thaumatin II are shown as different shape symbols on the graph markers.

Additional details of the methods used in these sensory evaluations and the results obtained are provided in
APPENDIX C, Section C-7 Determination of Sweetness Intensity).
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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA

Application rates of THAUMATIN II sweetener to food and beverages
When used as a sweetener, the incorporation of N. benthamiana-produced THAUMATIN II into various foods
is expected to include generally the same food categories and limits specified in the EU (EFSA 2015) andclaimed in GRN 738, and as specifically defined in follow-up communications with the Agency, in particular“Response to FDA's Dec 18, 2017 Request for Additional Information” (January 11, 2018).
The original maximum safe application or use rates listed in GRN 738 are reaffirmed in this submission, andare summarized in Table 3-1.
Table 3-1. Estimated application rates of THAUMATIN II (Thaumatin II) sweetener
THAUMATIN II Use Category: Sweetener (*ppm thaumatin = mg/kg or mg/L food or beverage)
Use level (ppm)*
GRN 738

Use level (ppm)*
This Notice

Wine, beer and otherfermented or distilled
beverages

55Jams, jellies, marmalades,
etc.

5

5

Potato-based and similarsnacks10

10

Breakfast cereals

10

10Chewing gum

1010

If product contains other sweeteners

Chewing gum

50

50

If thaumatin is the only sweetener

Ice cream / other edible
ices

50

50

Cocoa and chocolate
products

50

50

Breath mints and similar

50

50

Confectionary50

50

Decorations, coating andfillings

50

50

Fine bakery items

30

30

Food supplements(capsules, tablets, syrups
and similar)

400

400

For use in food supplements only asa non-dietary ingredient

Table-top sweeteners

Quantum satis(10 ppm)

Quantum satis(10 ppm)

Includes liquid, powder and tablet
formulations

Average usual level

50 ppm50 ppmBased on EFSA's safe use levels

Average maximum level

400 ppm

400 ppm

EFSA’s Quantum satis is based on 10
mg/kg application rate.

Food Category

Comments

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA

3.2.

Estimated dietary intake of THAUMATIN II in foods and beverages

Maximum estimated intake from all allowed uses
With respect to human dietary exposure, we expect no higher levels of consumption of Notifier's thaumatins
per weight or volume of treated food as those allowed for extracted thaumatins. The European Food Safety
Authority (EFSA) provides guidance on maximum permitted levels (MPL) of thaumatin on various foods andbeverages according to Annex II to Regulation (EC) No 1333/2008 (OJEU 2011).
The EU’s MPL for thaumatin food additive E 957 ranges from a low of 5 mg/kg in "flavored fermented milk
products" (e.g. yogurt) to a high of 400 mg/kg in "food supplements supplied in a syrup-type or chewableform" (EFSA 2015). The same regulations allow the unlimited addition of thaumatin (Quantum satis 1) when
the protein is used as a table-top sweetener. It is envisioned that THAUMATIN II, regardless of production
host, could be used at equivalent maximum safe application rates on various foods. Until GRN 738,
thaumatins had not been considered a sweetener per se in the USA. As a flavor enhancer or modifier, FEMA
cites use ranges from a low of 1 ppm for baked goods to a high of 150 ppm for chewing gum (Cohen 2016).
The application rates of thaumatins to various food products (maximum permissible level, MPL) were derived
by assessing the consumption of each food type and estimating the additive intake to reach a maximum
exposure of 1.1 mg thaumatin/kg body weight (bw) per day, or 77 mg/day for a 70-kg person. Consumption
values were derived from typical European diets, taking into consideration country specific dietary
preferences in Belgium, Bulgaria, Czech Republic, Denmark, Finland, France, Germany, Greece, Italy, Latvia,
Netherlands, Spain, Sweden and the UK and various population age groups (adult, infant, elderly).
The original application rates allowed by EFSA (2008) were later amended (EFSA 2011) to include more foodtypes and higher allowed thaumatin levels (OJEU 2011). Revision of statutes for expanded use was based on
thaumatin's functionality and post-market safety record. Based on this safety record and on physicochemical
equivalency, in GRN 738 Nomad claimed GRAS conclusion for its THAUMATIN product produced in edible
plants when consumed at these same levels of intake. Additional detail is provided in Section 6.2.

Moderate estimated intake from use as a sweetenerIn the USA, manufacturers are not required to provide information publicly regarding the quantity of lowcalorie sweeteners added to food, with the exception of saccharin; hence, accurate estimations of the use
and intake potential for THAUMATIN II are difficult. We therefore used an extreme model for estimating
consumption of Thaumatin II in the sweetener category. We estimated current per capita intake of sugar(sucrose) as a reference and used our data on maximum sweetness replacement potential (Section C-7
Determination of Sweetness Intensity) to estimate intake if Thaumatin II were to replace one-half of all thesugar. As unlikely and extreme as this example might be (sugar and other caloric sweeteners have bulk andother rheological properties not replaceable by thaumatin or other high-intensity sweeteners), it helps
illustrate an upper limit for THAUMATIN II’s use and intake in the sweetener category.
Briefly, net consumption of sucrose per capita in the USA in 2018, adjusted for losses, was 18.3 kg (USDA ERS
2019b). Based on the replacement potential defined in Section 3.1, consumption of Thaumatin II would beless than 1 g/person-year, or ~2.5 mg/person-day, which is <0.005% the amount of sucrose currently
consumed. This estimated consumption rate is 1/30th (<3.25%) the safe daily maximum permitted level (MPL)
1

With respect to food additives, the term “Quantum satis” indicates that no maximum level is specified. However, additives must be
used in accordance with good manufacturing practice, at a level not higher than is necessary to achieve the intended purpose andprovided that they do not mislead the consumer. The EFSA Quantum satis designation was based on 10 mg/kg application rate.

Page | 18

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
allowed for all uses of thaumatin by an adult person (i.e. 77 mg/person-day). Although Thaumatin II’s
sweetening potency was not assessed in sensory evaluations against corn syrups (e.g., high-fructose cornsyrup; HFCS), in 2018 the US per capita consumption of HFCS was ~10 kg, or about 1/2 of the refined
crystalline sucrose consumed (USDA ERS 2019a). To complete the estimate, we added 50% to the projecteddaily sugar replacement value, for an estimated total intake of 3.75 mg/person-day (2.5 mg replacing sucrose+ 1.25 mg replacing corn syrup), which is <5% of the total safety based MPL for all uses of thaumatin.
Although replacement of this level of caloric sweeteners is highly unlikely, even with an equally unlikely 100%
market capture by Notifier’s THAUMATIN II product, our “worst-case” model enables estimations of safety.

3.3.

Additional exposure to thaumatins (intake not related to THAUMATIN product)

On the basis of the estimates presented in Section 3.2 for THAUMATIN II used as a sweetener, 3.75 mg
thaumatin/person-day is expected to be the maximum intake contribution of Notifier’s product in an extreme
scenario where the product displaces one-half of caloric sweeteners but none of the existing thaumatins.
Notifier does not have market segmentation information for thaumatin’s various commercial uses, but
no/low caloric high-intensity sweeteners (HIS) collectively represent a small fraction of all sweeteners, as
reviewed by Sylvetsky et al. (2017). Assuming that 1/3 of thaumatin’s current maximum safe use level is forfood and beverage sweetening applications, the maximum safe daily per capita intake would be ~25 mg (1/3
of 77 mg). Hence, the additive consumer intake for thaumatin sweeteners (3.75 mg from THAUMATIN II plus
25 mg from existing sources) would be 28.75 mg.
A more likely scenario is one where Notifier’s product displaces some of the current thaumatins and some of
the current caloric sweeteners, although an accurate mix for market perturbation is difficult to predict. The
commercial introduction of Notifier's THAUMATIN II product, irrespective of the manufacturing host used, is
expected to displace, initially, thaumatin that is currently derived from extraction of the same proteins fromThaumatococcus. Should industrial thaumatin usage expand due to higher or more consistent availability oflower-cost THAUMATIN II, consumption of thaumatin would be expected to increase. Even if daily per capita
intake were to double from 25 mg/day to 50 mg/day for sweetener uses, or from 77 mg/day to 150-200
mg/day for all uses, thaumatin's safety factor of 1,300 based on chronic toxicity studies suggest that any
additional safety risk to consumers would be minimal (Hagiwara 2005; OJEU 2011; EFSA 2015). Additional
perspective on the safety risks of such intake from Notifier's THAUMATIN II product and from all sources ofthaumatin is provided in Section 6.2 (Safety in Relation to Dietary Intake of THAUMATIN II).

3.4.

Dietary Exposure to Host- and Process-Derived Impurities

Although the safety of thaumatin proteins has been documented (Notifier’s equivalency data and referenced
studies in GRN 738, plus this Notice), the use of non-food plant species as manufacturing hosts introduces
the potential for new risks not found in the original list of production plant varieties. A description of risk
factors and their mitigation is found in Section 6 of this Notice.
On a protein basis, the THAUMATIN II product consisting of Thaumatin II produced in N. benthamiana has apurity of >95% (typically ~98%); hence the Specification was set at >95%. The residual host-derived
constituents of chromatographically purified thaumatins are mainly proteinaceous, and there are no unusual
proteins in the host that would introduce risk, especially at such low levels (Leffingwell 1999).
Assuming a worst-case scenario where the purity of the product is only 95% (i.e. the Specification minimum),at the thaumatin adult MPL of 77 mg/day, proteinaceous host-derived impurities would amount to <4
mg/day (i.e. 5% of 77 = 3.85). With a US food consumption average of 0.56 kg/day, the maximum exposure
to host-derived proteins would be ≤7 ppm/day (3.85 mg protein impurity/0.56 kg-day food intake), which isPage | 19

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
an insignificant level and not expected to impact either safety or nutritional content. Extending thiscalculation to the moderate exposure from Thaumatin II used in foods only as a sweetener (i.e., 3.75
mg/person-day), a 95%-pure product would expose consumers to <0.335 ppm of host-derived proteins (i.e.
0.187 mg protein impurity/0.56 kg/day food).
Some elemental impurities derived from plant growth media could be co-purified with thaumatin proteins(see Section 6.1.4 Process impurities in N. benthamiana-based THAUMATIN II manufacture). The Specification
limit set for total Class 1 heavy metal content (sum of Pb, Cd, Hg, As) in N. benthamiana-purified Thaumatin IIof 5 ng/mg would expose consumers to <20 nanogram/person-day of these metals in sweetening uses (3.75
mg Thaumatin II/day) and to <400 ng/person-day at the thaumatin MPL. Both levels are well below the
55 micrograms/person-day combined oral permitted daily exposure (PDE) for Class 1 elements (ICH 2015).
The remaining inorganics include salts derived from the buffer (phosphate, NaCl) that are safe and allowed
for food use. Hence, we assess that non-alkaloidal host- and process-derived impurities in N. benthamianaproduced THAUMATIN II would pose little to no risk to consumers.
The main organic impurities of concern are the alkaloids nicotine and anabasine, given their presence in N.
benthamiana and their toxicity profiles. The other alkaloids potentially present, namely nornicotine andanatabine, have similar activities but are present at very low levels in the plant and pose no significant risk.
Hence, our focus has been on assessing risk from the two main alkaloids present. The multi-batch averagevalues for nicotine and anabasine (nanograms; ng) per milligram (mg) of Thaumatin II protein (i.e., ppm) were
determined as described in APPENDIX C Section C-5. The results were used to support Specification limits of
20 ng/mg and 5 ng/mg for nicotine and anabasine, respectively, with a combined limit for total alkaloid
established at 25 ng/mg protein. To estimate a maximum exposure level at the thaumatin adult MPL of 77
mg/person-day, the limit value (25 ng total alkaloid/mg protein) was multiplied by 77. Similarly, themoderate exposure from Thaumatin II sweetener was determined by multiplying the Specification limit value
by 3.75 mg/person-day. The per capita daily exposure maxima at both intake levels are shown in Table 3-2.
Table 3-2. Estimated exposure to host alkaloids from Thaumatin II produced in N. benthamiana
Nicotine
Thaumatin consumption andestimated alkaloid exposure

Anabasine

Total Alkaloid

Specification(ng/mg)Daily
exposure(ng/day)

Specification(ng/mg)Daily
exposure(ng/day)

Specification(25 ng/mg)

Thaumatin MPL (77 mg/d)

20

1,540

5

385

1,925

Sweetener exposure (3.75 mg/d)

20

75

5

18.75

93.75

Host alkaloid exposure potential was estimated from the maximum permitted level (MPL) from all uses of thaumatins (77mg/adult person-day) as well as from consumption of Thaumatin II sweetener (3.75 mg/person-day). The Specification limits for
nicotine (20 ng/mg) and anabasine (5 ng/mg) were multiplied by 77 mg (MPL) or by 3.75 mg Thaumatin II sweetener consumed
per person-day, to arrive at the daily exposure estimates, in nanograms (ng) per person-day.

At the limit values the total alkaloid exposures are estimated to be <2 µg/person-day and <100 ng/personday at the Thaumatin II MPL and sweetener use levels, respectively. These are comparable or lower exposuresthan from other sources of pyridine alkaloid-containing foods, consumer products and the environment. For
comparison, several studies have reported the average per capita daily food-borne exposure to nicotine fromconsumptions of common vegetables as ~1,000 ng (900-1,300 ng)/person-day (Andersson 2003; Davis 1991;
Domino 1993; Liu 2013; Moldoveanu 2016; Nielsen 2013; Siegmund 1999). Given Thaumatin II’s maximum
Page | 20

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
alkaloid Specification limit of 25 ng/mg protein, and assuming full systemic distribution of alkaloids uponingestion, a 70-kg person would experience <0.03 ppb level of alkaloids (Table 3-2, last column, ~2 µg total
alkaloid/70 kg body mass). Moderate consumption of 3.75 mg/day of Thaumatin II as a sweetener by a 70 kg
person would lead to 0.001 ppb concentration of total alkaloid.
For perspective, the amount of nicotine absorbed in second-hand (passive) smoke is 100 micrograms (µg)/dayfor those reporting exposure, and 20 µg/day for those not reporting exposure, due to the pervasive natureof environmental nicotine (Karačonji 2005). Even in a low-use state like California, 1/2 of consumers are
exposed to second-hand cigarette smoke, and 1/3 are exposed to second-hand nicotine and related alkaloids
emitted from vaping devices (Vuong 2019). Legally sold nicotine-containing products contain milligram (mg)levels of nicotine. A single cigarette may contain from 2 to >10 mg of nicotine, of which 85-90% is absorbed
into the blood stream through inhalation (Digard 2013). Because exposure is very rapid, up to ~9 mg nicotine
may be systemically absorbed within 5 minutes of smoking (Digard 2013). That level corresponds to up to
130 µg/kg nicotine exposure for a 70-kg person (130 ppb), which produces pharmacologic but not toxic
effects. Nicotine-containing gum has >4 mg nicotine per dose (4.2 mg for Nicorette) of which only 63% isabsorbed via the oral route over a 45 min period (Digard 2013). This translates to 2.65 mg nicotine/70 kg
person, or an exposure level of ~40 µg/kg (or ~38 ppb). This oral dose produces a mild, non-toxic
pharmacologic effect. And these values are from only a single exposure to nicotine-containing products;
multiple exposures per day are common.
Therefore, Thaumatin II alkaloid exposure is, at worst, thousands of times lower than from a single exposure
to nicotine-containing consumer products. Although the nicotine analog anabasine may be similarly bioactive
as nicotine, it is present at only a fraction of the level of nicotine in N. benthamiana-produced Thaumatin II,
and at that level it too is not impactful on safety.

4. Information on Any Self-Limiting Levels of Use
THAUMATIN II has been reported to be 2,000 to 3,000-times (Table 2-1) sweeter than sucrose; therefore, aself-limiting level of use might be expected on the bases of (a) sensory saturation and (b) organoleptic
tolerance. In addition, the cost of manufacturing Thaumatin II is higher than the cost of an equivalent
sweetening level of sucrose or HFCS; hence, (c) economics may also self-limit use of this product.

5. Experience Based on Common Use in Food Before 1958
Notifier's GRAS Notice is based on scientific procedures. Thaumatins from Thaumatococcus have been used
in indigenous human diets in West Africa for more than 200 years, as documented in British surgeon W.F.
Daniell's report when he discovered the native use of Thaumatococcus pods in Nigeria in 1839 (Kinghorn1986).

6. Basis for Conclusion of THAUMATIN II’s GRAS Status
Notifier has used scientific procedures to conclude that its N. benthamiana-produced THAUMATIN II
sweetener is GRAS under the conditions of intended use. Notifier has relied on detailed physicochemical
analyses of its Thaumatin II protein and on extensive comparisons of its properties to a large volume of public
information on thaumatins, including thaumatin's excellent safety record since these proteins were firstcommercially used as sweeteners and food flavor modifiers in multiple countries.
We conclude that the low residual levels of host alkaloids and elemental impurities in the N. benthamianaproduced THAUMATIN II product should not pose a risk to consumers, as those levels are lower than, or inPage | 21

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
the same range as, the levels consumers are exposed to in their present diets or daily environment. We
further assert that the additive content of alkaloids (THAUMATIN II-derived and dietary) should not expose
consumers to undue risk because of the small contribution of the product to total alkaloid intake.
Section 6.1 through Section 6.5 of this Notice summarize the data and information on N. benthamianaproduced Thaumatin II that were used by Notifier to support its GRAS conclusion. In Section 7 (Supporting
Data and Information), Table 7-1 presents a tabulated summary of this information from all sources.

6.1.

THAUMATIN II Overall Safety

Prior to their broad approval for consumer use in the EU, Japan and other nations, thaumatin proteins were
subjected to extensive safety assessments in animals and in humans, which have led to definition of
application rates and daily exposure limits. Guidelines for thaumatin use and dietary exposure limits havebeen published in a number of regulatory documents issued by the European Food Safety Authority (EFSA
2015), based on reviews conducted by the Joint FAO/WHO Expert Committee on Food Additives (JECFA)(JECFA 1983, 1986) and the EU Scientific Committee on Food (SCF 1985, 1989).
Thaumatin is considered safe for use in food and beverages. It is approved by EFSA in the EU (E 957) and inJapan, Australia, Israel and other countries as a food additive. In the USA thaumatin was granted GRAS status
by FEMA (GRASa No. 3732, Oser 1984; and GRASa No. 3814, Smith 1996) as a flavor modifier, but not as asweetener. FDA issued a “No Questions” letter to Nomad’s GRAS Notice GRN 738 on 18 April 2018, for
THAUMATIN (recombinant, plant-produced thaumatin I and/or II) as a food and beverage sweetener.
A discussion of thaumatin's safety from which a projection of Notifier's THAUMATIN safety can be drawn waspresented in GRN 738 and is summarized here, with an expanded discussion on the safety of the new
production host, the plant species Nicotiana benthamiana. Specifically, the safety profile of THAUMATIN II's
active ingredients can be deduced from (1) their equivalence to commercially available thaumatins, and (2)published results of detailed studies on the safety of thaumatin conducted over the last 40 years.
The safety of the gene expression system and of residual impurities from edible species of host plants that
Notifier uses to produce THAUMATIN were discussed in detail in GRN 593 (GRN 593 Section A.2.2; pp 25-28).
The impurities from N. benthamiana were discussed in detail in GRN 775 (GRN 775 Section 6.1.1 – 6.1.6; pp
28-39) and are summarized herein for convenience. Regardless of host plant, the manufacturing processes
defined in GRN 738 (THAUMATIN from edible species), GRN 593 (COLICIN from edible species) and GRN 775(COLICIN from N. benthamiana) are analogous and differ mainly in the higher downstream purification
applied for THAUMATIN II when using N. benthamiana as the host.
With respect to the thaumatin proteins themselves, studies by Higginbotham et al. (1983) and Hagiwara etal. (2005) have been pivotal to establishing safety and have helped craft regulations for the application rateand safe usage levels of these substances. Higginbotham et al. (1983) found that thaumatin, when used as asweetener/flavor enhancer, was unlikely to be hazardous at the anticipated level of consumption. They
studied the safety of thaumatin over a 7-year period when various purities and formulations of thaumatinwere under development and the initial product was being commercially manufactured. A summary of theirfindings includes:
Thaumatin digestibility. Thaumatin was readily digested prior to absorption in rats. Groups of 20
animals were split into 2 x 10 animals each, and fed diets containing no (0%) or 5.73 wt% thaumatin, for 10
consecutive days with sampling at two time periods. The results of metabolic analyses showed that thaumatin
was 89 to >90% digested when fed as a mixed additive to the diet. Digestibility was high even thoughthaumatins I and II have 8 intramolecular disulfide bridges.
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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
Subacute toxicity in rats and dogs. Thaumatin-containing diets were fed to groups of 20 male and 20
female rats, and to groups of 4 male and 4 female dogs, at concentrations of 0%, 0.3%, 1.0% and 3.0% (w/w),
daily for 13 weeks. Behavioral, food consumption, weight gain, clinical chemistry, cytology, ophthalmoscopy,
urinalysis, and histopathology observations were conducted on all animals at every dose group. No adverse
effects were found for any dose of thaumatin administered.
Teratogenicity. Thaumatin was not teratogenic when administered orally by gavage to groups of 20
pregnant rats at 0, 200, 600 and 2,000 mg/kg body weight/day from day 6 to 15 of gestation. No thaumatinor dose-related adverse effects on the fetus were found.
Mutagenicity. Thaumatin was evaluated for mutagenic potential using two standard assays, thedominant-lethal test, and the Ames' in vitro mutagenicity assay. In the first study, groups of 15 male miceeach of proven fertility were intubated daily for 5 consecutive days with thaumatin or negative or positive
controls. Thaumatin administration had no effect on the incidence of dominant lethal mutations whenadministered at doses of 200 and 2,000 mg/kg-day. In the second study, the lack of mutagenic potential was
confirmed in bacterial mutagenicity assays with Salmonella typhimurium (strains TA1535, TA1537, TA1538,
TA98 and TA100) and Escherichia coli WP2, at levels of addition of 0.05–50 mg thaumatin/plate. All results
were negative for mutagenicity.
No observed adverse effect level. The multi-species preclinical studies by Higginbotham et al. helped
establish the NOAEL (no observed adverse effect level) for thaumatins at 1.4 g/kg (dog) to 2.4-2.8 g/kg (Mand F rat). By conventional criteria, thaumatins are non-toxic. Allergenic risk potential was low.
Persistence of thaumatin proteins and genes. To complement Higginbotham (1983), Szwacka (2012)
studied digestibility of thaumatins and the uptake of the thaumatin genes in animals fed transgenic fruit. The
proteins were fully digested, and there was no gene transfer to the intestinal microflora.

6.1.1 Allergenic potential of THAUMATIN II
Amino acid sequences in Thaumatin II with similarity to known allergens
Because Notifier's Thaumatin II is identical in amino acid sequence and no less pure than commercial
compositions containing thaumatin II, the allergenic risk for the recombinant proteins is deemed to be nothigher than that of approved commercial products. Internal and external data were used to reach thisconclusion. Baniulis et al. (2008) had conducted a computational analysis of thaumatin II allergenicity andassessed potentially antigenic elements in thaumatin-like family proteins. Of the various candidate antigenic
domains in thaumatin II, only four amino acid residues (Thr12, Leu74, Gln133 and Thr161) were found topossess high surface exposure and antigenic propensity, in theory. Using a similar technique, Notifier
conducted an updated search in silico for potentially antigenic domains in Thaumatin II.
The results of Notifier’s sequence searches were included and discussed in detail in GRN 738 (Section 6.2,
Allergenic potential of THAUMATIN, pp 17-21). The majority of identity hits were for thaumatin-like proteins("TLP"), which are widely distributed across many genera. It is thought that TLP serve a protective functionin the plants that synthesize them, including protection from fungal attack and/or stress; TLP may also be
involved in fruit ripening (Barre 2000; Liu 2010). Table 6-1 summarizes the results of Notifier’s amino acidsequence analyses performed in silico.
TLP are polypeptides of about 200 amino acid residues that share sequence similarity with thaumatins fromThaumatococcus daniellii. Due to their inducible expression by stresses like pathogen/pest attack, plant TLP
are classified as the pathogenesis-related (PR) protein family 5 (PR5), which is one of 17 families of defensePage | 23

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
related PR proteins (Christensen 2002; van Loon 2006). The most closely matched amino acid sequences tothose of Thaumatococcus thaumatin II protein are found in TLP in banana fruit (Musa acuminata). Of further
interest is the finding that TLP, once thought to be present only in dicots, also appear in the fruits of monocots(e.g. banana) and may serve similar protective functions (Barre 2000; Liu 2010).
Table 6-1. Bioinformatic amino acid scan for allergenic sequences in Thaumatin II
Thaumatin
II>35% allergen similarity at indicated search granularity
Full seq

80-mer8-mer

35 identified

33 similar seq

17 matches

TLP Species
Prevalence

Allergenicity
Potential

Banana(17/17)Low

The published literature show that TLP are consumed from multiple sources in our daily diet. Like any foreign
protein, they have the potential for allergenicity. Yet, the lack of literature linking TLP to severe food allergies
or anaphylactic reactions suggests that TLP, including thaumatins, present a low allergenic risk relative toother allergens in common foods, such as peanut, shellfish or gluten.

Allergenicity of Thaumatococcus thaumatins determined in preclinical and clinical studies
Results of prior allergenicity studies in humans and animals were included and discussed in detail in GRN 738(Section 6.2, pp 19-21). Briefly, Higginbotham et al. (1983) performed detailed acute and sub-chronic
toxicological evaluations of Thaumatococcus-extracted thaumatins, including determining the allergenic andanaphylactic potential of these proteins in rodents, NHP (non-human primates), and human volunteers. Their
multi-species studies were comprehensive, and included evaluating the potential toxicity of purified
thaumatin proteins and plant-derived impurities from Thaumatococcus, as well as formulation excipients. For
example, they compared the safety of purified thaumatins I and II (95-98% protein) to that of less pure (7885%) formulations.
These studies showed no evidence of thaumatin-related toxicity. Extensive preclinical and two human clinicalstudies verified the low allergenicity of thaumatins. Some commercial thaumatins from Thaumatococcus
available even today may contain potentially allergenic and/or toxic impurities, including proteases,
thaumatin variants and thaumatin-like proteins (some of which are known allergens), aluminum salts andother impurities and contaminants (Asherie 2008; Nikolic 2014).
In contrast, Notifier's THAUMATIN II is devoid of impurities and contaminants associated withThaumatococcus extraction, and any residual host-derived proteins or other host components are found inlow concentration (see APPENDIX C THAUMATIN II Characterization, for detail).

6.1.2 Host impurities
Edible plant species hosts
The acceptability and safety of food species of plants, such as red or leafy beet, spinach or lettuce, as
manufacturing hosts for recombinant proteins was described in GRN 593 (Section A.2.2; pp 25-28), and thesafe use of the same species in thaumatin production was described in GRN 738 (Section 6.1, pp 15-16 ).
Edible food plants used in manufacturing recombinant thaumatins are in fact “food" and are therefore
generally recognized as safe for ingestion at unrestricted levels.

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA

Properties of Nicotiana benthamiana as a preferred manufacturing host
Although the original list of production hosts described in GRN 738 included only food (edible) species,
Notifier has determined that additional plant species could be included in the manufacturing host list, andthat these additional hosts also yield thaumatin proteins that are safe when produced using Notifier’s
manufacturing process, even though such species have not been traditionally consumed in human diets.
The concern in using non-food species derives from toxic components of the host that may remain in the final
thaumatin product at levels to cause a safety risk. Therefore, 100% pure thaumatins produced in food species
are expected to have equivalent safety features as 100% pure thaumatins produced in non-food species.
Since thaumatins expressed in various food and non-food plant hosts have the same amino acid compositionand do not undergo any differential postranslational modifications, an assessment of risk needs to beapproached not from the standpoint of the safety of the thaumatin proteins themselves (established in
GRN 738), but rather from potentially toxic levels of host and process impurities that may remain in the final
product.
Genetic homogeneity. Nicotiana benthamiana is an ideal plant host for producing a wide range of biologics,
including food additives. A thorough review of N. benthamiana was published by Goodin et al. (2008). Native
to Western Australia, specimens of the species were reportedly first collected and brought to England aboard
the HMS Beagle during the ship’s third voyage (1843-1846). The specimens were eventually transferred to
the Royal Botanic Garden, Kew, and the holotype first described as a new taxon in 1929 (Goodin 2008).
Despite N. benthamiana having become a popular tool to study plant-pathogen interactions, relatively few
accessions may have been the source of cultivars in use today.
The United States Department of Agriculture–Agricultural Research Service (USDA-ARS) National Plant
Germplasm System has two accessions (plant introduction [PI] numbers 555478 and 555684), the Botanical
Garden of Nijmegen (Netherlands) lists two accessions, the Institut fur Pflanzengenetik und Kulturpflanzenforschung Gatersleben (IPK, Germany) has one, and the Australian Plant Genetic Resource Information
Service (AusPGRIS) lists five accessions but only two are presently available for distribution. The Kentucky
Tobacco Research and Development Center (KTRDC) at the University of Kentucky presently maintains six
accessions of N. benthamiana, of which two are the USDA-ARS accessions and another two are almost
certainly redundant (Goodin 2008).
Goodin et al. (2008) also used amplified fragment length polymorphism (AFLP)-based cluster analyses in an
attempt to determine whether any genetic variation existed among 11 research accessions. They used DNA
obtained from plants grown from seed submitted by researchers in five countries. When grown to maturity,
no obvious differences in growth habit, foliage, or flowers were noted, with the exception of a single novel
accession of N. benthamiana in the KTRDC collection, which was a much larger, coarser plant with larger
flowers than the research accessions. Comparisons among the other 10 accessions averaged 0.924, indicatingthat they are very closely related and could possibly be derived from one source.
These published comparisons suggest that N. benthamiana accessions used in research and in the
manufacture of plant-made pharmaceuticals are very similar in genotype and phenotype, including metabolic
features. Therefore, importantly, with respect to the use of N. benthamiana for manufacturing food additives,
it can be concluded that the exact cultivar used should not be impactful on the plant's compositional makeup
or its inherent safety.
Virus and viral vector permissiveness. Nicotiana benthamiana is highly susceptible to infection by many plantviruses, at least in part due to a natural mutation in an RNA-dependent RNA polymerase gene (NbRdRP1m;
Page | 25

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
Yang 2004), making it a preferred species for studying plant-viral interactions. Nicotiana benthamiana EST(expressed sequence tags) are generally highly homologous to those of agriculturally relevant solanaceous
crops, such as tomato, potato, pepper, and petunia, which collectively had a 2016-17 farm-gate commercial
value of nearly US $7 billion in the United States alone (USDA ERS Cash Receipts by Commodity database,2015-2018; https://data.ers.usda.gov/reports.aspx?ID=17845). As suggested by these USDA data, functional
genomics projects concerned with host–pathogen interactions conducted in N. benthamiana will most likely
reveal genes that play similar roles in agronomically important crops.
More recently, N. benthamiana has been the preferred host for heterologous protein expression in plants
using plant virus-derived transient expression systems based on tobacco mosaic virus (TMV), potato virus X(PVX), and other vector backbones. A comprehensive review (Gleba 2014) catalogs efficient expression ofheterologous proteins using Agrobacterium-mediated import of viral replicons, typically using N.
benthamiana as the production host.
Nicotiana benthamiana as a biomanufacturing platform. The homogeneity of the host, the ease of viraltransduction for heterologous protein expression, and the high levels of accumulated protein productachievable after 7-10 days post infection, have made N. benthamiana a widely adopted "platform" species
for plant-made pharmaceutical (PMP) R&D.
PMP is a rapidly developing ﬁeld and its progress has been the subject of a number of reviews, many of which
cover N. benthamiana as the preferred host (Cañizares 2005a; Cañizares 2005b; Chen 2011; Daniell 2009;
Gleba 2004; Gleba 2005, 2007; Karg 2009; Klimyuk 2014; Komarova 2010; Lico 2008; McCormick 2008a; Mett
2008; Mortimer 2012; Pelosi 2012; Pogue 2010; Rybicki 2009; Sainsbury 2009; Saunders 2013; Smith 2009;
Tusé 2011; Whaley 2011; Yusibov 2008) and others.
It is poignant that most of the currently announced or planned clinical trials of PMP are based on
biopharmaceuticals produced using transient expression systems with N. benthamiana as the host. Those
include recombinant biosimilars, antigens, and immunoglobulins produced using agroinfection (Icon
Genetics/Nomad Bioscience, Germany; Mapp Biopharmaceutical, USA; Fraunhofer Institute for Molecular
Biotechnology (IMB), USA; iBio, USA; University of Louisville/Intrucept Biomedicine, USA; PlantForm, Canada,etc.) or a non-viral transient system (Medicago, Canada).
Ease of scale-up and regulatory compliance. The popularity of transient systems reﬂects their speed andyields, as well as their scalability, and importantly, acceptance by the regulatory agencies. There are several
plant-based cGMP-compliant or GMP-certiﬁed manufacturing facilities currently in operation, including thoseof Kentucky Bioprocessing (KBP), USA; iBio CMO, USA; Medicago, Canada; Icon Genetics, Germany; andFraunhofer IMB, Germany. Manufacturing processes for biopharmaceuticals using N. benthamiana as thehost are in compliance with quality and safety criteria specified in various FDA guidance documents and theCode of Federal Regulations (CFR), specifically CFRs treating GACP, cGMP and GLP.
Most of the PMP products that have or will enter clinical studies are manufactured in N. benthamiana;
notably those manufactured by MAPP, KBP, Fraunhofer IMB, iBio CMO, University of Louisville/Intrucept
Biomedicine, Icon Genetics and Nomad Bioscience. Universally, the products produced in Nicotiana haveshown a high level of safety in clinical studies, even products whose release specifications for purity were set
at only ≥90% (Tusé 2011). This means that Nicotiana host-derived impurities allowed in clinical products at
up to 10% levels have failed to produce adverse events in clinical studies, even when the products were
injectable vaccines co-administered with an adjuvant (McCormick 2008b; Tusé 2015).

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA

Host impurities from Nicotiana species and their impact on safety
Humans and other animals consume a wide range of plants. Historical (evolutionary) trial and error, safety,
nutritional value, ease of cultivation, and organoleptic qualities no doubt contributed to the selection ofpreferred species for dietary consumption. Plants of the genus Nicotiana share the main structural,
physiological and biochemical constituents with all other land plants.
As extensively reviewed by (Leffingwell 1999), such constituents include (a) carbohydrates, including starch,sugars, sugar esters, cellulose and pectin; (b) nitrogenous constituents, including protein, soluble amino acids,
nitrate, and certain alkaloids; (c) plastid pigments, including chlorophyll and carotenoids; (d) and isoprenoids
and diterpenoids (both carotenoid-derived and non-carotenoid-derived), cembranoids and labdanoids; (e)phenolics, including polyphenols, lignin and various other phenolics; (f) sterols such as cholesterol andstigmasterol, and (g) various inorganics, including calcium, potassium, magnesium, sodium, chloride, andvarious other minerals that are absorbed from the soil (Leffingwell 1999).
These major structural, proteinaceous and biochemical components of Nicotiana species, including N.
benthamiana, are shared with other plant species, including edible species, and are not considered inherently
toxic. In fact, several studies have appeared in the peer-reviewed literature where members of the genus
were assessed as a source of nutritional protein and other valuable biochemicals.
For example, in the proceedings of an Office of Technology Assessment conference on alternative uses oftobacco in agriculture, Wildman (1983) demonstrated from analytical studies that tobacco-derived protein
fractions could be used as animal feed, with calculated nutritional value comparable or exceeding that ofalfalfa. Wildman designed and described the agronomics for producing tobacco strictly for nutritional uses,
including an extraction scheme for major plant components, each with directed end-uses. Even earlier, Kunget al. (1980) evaluated the nutritional content of tobacco leaf protein through analytical comparisons as wellas animal feeding studies (rodent models). Kung fractionated total leaf protein from N. tabacum cv Maryland
609 into fraction I (FI), which contained ribulose 1,5-bis-phosphate carboxylase-oxygenase, and FII, comprisedof soluble proteins from the cytoplasm and chloroplast. They compared the amino acid composition andnutritional value of FI and FII to milk-derived casein. Both Nicotiana protein fractions contained a fullcomplement of essential amino acids, with only slight deficiencies in the sulfur-containing amino acids.
Further, the protein efficiency ratio (PER; defined as animal weight gain per amount of protein consumed in
feeding studies) of both FI and FII were comparable (within 10%) to the PER of casein.
In their discussion, Kung et al. (1980) cite similar nutritional studies with tobacco-derived FI and FII protein
fractions, which showed that the Nicotiana-derived protein was similar in amino acid composition andnutritional value to soy protein (Kung 1978) and alfalfa leaf protein (Bickoff 1975), and better than other plant
proteins, including rice, wheat and corn (Block 1951).
The above-cited studies did not report toxic effects as they were nutritional studies designed to assess animal
weight gain. Conceivably, toxic metabolites from Nicotiana leaf preparations were removed in the extraction
process or at least were reduced in concentration so as not to induce overt toxicity.
Notably, interest in Nicotiana as a source of nutritional protein occurred mainly in the 1970s and 1980s. The
presence of toxicants and the availability of many other sources of vegetable protein may have discouraged
additional research. This concept is supported by Siceloff (1981), who reviewed the impact of public policies
on support for alternative uses of tobacco. However, Fu (2010) developed a modern tobacco purification
process, as there may be renewed interest in low-cost plant proteins as meat analogs.

Page | 27

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANAOf the minor constituents of Nicotiana species, it is the alkaloids that could have an impact on safety. Hence,
potential toxicants need to be removed or diluted to ensure that the final product is safe. Nicotiana species
synthesize a number of bioactive substances, some of which are toxic in sufficiently high doses. The major
bioactive alkaloids in the genus include nicotine, nornicotine, anabasine and anatabine. Due in large part totobacco variety improvement and tobacco safety research, the synthesis, accumulation and biological effectsof these alkaloids have been extensively studied.
Wang et al. (2015) reported on genetic factors in Nicotiana associated with nicotine content. In commercial
tobacco cultivars, nicotine represents 90–95% of the total alkaloid pool and is synthesized exclusively intobacco roots, transported to leaves, and stored in leaf cell vacuoles by a multidrug and toxic compound
extrusion (MATE) transporter. They reviewed various factors that control nicotine biosynthesis andaccumulation, including plant genetics, environmental conditions, insect predation, mechanical injury andagronomical management measures, such as topping (decapitation of the apical meristem at the early stageof flowering) and suckering (removing the axillary buds of plants activated by topping).
Nicotine has two ring moieties, a pyrrolidine ring and a pyridine ring, derived from two branched pathways.
The pyrrolidine ring is formed from the amino acids arginine or ornithine via putrescine andmethylputrescine. The pyridine ring is derived from nicotinic acid, an intermediate of the pyridine nucleotide
cycle. Lysine gives rise to piperidine, which with nicotinic acid gives rise to anabasine. The biosynthetic
pathway of nicotine, nornicotine, anatabine and anabasine is summarized in Figure 6-1.
id
Gly-3-~

Omithine + - - - Arginine! ADC
Agmatine
ODC

li/minosuccinic acid

Lysine!

116

Cadaverin e

fl 1-

l
"' "Y

!
piperideineNA{lAnabasine}

4-methylaminobutanal

!

cycle

T~'

0

6

....__! A622?
BBi:? 3,6 dihydronicotinicacid

BBL? /✓---.D ✓V ~·Anatabine

Spermidine,

Spermine

MPO !
Pyridine
nucfeotide

Nicot1nic Acid/
2,5 dihydropyridine.

N-methylputresc,ne

i OPT
NAMN

5-aminopentanal._. N-Carbamoyl putrescine

Putrescine

PMT

Quinolinic acid•!-----

H N............... Hi""' BBL?
,

N-melhyl- 6 1- pyrrolinium/~

V iH, ~

Nicotine

vY

Nornicotine

Figure 6-1. Biosynthetic pathway of major alkaloids in Nicotiana species
Schematic diagram of alkaloid biosynthesis in Nicotiana spp. Enzymes identified for key metabolic conversions are shown. A622:
isoﬂavone reductase-like protein; ADC: arginine decarboxylase; BBL: berberine bridge enzyme-like; MPO: N-methylputrescineoxidase; NND: nicotine N-demethylase; ODC: ornithine decarboxylase; PMT: putrescine methyltransferase; QPT: quinolinate
phosphoribosyltransferase (Dewey 2013).

Because the biosynthetic pathways for these alkaloids have been largely defined (Katoh 2005), it may bepossible to knock out or suppress key genes in the pathway to yield varieties of Nicotiana having either verylow (e.g. RNAi) or no nicotine and related alkaloids (e.g. gene knock-out) (Gavilano 2006; Hibi 1994; Hildreth
2011; Hung 2013; Kajikawa 2009; Shoji 2013; Takizawa 2007; Todd 2010; Wang 2008). Interspecific Nicotiana
hybrids are also under development with the same goal (e.g. Ling 2012).
Page | 28

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANAThis opens the possibility of developing new varieties of Nicotiana that are devoid of potentially toxic
constituents. These future hosts could be used to produce cruder (less purified) product formulations, akinto the purities currently acceptable when food species such as beet, spinach and lettuce are used to produce
food additives and processing aids. Notifier is also working on controlling host alkaloids "upstream" byinterfering with alkaloid biosynthetic pathways. These studies will continue with the goal of further reducing
alkaloid content to below the level found in vegetables. In the meantime, Notifier has successfully reduced
the levels of host alkaloids by "downstream" process controls (APPENDIX B). This Notice focuses on
Thaumatin II purified from N. benthamiana using only downstream process controls.
The need for purification is borne from the pharmacological activity of residual host alkaloids. These alkaloids
exert neuroactive effects and gathering evidence support their role in protecting the plant from insect
predation (Steppuhn 2004; Todd 2010). Tobacco (N. tabacum) has a long history of human use for its
neurostimulant effects (nicotinic acetylcholine receptor agonist) derived from these alkaloids, principally
nicotine, which represents more than 90-95% of the total alkaloid content (Wang 2015). While several species
and varieties of “tobacco” have been cultivated, there is no evidence that N. benthamiana was ever cultivatedfor any such purposes, probably because of the plant’s morphology, its poor field performance, and theavailability of hardier, larger species of tobacco with higher nicotine content.
The toxicities of Nicotiana alkaloids have been extensively studied, with variance due to multiple factors,
including age (Okamoto 1994). For nicotine and its related analog nornicotine, the mouse and rat oral LD50
are in the range of 3-4 mg/kg and 50-60 mg/kg, respectively (see IPCS INCHEM 2012
http://www.inchem.org/documents/pims/chemical/nicotine.htm for comprehensive toxicity review).
Human data from controlled toxicity studies are of course lacking, but the lower range of a human lethal doseis estimated to be 0.5 to 1.0 grams (~7-14 mg/kg), based on records of clinical overdoses (Mayer 2014).
Table 6-2 from Baumung et al. (2016) summarizes some pharmacological threshold values for nicotine.
Table 6-2. Estimated pharmacological thresholds of nicotine
EffectType of endpoint

Humans, i.v., acuteHeart rate acceleration

LOAEL

Value [mg/kgbw]
0.008'EFSA 28 based on Lindgren et al.26

Reference

Humans, i.v., acuteHeart rate acceleration

BMDL for BMR= lSD

0.013

Own modelling" based on datafrom Lindgren et al. 26

Humans, chronic cigarette use

Addiction

Threshold0.07b

Benowitz and Henningfield 29

Humans (Children) , dermal, acute

Various symptoms of intoxication

LOEL

0.01

EFSA 28 based on Woolf et al. 34

Humans (Children), dermal, acute

Various symptoms of intoxication

BMDLl00.004

Own modelling" based on datafrom Woolf et al. 34

Various animal species, acuteMortality (LOSO studies)

BMDLl0d3Lachenmeier and Rehm 24

Rats, 10-day study

Liver: fatty change

BMDLl00.27

Own modelling" based on datafrom Yuen et al. 10

Rats, 10-day study

Liver: focal necrosis

BMDLl00.24

Own modelling" based on datafrom Yuen et al. 10

Rats, 10-day study

Liver: dark cell change

BMDLl00.21

Own modelling" based on datafrom Yuen et al. ' 0

Rats, 10-day study

Pathological changes in liver

NOAEL

1.25

US EPA61 based on data from
Yuen etal. 10

A key metric in Table 6-2 is the minimum dose of nicotine reported to induce a measurable pharmacologic
effect in humans (increase in heart rate), considered to be the Lowest Observed Adverse Effect Level (LOAEL),
which ranges from 8 to 13 µg/kg Baumung 2016. Presumably, the No Observed Adverse Effect Level (NOAEL)
in humans via the i.v. route would be <8 µg/kg. However, and importantly, these values are for intravenous
bolus injection of nicotine, and hence due to ADME differences the NOAEL and LOAEL are both expected tobe higher values via the oral route relative to the i.v. route.
Page | 29

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
Uptake of nicotine after oral administration, as would occur from food, cannot be directly compared with theuptake from pulmonary exposure. Oral exposure to nicotine leads to molecules reaching the gastrointestinal
tract and being absorbed through the gut mucosa. However, in the acidic pH of the stomach, little nicotine isabsorbed because the molecule is in its ionized state (Andersson 2003; Karačonji 2005). Intestinally absorbed
nicotine molecules are transported via the hepatic portal vein to the liver, where extensive metabolism
occurs (60-80% to cotinine; Benowitz 1999) before the alkaloid reaches the systemic circulation. Thus,
nicotine will to some extent be metabolized by the bacteria and enzymes in the gastrointestinal tract, and toa large extent by the hepatic enzymes, before circulatory distribution. It has been calculated that 60-70
percent of orally supplied nicotine is metabolized by this type of first-pass metabolism before ever reaching
the systemic circulation (Benowitz 1991).
Figure 6-2 is a pharmacokinetic profile showing nicotine bioavailability in human subjects through threedifferent routes of administration, namely, inhalation via cigarette, intranasal spray (transmucosal), andorally from nicotine-containing chewing gum. The delay in absorption and the lower bioavailability of orally
ingested nicotine is evident (Russell 1983).

165
145
125
10
Plasmanicotine(nmol/1)

85

45
25
5
0

10

20

30

40

50

60

Time (min)NNS

Cigarette
Nicotnegumt

LJ_______tt

Figure 6-2. Bioavailability of nicotine in humans as a function of route of administration
Average plasma nicotine concentrations of three subjects after smoking a cigarette, taking nasal nicotine solution (NNS), andchewing nicotine gum. Doses of nicotine were 2 mg for nasal nicotine solution and nicotine gum and averaged 1.97 mg for thecigarette. Conversion: SI to traditional units-Nicotine: 1 nmol/ ~0.16 ng/ml (from Russell 1983).

There can be significant interindividual differences in the rates of nicotine metabolism in humans (Benowitz
1982). In studies with larger numbers of subjects, the half-life of systemically absorbed nicotine averaged 23 hours and that of cotinine, its major metabolite, about 17 hours (Benowitz 1996).
Because nicotine is rapidly metabolized and despite nicotine’s physiological effects, it is unlikely that a person
would overdose on nicotine through smoking, or nicotine replacement therapy (NRT) alone. The FDA statedin 2013 that "There are no significant safety concerns associated with using more than one nicotine
replacement therapy product (nicotine “patch” or nicotine gum) at the same time or using an NRT at the sametime as another nicotine-containing product, including a cigarette” (FDA 2013).
Page | 30

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
Anabasine (neonicotine) is a structural isomer (analog) of nicotine primarily found in the tree tobacco (N.
glauca); it is present in N. benthamiana albeit at very low levels. It’s acute oral LD50 is in the range of 200-300
mg/kg, rat (Toxnet: https://chem.nlm.nih.gov/chemidplus/rn/53912-89-3). Anabasine has been used
medicinally to treat rectal prolapse in Russia, and as a urine biomarker for smoking cessation (Jacob 2002).
The concentration of nicotine and other alkaloids varies substantially among Nicotiana species and is
impacted by genetics, cultivation practices, and biotic and abiotic stresses. The same is true for other species
that biosynthesize these alkaloids. Several original articles and reviews have been published on the genetics
and metabolism of alkaloid biosynthesis and accumulation. Saitoh et al. 1985 reported the alkaloid content
of 60 Nicotiana species, including N. benthamiana. Similarly, Sisson and Severson (1990) reported on thealkaloid content across Nicotiana species.
Table 6-3 summarizes levels of nicotine and anabasine found in N. benthamiana by Notifier's own analyses
compared to levels reported in the literature. Nicotine is the most abundant bioactive alkaloid in
N. benthamiana, followed distantly by anabasine. Nicotine constitutes 80-90% of the total alkaloid content
of N. benthamiana and anabasine 8-12% of the total; the typical ratio of nicotine to anabasine is ~10:1 (Sisson
1990). Nornicotine and anatabine may be present in trace amounts (<1% of total alkaloids each), and at the
projected use levels of thaumatins these two alkaloids are not a risk. The genetic homogeneity of N.
benthamiana cultivars suggests that alkaloid levels and ratios will remain consistent (Goodin 2008).
Table 6-3. Levels of nicotine and anabasine in N. benthamianaA

Notifier's Internal Data
SampleN. benthamiana
Buffer 1

N. benthamiana
Buffer 2

B

Nicotine content
ng/g fresh weight

Nicotine content

61,830 ± 29,590

690,000 ± 340,000

103,330 ± 15,610

1,140,000 ± 190,000

ng/g dry weight

Notifier's Internal Data
SampleN. benthamiana
Buffer 1

N. benthamiana
Buffer 2

Data From Literature
Nicotine content
ng/g fresh weight~soo,ooo(Todd 2010)

Nicotine content

ng/g dry weight~14,000,000(Sisson 1984)~3,000,000(Saitoh 1985)

Data From Literature

Anabasine content

Anabasine content

Anabasine content

Anabasine content

ng/g fresh weightng/g dry weightng/g fresh weightng/g dry weight

7,070 ± 1,290

80,000 ± 20,000

12,930 ± 400

140,000 ± 10,000~1,300,000(Sisson 1984)(9 .3% of nicotine)

Panels A and B show the levels of nicotine and anabasine, respectively, found by Notifier in analyses of N. benthamiana using
two different buffer extraction systems, compared to results reported in the literature.

6.1.3 Process modification to lower host alkaloid content in THAUMATIN II productThe downstream process used to reduce the level of nicotine and anabasine in the purified Thaumatin II
protein expressed in N. benthamiana is an adaptation of the method described in GRN 738 for thaumatinsproduced in edible plants. The general approach was first described by Stephan et al. (2017) and is further
detailed in APPENDIX B of this GRN, with adaptations for the properties of thaumatin proteins.
The process successfully reduces the level of host alkaloids in purified proteins relative to initial levels in theplant. Table 6-4 summarizes results from 3 batches showing average residual level of host alkaloids that are
currently achievable. The process is subject to additional optimization that is expected to further reduce
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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
alkaloid residues. Determination of alkaloid levels is discussed in APPENDIX C, Section C-5. The results wereused to develop the upper alkaloid limits in the Specification, namely, 20 ng/mg protein for nicotine and 5ng/mg protein for anabasine, for a combined limit of 25 ng alkaloid/mg Thaumatin II.
Table 6-4. Residual levels of N. benthamiana alkaloids in purified Thaumatin II
Residual alkaloid level in purified protein samples
Nicotine(ng/mg protein)
ProteinSingle Batches

Anabasine(ng/mg protein)

Mean ± SD

Single Batches

14.09 aThaumatin II

2.52 ± 1.55

3

3.37 a13.78 a14.41 ± 0.67

<3.84 a15.37 ba

Mean ± SD

No. ofbatchesanalyzed

0.34 bBatches produced at bench scale at Notifier’s facilities. b Batch produced at pilot-scale by Notifier’s CRO.

For perspective, humans chronically ingest low levels of nicotine from their diet. Table 6-5 from Andersson(2003) summarizes the nicotine content of common vegetables as reported in multiple published studies.
Table 6-5. Average nicotine content in common vegetablesfrom literature

SampleCherry tomato,
fruit

Nicotine content
ng/g dry weight(Moldoveanu 2016)

Nicotine content
ng/g dry weight

181.9(tomato)79-98 (a)78-148 (b)28-115 (c)174.3

1,000 (a)52-150 (e)42.6

71+/ -59 (d)76 (b)
45-123 (c)Refe rences(a) Castro and M onj i 1986(d) Domino et al. 1993(b) Davis et al. 1991

Eggplant,
fruit(e) Davis et al. 1986(c) Liu et al. 2013

Potato (whol e),
tuber

Cauliflower

3.8 (d)Sweet pepper

5.4 (g)(f) Siegmund et al. 1999(g) Andersson et al. 2003

Hence the natural dietary intake of nicotine can be calculated. Table 6-6 (Andersson 2003) summarizes theestimated food-borne nicotine exposure in Scandinavia; although daily consumption values for eachvegetable sampled may differ from US consumption estimates, nicotine ingestion ranges from 900-1,300
ng/person-day depending on dietary habits.

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
Table 6-6. Consumption, nicotine content and dietary nicotine exposure from common vegetables
Food Source

Average daily

Average nicotine

Average daily dietaryconsumption (g/day)content (µg/kg)

nicotine exposure(ng/day)

TOMATO- Sweden- Denmark

21 .6
62

4.4

95.0
273

POTATO- Sweden- Denmark

168.6
156
0.71 °
2.7

5.8

977 .9905

EGGPLANT - Sweden- Denmark

34.4

24.4
93
SWEET
- Sweden
1.5
0.27*
5.4
PEPPER- Denmark5.5**30
a Swedish import of eggplant in 2000 (2 297 000 kg) divided with nlllllber of citizens in 2000 ( 8872294) and 365 days; * only
green pepper; ** green, yellow or red pepper

In Section 3.4 of this Notice, Table 3-2, we presented the estimated maximum intake of alkaloids at the daily
adult thaumatin MPL of 77 mg/person. At the Specification limit for total alkaloid (25 ng/mg protein), theestimated per capita daily exposure to all host alkaloids in that "worst case" scenario was <2 µ g/person-day,
which is in the same range of the level consumed daily in common vegetables.
Importantly, when THAUMATIN II is used only as a sweetener, with per capita consumption estimated at 3.75
mg per day, the total alkaloid intake would be <100 nanograms/day, or <10% of the level in vegetables.

6.1.4 Process impurities in N. benthamiana-based THAUMATIN II manufacture
Other than the low risk from natural alkaloids present in N. benthamiana discussed above, the only remaining
potential risks would come from the vector or induction process used to express the thaumatin genes and/or
any residual reagents added in the manufacturing process.

Agrobacterial vector and ethanol induction
Gene expression and thaumatin synthesis can be induced via infection of non-GM plants with agrobacteria
or via chemical induction (e.g. ethanol) in transgenic hosts, as first defined in GRN 593 and subsequently
described in APPENDIX B of GRN 738 (THAUMATIN from edible species). When agroinfection is used, thebacterial vectors carrying viral amplicons (TMV or PVX) with the thaumatin genes are recombinantly
produced and become part of the manufacturing process. When ethanol induction is used, the thaumatin
genes are a stable but silent trait in the host and are expressed when a dilute solution of ethanol is applied
to the plant (Werner 2011). The ethanol is removed during thaumatin purification (data not shown).
Either agrobacterial induction process yields non-viable vector after the plant biomass is treated and thethaumatin protein is extracted. The question can be asked about potential safety risks from vector that may
remain viable after bioprocessing. This issue can be addressed by noting that the Agrobacterium industrial
strain used in the production of thaumatins is very similar to wild-type strains of Agrobacterium from which
it was derived. Notifier employs a weakened strain of the vector that contains mutations that severely
constrain its environmental viability and competitiveness, so it is environmentally safe.
Agrobacterium tumefaciens and related agrobacteria are natural soil inhabitants, ubiquitous plant colonizers,
can effect lateral gene transfer in plants, and are present at levels as high as 30 million CFU/g of soil (Bouzar
1993; Krimi 2002; Mougel 2001; Penyalver 1999; Stockwell 1993; Vicedo 1993). A. tumefaciens builds
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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
cellulosic bridges to plant structures, sticking to the plant during washing and consumption. Humans,
therefore, unavoidably consume agrobacteria in their natural diet. No ill effects have been reported fromconsumption of agrobacteria present in produce (i.e. it is not an animal or human pathogen), even at levels
far exceeding those that might result from the current manufacturing process.
Lastly, the agrobacterial vector carries genes that encode RNA amplicons, which replicate in the plant cells’
cytoplasm and are translated by cellular machinery to yield thaumatin protein. The RNA replicons cannot
integrate into the plant genome or be passed on via seed. They cannot integrate into the human genome or
replicate in mammalian cells.
Humans are routinely exposed to significant amounts of TMV and PVX through consumption of uncooked
vegetables (e.g. spinach, beets, collard greens, tomato, cucumber, etc.). A large percentage of smokers haveserum antibodies against TMV (Liu 2013) with no ill effects.
Neither virus as used in these expression systems (Gleba 2014) has been implicated as a health risk.
Therefore, even if viable vector became an impurity in the final product, the levels would be lower than what
humans naturally consume from ingesting produce.

Abiotic process impurities
Purified Thaumatin II protein obtained from two bench-scale developmental batches and from one pilot-scale
manufacturing batch have been analyzed for the elemental impurities of most concern in food (i.e. Class 1
heavy metals due to their high toxicities; see APPENDIX C, Section C.6 Analysis of Elemental Impurities). The
results are summarized in Table 6-7.
These values were used to set Specification limits of 1 ng for Pb and 5 ng total Class 1 metals per mg
Thaumatin II protein. Hence, hypothetical exposure to these most toxic metals (Pb, Cd, Hg, As) fromconsumption of Thaumatin II as a sweetener (3.75 mg/day) and at the maximum permitted level (MPL; 77
mg/day) are projected to be <20 nanograms/person-day and <400 nanograms/person-day, respectively.
Considering that the cumulative PDE for this combination of Class 1 elements is 55 micrograms/person-day(ICH 2015), we conclude that the intake of heavy metal impurities from consumption of THAUMATIN II ateither sweetener or MPL quantities will be inconsequential and will constitute a very low safety risk.
Table 6-7. Estimated exposure to elemental impurities in Thaumatin II
Heavy Metal Specification, Thaumatin II
Element

Class

Estimated Exposure

PDE

Spec. Limit

Sweetener

MPL(Oral/Daily)(ng/mg protein)(ng/day)(ng/day)(µg/day)

Pb

1

1

3.75

77

5

Sum of Pb, Cd, Hg, As

1

5

18.75

385

55

Specification limits for Pb and for all Class 1 heavy metals (Pb, Cd, Hg, As) were set at 1 ng/mg protein and 5 ng/mg protein,respectively, based on results of multi-batch analyses described in APPENDIX C, Section C-6. The Specification limits were
multiplied by 77 or by 3.75, to estimate total potential exposure from consumption of Thaumatin II at the thaumatin MPL orat the level used as a sweetener; values are expressed as nanograms/person-day (ng/day). The estimated elemental intake
values at each level of consumption are compared to the permitted daily exposure (PDE) for oral intake of Class 1 elements
and are expressed in micrograms/person-day (µg/day; last column in the table; ICH 2015).

Page | 34

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
The conclusion that can be drawn with these new data is that sweetener consumption at 3.75 mg Thaumatin
II/day would expose consumers to 3.75 x 1 = 3.75 ng/day of Pb and to 77 x 1 = 77 ng Pb/day from all uses.
Likewise, exposure to all Class 1 heavy metals would lead to consumption of 3.75 x 5 = 18.75 ng heavy
metals/day for sweetener uses and to 77 x 5 = 385 ng heavy metals/day from all uses.
Compared to the 5 micrograms/day (Pb) and 55 micrograms/day (sum of Class 1 elements) permitted daily
exposures to these same heavy metals, respectively, exposure from Thaumatin II consumption would lead toless than 0.1% and ~1.5% Pb exposure from sweetener uses and from all uses, respectively, relative to the
PDE. Similarly, consumption of Thaumatin II would expose consumers to <0.035% and ~0.7% of the Class 1
element PDE from consumption of the protein as a sweetener and from all uses, respectively.
For perspective, using the NHEXAS database, Ryan (2001) estimated the actual oral daily intake of heavy
metals in Maryland residents as 28 mg for arsenic, 10 mg for cadmium, and 8 mg for lead. ASTSDR(1999) states that the mercury intake through food is approximately 3.5 mg/day. Compared to thesereported levels of heavy metal intake and even the guidance limits (PDEs), the level of exposure to theseelements from THAUMATIN II should be of little consequence with respect to health risks.
With respect to abiotic safety risks from process reagents, all processing aids, aqueous buffers/salts andsolvents are selected from lists of approved food additives and food processing aids. Any residual levels ofprocessing reagents would introduce no more risk than those derived from the consumption of other foodproducts that also utilize them.

6.2.

Safety in Relation to Dietary Intake of THAUMATIN II

We expect the same maximum levels of consumption of Notifier's Thaumatin II, whether produced in edible
hosts or in N. benthamiana, per weight or volume of treated food as those allowed for all uses of extracted
thaumatins. We also expect a much-reduced, moderate level of thaumatin consumption when Thaumatin II
is used as a sweetener.
EFSA provides guidance on maximum permitted levels (MPL) of thaumatin on various foods and beverages
according to Annex II to Regulation (EC) No 1333/2008 (OJEU 2011).
Table 6-8 lists the authorized original and expanded-use levels for thaumatin (E 957) in mg/kg food (w/w)
and/or mg/L for beverages or non-solid foods (w/v) (EFSA 2015).
The same regulations allow the unlimited addition of thaumatin (Quantum satis) when the protein is used asa table-top sweetener. Although it excludes sweetener applications, FEMA's GRAS List (Cohen 2016; Cohen2018) provides application rates for thaumatin in the USA.

Page | 35

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
Table 6-8. EFSA Currently authorized and extended use levels (2015) for thaumatin (E 957)
FCS
catego ry
numbe rFood categories

authorised
MPL (mg/ Lor mg/kg asappropriate)

Currently authorisedand change of use
levels and proposed
ex tension of use(mg/Lor mg/kg as

ae2ro2 riate2

1.4

Flavoured fermented milk products
including heat-treated products

5

5

3

Edible ices

50

50

4.2.5
5.1

Jams, jelDes and marmalades andsimilar products
Cocoa and chocolate products ascovered by Directive 2000/3 6/EC

5
50

50

5. 2

Other confectionery i nc:luding
breath refreshening microsweets

50

50

5.2

Other confectioneiy including
breath refreshening microsweets

50

50

5 .3

Chewing gum

10

10

5.3

Chewing gum
Decorations, coa tings and fillings,
except frnit-based flllings coveredby category 4 .2.4
Decorations, coa tin115 and fillin115,
except frnit-based fillings coveredby category 4.2.4

50

50

50

50

50

50

5.4

5.4
6.3

Breakfast cereals

10

7.2

30

12.1.2
12.5
12.6

Flne bakeiy wares
Table-top sweeteners in liquid
form
Table-top sweeteners in powder
form
Table-top sweeteners in tablets
form
Salt substitutes
soups and broths
Sauces

14.1.4

Flavoured drinks

14.2. 1

Beer and malt beverages
Wine and other products definedby Regulation (EEC) No 1234/ 2007
and alcohol-free counterparts
Fruit wine and made wine
Spirit drinks as defined inRegulalion ( EC) No 110/2008
Aromatised wine-based products
as defined by Regulation (EEC) No
1601/91
Other alcoholic dri nks iocluding
mixtures of alcoholic drinks with
non-alcoholic drinks and spirits
with less than 15 % of alcohol
Potato-, cereal-, flour- or starchbased snacks
Desserts excluding products
covered in cat~O!J: 11 3 and 4
Food supplements supplied in asolid form, iocl uding capsules andtable13 and similar forms excluding
chewable forms

11.4. 1
11.4.211.4.314.2.214.2.4
14. 2.6
14.2.714.2. 8

15.1
16

17.1

17.2
17.3

18

Food supplements supplied in aliq uid form
Food supplements suppDed in asyrup-type or chewable form
Processed foods not covered bycategories l to 17, excluding foods
for infants and ~!!!l children

Quantumsati:f•>

QUantum sati:f•>

Quantum

Quantum sati1-•>sam•>

Only as a flavour enhancer
Only energy reduced or withno added sugar
Energy red uced or with noadded sugar
Only energy reduced or withno added sugar
Only cocoa or dried fruit
based, energy reduced orwith no added sugar
Only confectionery with noadded sugar
Only with added sugar or
polyols, as flavour enhancer
Only with no added sugar
Only confectionery with noadded sugar
Only cocoa or dried fruitbased, energy-reduced orwith no added sugar
Energy red uced or with noadded sugar

Quantum saos-•>

Quantumsati:J.•>

Restrictions/ except ion

15
15
15
0.5

5

Only water-based flavou1l!d
non-alcoholic drinks. As
flavour enhancer only

5
5
5
5

5

s
10
5

s

As flavour enhancer only

400

400

400

400
10

Ready mea Is {chilled, fro ten,dried) only

FCS: Food categorisalion System.
(a): AlthDU!lh permitted at quantum satis levels, a level of use of 10 m~/kg in table-top sweeteners (liquid, powder and tablet
form) Is considered an approprtate current level of use by the applicant and was used In the Intake assessment.

Page | 36

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
Table 6-9 lists the original and expanded-use levels of extracted and recombinantly produced thaumatinsfor US food products. Notifier expects that THAUMATIN II can be used at equivalent application rates on
various foods, as we claimed in GRN 738.
Table 6-9. FEMA Updated thaumatin use levels (2018)

..
Ill

E
:I

Ill

J:

I-

FEMANO.
GRAS
PUBLICATION

..

.m:ac
C

.5

Ill

C

Ill

E

Ill

E E
:I

Ill

J:

:I

Ill

0
u

J:

QI

I-

I- llii:

3732

3814

13

17

CATEGORY

FEMANO.
GRAS
PUBLICATION

.m:ac
Ill

C

C

Ill

E E
:I

Ill

J:

0
u

QI

I- llii:

3732

3814

13

17

CATEGORY

Baked goods

7•17•

1/1

Jams andjellies

7•17•

2/5

Beverages,
non-alcoholic

1•11•

5/7•

Meat
products

1•11•

2/2

Beverages,
alcoholic

1•11•

5/7•

Milk products

1•11•

3/6

Breakfastcereals

7•/7•

1/2

Nut products

7•/7•

5/7·

Cheeses

7•17•

7•17•

Other grains

Chewing gum

150•/ 150•

150/150•

Poultry

7a17a
1•11•

2/5

Condiments
and relishes

7•/7•

1/2

Processed
fruits7a17a

2/5

Confections
and frostings

7•17•

2/5

Processed
vegetables

7a17a

2/5

Egg products

7•17•

2/5

Reconstituted
vegetables

7a17a

2/5

Fats and oils

7•17•

Seasonings
and flavors

7•17•

0.5/1

Fish products

1•11•
7•17•
7•17•

5/7·

Snack foods

1/2

1/2
2/5

Soft candy

1•11•
7a17a
7•17•

7•/7•

1/2

Frozen dairy
Fruit icesGelatins andpuddings
Granulated
sugar
Gravies
Hard candy
Imitation dairy
Instant coffee
and tea

Soups
Sugar
substitutes
Sweet sauces

7•17•
1•11•
7•17•

1•11•

7•17•

2/5

2/5
2/5

o•fo•

7a17a

2/5

2/ 5
2/5

Abstracted from FEMA GRAS™ 28 List (July 2018). Columns show allowed use levels for extracted and recombinant thaumatinsin various food products. FEMA Updated average usual use levels (ppm)/average maximum use levels (ppm) for flavoring
substances previously recognized as FEMA GRAS. Superscript “a” represents a new use level (Cohen 2016; Cohen 2018).

Page | 37

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
Consumption values used to develop current regulations in the EU for the safe daily intake of thaumatin were
derived from typical European diets, taking into consideration country specific dietary preferences as well as
various population age groups (infant, adult, elderly). In surveying the types of sweeteners applied to foods,
and the consumption statistics for those foods in typical US diets (derived from USDA sources, including Haley
2011, and McConnell 2016), it was concluded that US and Western European diets were not sufficiently
different to warrant separate calculations of consumption. Hence, the safety upper limits (MPL) found inextensive studies in the EU are applicable to domestic (US) consumption of thaumatin.
While near-term THAUMATIN II dietary intake levels are easier to project based on currently allowed
application rates in many countries, future trends are more difficult to forecast and are the topic of several
recent studies. Ng et al. (2012) tried to quantify the use of caloric and non-caloric sweeteners in US consumer
packaged foods. They reported that of the 85,451 uniquely formulated foods purchased during 2005–2009,
75% contained sweeteners (68% with caloric sweeteners (CS) only, 1% with non-caloric sweeteners (NCS)
only, 6% with both CS and NCS). CS were used in >95% of cakes/cookies/pies, granola/protein/energy bars,
ready-to-eat cereals, sweet snacks, and sugar-sweetened beverages. NCS were in >33% of yogurts andsports/energy drinks, 42% of waters (plain or flavored), and most diet sweetened beverages.
Across unique products, corn syrup was found to be the most commonly listed sweetener, followed bysorghum, cane sugar, high fructose corn syrup and fruit juice concentrate (FJC). Ng et al. also reported that
77% of all calories purchased in the US in 2005–2009 contained CS and 3% contained NCS, while 73% of thevolume of foods purchased contained CS and 15% contained NCS. Trends during this period suggest a shift
towards the purchase of NCS-containing products.
More recently, a survey by Sylvetsky et al. (2017) that tracked US consumption of low-calorie sweeteners(LCS) showed that intake has increased significantly during the past several decades. The goal of the Sylvetsky
et al. study was to describe LCS consumption in the United States and to characterize consumption bysociodemographic subgroups, source, frequency, eating occasion, and location. The sources of information
were the National Health and Nutrition Examination Survey (NHANES) data from 2009-2010 and 2011-2012.
Sylvetsky et al. found that total of 4,981 unique food and beverage items were consumed by participants in
NHANES 2009-2010. Of these items, 126 contained LCS, including 57 beverages, 61 foods, and 8 packets.
Similarly, 5,192 unique food and beverage items were reported in NHANES 2011-2012. Of these items, 147
contained LCS, including 74 beverages, 65 foods, and 8 packets.
Importantly, Sylvetsky et al. were not able to quantify the amount of LCS in LCS-containing products because
manufacturers are not required to provide information regarding the quantity of LCS added, with the
exception of saccharin. Due to the inability to quantify the amount of LCS in foods and beverages, intake (ingrams) of LCS-containing products was estimated as a proportion of an individual’s total intake of the specificproduct category (e.g., yogurts, desserts) reported in NHANES (Sylvetsky 2017).
The food categories in NHANES surveyed by Sylvetsky (2017) are very similar to the categories surveyed in
the EU, with the possible exception that serving portions of some foods tend to be larger in the USA thanthey are in Europe; nevertheless, the EFSA application rates provide an accurate basis for estimating potential
US intake of thaumatins based on the types of foods consumed.
Therefore, in GRN 738 Notifier assumed a "highest use" scenario for intake of thaumatin in the diet, andderived use levels from the broadest groups of foods that could be treated. Not coincidentally, the EFSA/EU
database and use levels by food group presented the most comprehensive source of information, because
thaumatin is already approved for such uses in the EU.

Page | 38

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
To summarize, an assessment of the potential dietary intake of thaumatins can best be derived from EU usage
data, which show that, from all dietary sources, continuous thaumatin ingestion levels of up to 1.1 mg/kg(bw)-day in all age populations, or 77 mg/person-day for a 70-kg adult, are safe. The commercial introductionof Notifier's THAUMATIN II product, irrespective of the manufacturing host used, is expected to displace,
initially, thaumatin that is currently derived from extraction of the same proteins from Thaumatococcus.
Should industrial thaumatin usage expand due to availability of THAUMATIN II, consumption of thaumatinwould be expected to increase. Even if daily per capita intake limit were to double to 150-200 mg from thecurrent 77 mg MPL, thaumatin's safety factor of 1,300 based on chronic toxicity studies suggest that any
additional safety risk to consumers would be minimal (Hagiwara 2005; OJEU 2011; EFSA 2015).

6.3.

Occupational safety

With respect to occupational safety, Notifier's Thaumatin II protein is considered non-toxic. No higher-level
precautions for personnel protection are suggested for handling, preparing or disposing of THAUMATIN II
powder or liquid solutions. Only general precautions for handling powdered bulk substances and proteincontaining waste solutions are suggested. A draft SDS for THAUMATIN II is appended to this Notice(APPENDIX A), where additional recommendations for storage, handling and disposal of this product arelisted.

6.4.

GRAS conclusion for N. benthamiana-produced THAUMATIN II sweetener

The results of clinical studies by Higginbotham et al. (1983) assessing the effects of occupational pulmonary
exposure to thaumatin-containing substances, as well as the effects of oral ingestion of thaumatins,
corroborate the results they obtained with multiple animal species that show that thaumatins are digestible,
show no systemic toxicity when administered orally, and have low allergenic potential if delivered
systemically but none if delivered orally.
The sub-acute (28-day with 14-day thaumatin plus 14-day lactose cross-over) ingestion study with humanvolunteers administered a total of 1.4 grams of thaumatin over a two-week period, with no ill effects.
Similarly, the multi-year occupational exposure to low levels of thaumatin-containing dust in the workplace
had no obvious toxic effects and only low measurable sensitivity, which was lower in prevalence than allergy
to dust mites in the surveyed population.
Subsequently, Hagiwara et al. (2005) reported results of a 13-week (sub-chronic) feeding study of thaumatinin Sprague–Dawley rats. Thaumatin was administered at dietary levels of 0% (control), 0.3%, 1.0% and 3.0%
to groups of 10 male and 10 female Crj:CD (SD) IGS rats. There were no treatment-related clinical signs oradverse effects on the survival rate, body weight, food consumption, water consumption and urinalysis,
ophthalmology, hematology, or blood biochemistry data. No treatment-related alterations in gross pathology
or organ weights were found in any group. On histopathological examination, sporadic spontaneous lesions
known to occur in this strain of rats were the only findings, with no specific relation to the test substance.
Safety data available at the time helped support extensive reviews conducted by the EU Scientific Committee
on Food (SCF 1985, 1989) and by the Joint FAO/WHO Expert Committee on Food Additives (JECFA 1983,1986). Subsequently, Hagiwara et al. (2005) calculated the no-observed-adverse-effect-level (NOAEL) in rats
to be a dietary intake of 2,502 mg/kg body weight-day for males and 2,889 mg/kg body weight-day forfemales. A study in dogs established a lower average NOAEL of 1,400 mg/kg body weight/day. Collectively,
these studies have helped update the application rates of thaumatin. The levels of usage of thaumatin on
various food groups, the consumption of each food type, and the anticipated total exposure to thaumatin asa food additive have since been aggregated and updated (EFSA 2015).
Page | 39

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
Based on the lowest NOAEL of 1,400 mg/kg bw-day for dogs calculated from results by Higginbotham et al.
(1983) and Hagiwara et al. (2005) and others, EFSA applied a safety factor of ~1,300 for E 957 (1,400 mg/kg
bw-day in dogs and the then-current human maximum daily intake level of 1.03 mg/kg body wt-day) to arriveat an MPL for thaumatin by food product group, including solids, semi-solids and liquid beverages, for all age
groups. The cumulative daily limit for chronic human consumption was set at 1.1 mg/kg-day, or 77 mg/day
for a 70-kg adult person. At this intake level, thaumatin protein would represent <0.13% of the total adult
daily protein intake (i.e. nutritionally insignificant). Even at 2x the current intake level, the safety factor would
be nearly 700:1.
The safety of Thaumatin II protein comprising Notifier's THAUMATIN II product can be deduced by analogy
to the thaumatin products evaluated in the studies cited herein. Higginbotham et al. (1983) evaluatedthaumatin compositions of various degrees of purity, including commercial thaumatin (Thalin) that
contained 3% residual host impurities, including non-thaumatin proteins, carbohydrates, pectin, andglucuronoxylan from the arils of the source fruit. Ramsohoye (1989) found non-thaumatin protease andesterase impurities in commercial thaumatin products. In addition, because the original Tate & Lylethaumatin extraction process involved the use of aluminum salts to assist with purification, the final productcontained small amounts of the heavy metal. Importantly, in spite of these findings, the toxicity studies ofHigginbotham et al. included evaluation of isolated host and process impurities, which proved to beinnocuous with respect to safety. Since Notifier’s submission of GRN 738 (THAUMATIN from edible plant
species; 16 October 2017), no new literature was identified citing thaumatin safety concerns.
In the current Notice, Notifier has concluded using scientific procedures that its Thaumatin II protein
produced in the host Nicotiana benthamiana is generally recognized as safe when used at up to 100% of the
MPL allowed by current statutes for commercially available thaumatins. When used as a sweetener,
Thaumatin II would be ingested at not more than 5% of the daily MPL (i.e., 3.75 mg/77 mg person-day). To
ensure a high safety margin, host-derived alkaloids are monitored and controlled to minimize risk. In ourtarget Specification for Thaumatin II, we have set 20 ng/mg and 5 ng/mg as the maximum nicotine andanabasine levels, respectively, and total alkaloids of <25 ng/mg protein in a final product. At the Specification
limit, ingestion of the adult thaumatin MPL of 77 mg/day would lead to total alkaloids consumed of~2 µg/person-day, or the equivalent of 1-2 extra servings of vegetables at the current average rate of US foodconsumption (0.56 kg food/day). At the same Specification limit, consumption of 3.75 mg Thaumatin II
sweetener/person-day would result in exposure to <100 ng total alkaloid.
We present data from multiple public sources that these levels of pyridine alkaloids are below the thresholdof pharmacologic or toxic activity even for intravenous administration. We reiterate that these low exposuresfrom ingestion of residual alkaloids from Thaumatin II are exaggerated by the assumption of 100% adoptionof Notifier’s product in all food categories claimed and at the maximum permitted level (MPL) of the product.
Notifier describes a process modification introduced into its manufacturing method when using
N. benthamiana as the expression host for thaumatins (APPENDIX B). The modification involves cool ambient
temperature neutral extraction of biomass, heat treatment, and cation-exchange chromatography followedby diafiltration. These downstream steps effectively reduce the levels of host alkaloids and other impurities
in the purified Thaumatin II protein.
We also present the content of Class 1 elemental impurities in purified Thaumatin II derived from analyses ofmultiple batches. Our process is undergoing optimization, yet current results enabled us to set a Specification
limit of 5 ng total Class 1 metals (sum of Pb, Cd, Hg, As) per mg Thaumatin II protein. At that limit, total heavy
metal exposure resulting from the consumption of Thaumatin II at the adult MPL (77 mg/day) would be <400ng/person-day, whereas <20 ng/person-day elemental intake would be realized from Thaumatin II’s use only
Page | 40

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
as a sweetener (3.75 mg/day). These levels pose no undue risk to consumers relative to the cumulative Class 1
metal PDE of 55 micrograms/day. Preliminary analyses of the less toxic Class 2 and Class 3 metals in a pilotscale manufacturing batch of Thaumatin II have yielded very low impurity levels (sum of Ag, Bi, Cu, Mo, Sb
and Sn = <1 ng/mg protein), and hence these additional elements are not included in the Specification.
There are no other identifiable toxic entities in N. benthamiana that we could find from extensive literature
surveys or from our own analyses. The protein, lipid, carbohydrate and mineral components of the plant havebeen studied and are found in the public record, and reveal no unusual properties of the plant that are notalso present in solanaceous plant species consumed as food.
Similarly, no allergenic or hypersensitivity inducing components have been reported for this species. In an
average diet, exposure to residual N. benthamiana proteins would be ≤ 15 ppm/day (worst case); hence, hostproteins are not a risk factor in the product.

Conclusion
The totality of evidence, from Notifier's internal studies and from the extensive public record cited herein
and in GRN 738, suggests that consumer risk associated with ingestion of thaumatins themselves, or withingestion of trace amounts of impurities derived from manufacturing Thaumatin II protein in N. benthamiana,
is low. Consumers already ingest equivalent or even higher levels of the same impurities in their daily diet,and any contribution of additional alkaloids or elemental impurities from the use of THAUMATIN II as asweetener is not expected to significantly raise dietary risk.

7. Supporting Data and Information
Multiple sources of information were used to support the conclusion that Notifier's THAUMATIN II productproduced in the host N. benthamiana is GRAS. Table 7-1 lists the various data and other information discussed
in this Notice and used in reaching this conclusion. Also listed in the table is whether the specific information
cited was generated by Notifier and/or obtained from databases or references in the public domain.
The active ingredient Thaumatin II in Notifier's THAUMATIN II product is of the same molecular composition
as the active ingredient in thaumatin II-containing products derived from extracts of Thaumatococcus orrecombinant versions thereof, and has the same functional properties. Therefore, we draw analogies to thosereferenced products with respect to sweetening functionality, dietary use levels, and safety.
Table 7-1. Information supporting THAUMATIN II sweetener GRAS conclusion
Topic

Location in this GRN

Source of data or information

Availability

Thaumatin identity

Section 2.1

Suami 1997; van der Wel 1972; Lee 1987;
Hough 1993; Zemanek 1995; Greenly 2003

PublicThaumatin structureand function

Section 2.1

Suami 1997; de Vos 1985; Ogata 1992; van der
Wel 1984; Hough 1993; Faus 2008

PublicSection 2.1

Bartoszewski 2003; Faus 2008; Glaser 2000;
Greenly 2003; Hellekant 1996; Kaneko 2001;
Kinghorn 1986; Ledeboer 1984; Lim 2012;
Masuda 2018; Nagarajan 2017; Ohta 2011;
Sardesai 1991; Schiffman 1995; Suami 1997;
Tinti 2000; van der Wel 1972; Witty 1990

PublicMode of action andmechanisms of taste
perception

Page | 41

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
Topic

Location in this GRN

Source of data or information

Availability

Method ofmanufacture and QC

Section 2.2;
APPENDIX B

Nomad Bioscience GmbHThis GRN

Host, vector andprocess impurities

Section 2.2;
APPENDIX B;
APPENDIX C

Nomad Bioscience GmbHGRN 738 andThis GRN

Composition andSpecification

Section 2.3
Table 2-2

Nomad Bioscience GmbHThis GRN

Allowed maximum
and moderateapplication rates;
Estimated dietaryintake; Additional
exposure not related
to Notifier's productSections 3.1, 3.2 and3.3

OJEU 2011; EFSA 2008; EFSA 2011; EFSA 2015;
Cohen 2016; Codex Alimentarius 2016; Nomad
Bioscience GmbH

Public andThis GRN

Dietary exposure tohost- and processderived impurities

Section 3.4

Nomad Bioscience GmbHThis GRN

Thaumatin overall
safety; digestibility;
potential for genetransfer to intestinal
microflora

Section 6.1

JECFA 1983, 1986; EFSA 2015; SCF 1985, 1989;
EFSA 2011; Higginbotham 1983; Hagiwara
2005; Szwacka 2012

PublicEstimation ofthaumatin's NOAEL

Section 6.1

Higginbotham 1983; Hagiwara 2005

PublicDetermination ofthaumatin's
allergenic potential

Section 6.1.1

a. Computational estimates / in silico
modeling: Baniulis 2008; Nomad BioscienceGmbHb. Empirical studies, preclinical and clinical,
Higginbotham 1983

a. Public and
GRN 738
b. Public

Commonly
consumed andenvironmentallycontacted
thaumatin-likeproteins (TLP)

Section 6.1.1

Barre 2000; Liu 2010; Christensen 2002; vanLoon 2006

Public and
GRN 738

Allergenicity andoccupational safety

Sections 6.1.1 and
6.4

Higginbotham 1983

PublicSafety in relation toallowed dietaryintake of thaumatin

Sections 3.1, 3.2, 6.1
and 6.2

EFSA 2015; OJEU 2011; Codex Alimentarius
2016; Oser 1984; Smith 1996; Cohen 2016;
Nomad Bioscience GmbH

Public andThis GRN

Page | 42

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
Topic

Location in this GRN

Source of data or information

Availability

Potential additional
exposure if
consumption of
low/no calorie
sweeteners increases

Section 6.2

NHANES 2009-10, 2011-12 in Sylvetsky 2017;
USDA ERS in Haley 2011, McConnell 2016; Ng
2012

Public

THAUMATIN II Safety
Data Sheet (SDS)

APPENDIX A

Thaumatin II analysis
and characterization

APPENDIX C

a. Nomad Bioscience GmbHb. Sigma-Aldrich 2017; SFC 2006; SCBT 2015

a. This GRN
b. Public

Nomad Bioscience GmbHThis GRN

Page | 43

THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA

References
Adesina SK and JD Higginbotham. 1977. Studies on a novel polysaccharide gel from the fruit ofThaumatococcus daniellii (Benth). Carbohydrate Research 59(2):517-524.
Adesina SK and JB Harborne. 1978. Occurrence and identification of falvonoids in Thaumatococcus daniellii
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Wildman S. 1983. An Alternate Use for Tobacco Agriculture: Proteins for Food Plus a Safer Smoking
Material. In: Plants: The Potentials for Extracting Protein, Medicines, and Other Useful Chemicals Workshop Proceedings. U.S. Congress, Office of Techology Assessment, OTA-BP-F-23, Washington, D.C., p
63-77.
Witty M. 1990. Thaumatin II — a palatability protein. Trends in Biotechnology 8:113-116.
Witty M. 1998. New technologies for taste modifying proteins. Trends in Food Science & Technology
9(7):275-280.
Yang SJ, SA Carter, AB Cole, NH Cheng and RS Nelson. 2004. A natural variant of a host RNA-dependent RNA
polymerase is associated with increased susceptibility to viruses by Nicotiana benthamiana. Proc Natl Acad
Sci U S A 101(16):6297-6302.
Yusibov V and S Rabindran. 2008. Recent progress in the development of plant derived vaccines. Expert Rev
Vaccines 7(8):1173-1183.
Zemanek EC and BP Wasserman. 1995. Issues and advances in the use of transgenic organisms for the
production of thaumatin, the intensely sweet protein from Thaumatococcus danielli. Crit Rev Food Sci Nutr
35(5):455-466.

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANAAPPENDIX A. THAUMATIN II Safety Data Sheet
DRAFT- THAUMATIN II SAFETY DATA SHEETVe rsio n 2.0
Revision Dat e 10/30/2019
Print Dat e 10/30/2019

D
1. PRODUCT AND COMPANY IDENTIFICATION
1.1

Product identifiers

Product name

1.2

THAU MATIN II from plant s (e.g. lettuce, red beet, spinach, Nicotiana benthamiana)
CAS Nu mber 53850-34-3 (extracted); CAS Num ber 553850-34-3 (recom binant)Consists of Thau mat in II of identica l amino acid sequence t o t ha umat in II fromThaumatococcus dan ielfii

Functional uses

Identifi ed uses
1.3

Sweet ener fo r use in fo od and beverages

Manufacturer

Comp any

Nom ad Bioscience GmbHWei n bergweg 22
Halle 02160, Germ any+49 345 1314 2606
+49 345 1314 2601

Telepho ne
Fax

1.4

Emergency telephone number

Emergency Phone Number+49 3451314 2424 in the EU (U S emergency phone number to be provided)

2. HAZARDS IDENTI FICATION
2. 1

Class ification of the substance or mixture

2.2

GHS Labe l e lements, including precau tionary statements

Not a hazardous subst ance or mixt ure
Not a hazardous subst ance or mixt ure

2.3

Hazards not otherwise classified (HNOC) or not covered by GHS

None

2.4

HMIS Classif ication

Health hazard :Flammabi lity:
Physical hazards:

2.5

NFPA Rat ing

Health hazard :Fire:
Reactivity hazard:

2.6

0
0
0
0
0
0

Potential Health Effe.cts of Bu lk Powde r/Concentrate

In ha lati on
Skin
Eyes
Ingest ion

May be harmf ul if inhaled . May ca use resp irat ory t ract irritatio n
May be harmful if absorbed th rough skin. May cause skin irritati on
May cause eye irritation
May be harmful if swa llowed

3. COMPOSITION/INFORMATION ON I NGREDIENTS

3 .1

Substances

CAS Reg istry Num ber
53850-34-3
No ingredient s are hazard ous according t o OSHA criteria
No com ponents need t o be discl osed according t o t he applica ble regu lations

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANAII Safety Data Sheet (DRAFT)

Version 2.0

4. FIRST AID MEASURES

4.1

Description of first aid measures
If inhaled
If breat hed in, move person into fresh air. If not breat hing, give artifi cial respirati on
In case of skin contact
Wash off w ith soap and plent y of water
In case of eye contact
Flush eyes with water as a preca ution

If swallowed
Never give anything by m outh t o an unconscious person. Rinse mout h with water

5. FIREFIGHTINGMEASURES
5.1

Extinguishing media - Suitable extinguishing media
Use water spray, alcohol-resi stant foam, dry chemica l o r carbon dioxide

5.2

Hazardous combustion productsHaza rdous decomposition products formed under fi re conditions - Ca rbon oxides

5.3

Advice for firefighters
Wear self-<:onta ined breath ing appa ratus for fi refighti ng if necessary

5.4

Additional information
No additional information is availabl e

6. ACCIDENTALRELEASEMEASURES
6.1

Personal precautions, protective equipment and emergency procedures
As with an y concentrated protein, avoi d dust format ion and inhalation of pa rtic ulat es or aerosols. For personal prot ecti on
see Sect ion 8

6.Z

Environmental precautions
Product active ingredients are biodegradable. No speci al environmental precaut ions are necessary

6 .3

Methods and materials for containment and cleaning up
Sweep up and shovel solid. Water-w ash surfa ces. Use cl osed conta iners for dispos al of any unused prod uct

6.4

Reference to other sections
For disposal see Secti on 13

7. HANDLING AND STORAGE

7 .1

Precautions for safe handling
Provide appropriate exhaust ve ntilati on du ring preparati on and use. Provide appropr iate exhaust ve ntilation at places
whe re dust is formed. Normal measures for preve ntive fire protect io n

7 .Z

Conditions for safe storage, including any incompatibilities

Keep conta iner t ightly closed in a dry we ll-venti lat ed place. Recomm ended storage t emperatu re 2-8 •c

8. EXPOSURE CONTROLS/PERSONAL PROTECTION

8.1

Control parameters
Components with workplace control parameters
Conta ins no substa nces with occupational expos ure limit va lues

8.Z

Exposure controls
Appropriate engineering controls
General indust rial hygiene pract ice

Page 2

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANAII Safety Data Sheet (DRAFT)

Version 2.0

Personal protective equipment
Eye/face protection
Use government tested and approved eye protection devices (e.g. NIOSH - US or EN 166- EU)
Skin protection
Hand le with gloves that are inspect ed prior to use. Use proper glove removal t ech nique to avoid skin contact. Dispose ofused gloves in accordance with app licab le laws and good laboratory practices. Wash and dry hands
Body Protection
Wear lab coat or similar cover during preparation, application and disposal of product in keeping with specific practices in
the work environ ment
Respiratory protection
Use type N95 (US) or type Pl (EN 143) dust masks, or res pirators, depending on t he product formu lation and prepa ration
and use environment. Use devices approved under appropriate govern ment standards such as NIOSH (US) or CEN (EU)Control of environmental exposure
Product component s are biodegradable and wil l be diluted du ring use. No special procedu res fo r cont rolling environ mental
exposu re are recommended

9. PHYSICAL AND CHEMICALPROPERTIES

9.1

Information on basic physical and chemical properties

a)
b)
c)
d)
e)

Appe arance form:

Solid; yellowish-whit e to light t an

Odor

No specific odor

Odor t hres holdpH

No odor th reshold identifi ed
pH 2.7 - 6, depending on the formu lation

Melting point/freezing point

No data availablef) Initial boiling point and boi ling rangeg) Flash pointh) Evaporation rat e

No data available

i)
j)
)
I)

Upper/lower flammabi lity or explosive limits

No data avai lable
No data available

Vapor pressure
Vapor density

No data available
No data availablem) Relative density

No data available>25 g/L

Flammability (solid, gas)n) Wat er solubilityo) Partition coeffi cient n-oct anol/water
Auto-ignition temperat ure

No data available
No data availableq) Decomposition t emperat ure

No data available

Plr)

Viscositys) Explosive propertiesI) Oxidizing propert ies
9.2

No data available
No data available

No data available
No data availa ble
No data available

Other safety information
No additional information available

10 . STABILITY AND REACTIVITY
10 .1

Reactivity
No data available

10.2

Chemicalstability
Stable under recom mended st orage conditi ons

10.3

Possibility of hazardous reactions
No dat a available
Page 3

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANAII Safety Data Sheet (DRAFT)
10 .4

Conditionstoavoid
Strong oxidizing agents

10 .s

Incompatible materials
No dat a available

10 .6

Hazardous decomposition products
Other decomposition products - Carbon oxi des. In th e event of fire : See Section 5

11 ,

11.1

Version 2.0

TOXICOLOGICALINFORMATION
Information on toxicological effects
Acute toxicity
No data available
Inhalation
No data available
DermalNo data available
Skin corrosion/irritation
No data available
Serious eye damage/eye irritation
No data available
Respiratory or skin sensitization
No dat a available
Germ cell mutagenicity
No data available
Carcinogenicity
IA RC :

No component of this prod uct present at levels greater than or eq uaI to 0.1% is identified as a probable, possible orconfi rmed human carcin ogen by IARC

AC GIH: No component of this product present at levels greater than or equal t o 0.1% is identified as a card nogen or
potential carcinogen by ACGIH
NTP:

No component of th is product present at levels greater than or equal t o 0.1% is identified as a known or anticipatedcarcinogen by NTP

OSHA :

No component of this product present at levels greater than or equal t o 0.1% is identified as a carcinogen or
potential carcinogen by OSHA

Reproductive toxicity
No data available
Specific target organ toxicity- single exposure
No data avai lable
Specific target organ toxicity- repeated exposure
No data avai lable
Aspiration hazard
No data avai lable
Additional Information
RTECS: Not available. Product is not a hazardous substance or mixture

11.2

Potential health effects
Inhalation:
Ingestion:
Skin:
Eyes:

May be harmfu l if inha led. May ca use respirato ry tract irritation
May be harmfu l if swal lowed
May be harmfu l if absorbed thro ugh skin. May cause skin irritation
May cause eye irritation
Page 4

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANAIN II Safety Data Sheet (DRAFT)

Version 2.0

12 . ECOLOGICALINFORMATION
12 .1

Toxicity
No dat a available

12.2

Persistence and degradability
Active ingred ient s are destroyed by heat, acid, and by digestive and microbial enzymatic act ivity

12 .3

Bioaccumulative potential
None anticipated

12.4

Mobility in soil
No dat a available

12.5

Results of PBT and vPvB assessment
PB T/vPvB assessment not available as chemical safety assessment not requ ired/ not cond uct ed

12 .6

Other adverse effects
No dat a available

13 . DISPOSALCONSIDERATIONS

13 .1

Waste treatment methods
Product
Offer surpl us and non-recyclable sol utions t o a licensed disposa l company
Contaminated packaging
Dispose of any unused prod uct

14 . TRANSPORTINFORMATIONDOT(US)Not dangerous goods
IMDG
Not dangerous goods
IATA
Not dangerous goods
15 . REGULATORYINFORMATIONOSHA
No known OSHA hazards
SARA 302 Components
SARA 302: No chemicals in this mat erial are subject to the reporting require ments of SARA Title Ill, Section 302
SARA 313 Components
SARA 313: This material does not cont ain any chemical components with known CAS numbers t hat exceed the threshold (DeMinimis) report ing levels establ ished by SARA Title Ill, Section 313
SARA 311/312 Hazards
No SARA Hazard s
Massachusetts Right to Know Components
No components are subject to the Massachusetts Right to Know Act
Pennsylvania Right to Know Components
Th au matins from plants. See Section 1.1
New Jersey Right to Know Components
Th au matins from plants. See Section 1.1
California Prop. 65 Components
This prod uct does not contain any chemicals known to St at e of Ca lifornia t o cause cancer, birth defects, or any ot her
repro ductive harm

Page 5

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANAII Safety Data Sheet (DRAFT)

Version 2.0

16 , OTHER INFORMATION

Additional information
Copyright © 2019 Nomad Bioscience GmbH. Al l Rights Reserved Worldwide.
Th e information co nta ined in th is Safet y Dat a Sheet is believed t o be correct as of t he tim e of its re lease. It shou ld be used
as a guide for safe handling, st orage, preparati on, and disposa l of th e prod uct. Assess ment of prod uct safety underco nditions of norma l use is based on informat ion ava ilable at th e time . Because information in som e cat egories is lacking,th is SDS is not all-incl usive and is subject t o periodic updat es. Nomad Bioscience GmbH and its Affi liat es sh all not be held
liable fo r any damage resu lting from ha ndling, use, disposal or from conta ct w ith the above product.

Page 6

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA

In-Process controls and quality assurance
Notifier applies rigorous in-process controls to manage the quality of process intermediates and final
products throughout the manufacturing process. Materials not meeting pre-determined specifications are
rejected. Product release is done after each batch passes identity and purity tests. A Quality Management
system is in place to ensure conformance with industry standards and federal and local regulatory guidelines.

B.4.

Manufacturing Facilities

Notifier can manufacture THAUMATIN II product at various locations in Europe and the United States. Forcommercial manufacture, semi-automated plant cultivation, inoculation, incubation and harvesting systems
can be applied. Depending on the scale needed, Notifier can manufacture at its own facilities or use acontract manufacturing organization to produce and formulate thaumatin proteins meeting Notifier’s
specification. Features of an existing US facility’s upstream and downstream processing capabilities include:

Upstream80,000 sq ft of controlled growth space with 672 tables holding 30,240 plant trays in 3 levels. Eachtray holds 104 plants; controlled conditions for the growth and harvest of transfected plants
An automated plant transport system allowing movement, irrigation, lighting and environmental
control (temperature and humidity) of trays for plant growth

Downstream








32,000 sq ft manufacturing area
Linear scalability: 1 metric ton (mt)/shift pilot scale – 68 mt/shift commercial scale
75 L of Green Juice (post-grind/pre-clarification extract) per minute; continuous processing up to UF
35,000 L of tank storage capacity; heating and cooling control of in-process material
Manufacturing clean rooms with controlled environments
Computer-controlled processing and data collection
Clarification options (UF/DF/Microfiltration/Nanofiltration/Reverse Osmosis)

Regardless of manufacturing venue, all substances, materials and reagents used in manufacturing
THAUMATIN II by Notifier’s process conform to food grade or higher standards. All processing equipment ishigh-grade stainless steel meeting food-industry criteria. All cleaning and sterilization procedures arevalidated in accordance with FDA guidelines for food-grade materials.

B.5.

Waste Handling and Disposal

Waste streams containing plant-derived residuals are treated per local regulations and discarded. No byproducts of the process are used in food or feed products, supplements, additives or processing aids.

B.6.

Justification of Specification

A Specification for the plant-expressed THAUMATIN II product (final bulk) produced through the
manufacturing process described herein and using N. benthamiana as the host plant is shown in Table 2-2.
The current values for parameters used to establish and justify the target Specification for N. benthamianaproduced THAUMATIN II sweetener containing Thaumatin II as the active ingredient are shown in Table B-1.
The Specification limits for alkaloid impurities in the final product shown in Table B-1 were derived fromanalyses of two bench-scale and one pilot-scale Thaumatin II batches (discussed in APPENDIX C, Section C-5).

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANAThe Specification limits for lead (Pb) and the sum of Class 1 (Pb, Cd, Hg, As) elemental impurities were alsoestablished from results of analyses of two bench-scale developmental batches and one pilot-scale
manufacturing batch (APPENDIX C, Section C-6). Limits for the less toxic Class 2 and Class 3 metals were notincluded in the Specification because analyses of the pilot-scale manufacturing batch yielded very low levelsof these impurities (i.e. sum of Ag, Bi, Cu, Mo, Sb and Sn = < 1 ng total metal/mg Thaumatin II protein).
Table B-1. Justification of Specification Release Limits for THAUMATIN II ProductSummary of current THAUMATIN II Sweetener Conformance with Target Specification
Parameter

MethodSpecification limit

Results of analyses 1

Appearance

VisualPowder, yellowish-lt. tan

Conforms

Minimum total thaumatin content

CGE> 95% of total protein

Ave: 97.7 ± 1.19%
Range: 96.1% – 99.4%

Solubility (DI water)

Visual> 200 mg/mL

Conforms

pH of a 1% solution in water

Potentiometric

2.7 – 6.0

Conforms

Heavy metals (sum of Class 1 metals
Pb, Cd, Hg, As) 2

USP38<233>< 5 ng/mg thaumatin

Ave: 0.232 ± 0.048 ng/mg

Lead 2

USP38<233< 1 ng/mg thaumatin

Ave: 0.126 ± 0.22 ng/mg

HPLC/MS< 20 ng/mg thaumatin

Ave: 14.41 ± 0.67 ng/mg

HPLC/MS< 5 ng/mg thaumatin

Ave: 2.52 ± 1.55 ng/mg

Bioburden

USP32<61>< 5,000 CFU total per g

0 (absent)

Agrobacterium (CFU/10 g sample)

Selective platebased assay

0 (absent)

0 (absent)

Undesirable microorganisms:
Escherichia coli, Pseudomonas
aeruginosa, Salmonella spp. or
coagulase-positive Staphylococcus
spp., per 25 g final product

USP32<1111>

0 (absent)

0 (absent)

Stability of dry product (0-20°C) 3 as
<3% loss of thaumatin protein

CGE; THM peak at
Tm vs. T0> 6 months> 11 months (at time of
GRN submission)

Nicotine (per total thaumatin)

2

Anabasine (per total thaumatin)

1

2

Results of analyses of dry, purified Thaumatin II protein were obtained from three non-consecutive batches. Methods ofanalysis and results to support the Specification are discussed in APPENDIX C.

2Elemental and alkaloid impurity Specification limits were derived from results of analyses of 3 non-consecutive developmental
batches, including two bench-scale batches and one pilot-scale manufacturing batch.

3

Stability results were interim at the time of submission and were based on samples in an on-going stability program. Stability
is calculated from Thaumatin II protein peak determined by CGE for 1% (w/v) Thaumatin II solution in water at T0 and at various
durations of storage expressed in months (Tm) using samples from a minimum of 3 non-consecutive developmental batches.
Product is considered stable if there is <3% loss of Thaumatin II protein at the time of sampling.

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
Thaumatin II was stable during storage under the conditions indicated. The percent purity values shown in
Table C-1 are averages of two replicate analyses. The active ingredient in THAUMATIN II sweetener,
Thaumatin II, was found stable for 12 months when stored at 4°C, with less than 3% loss of purity over thatstorage time. Some batches of Thaumatin II were also stable at room temperature (RT) for up to 11 months.
Generally, cold storage (4°C-10°C) is expected to provide greater stability, hence enabling longer duration of
product storage. Storage conditions will continue to be defined and optimized during product development.
Table C-1. Stability of Thaumatin II during storage
Thaumatin II Purity During Storage
Protein

Storage at 4°C

Storage at RT (~22°C)

Time of storage (months)

Time of storage (months)

0

9

11

12

0

9

11

12

Thaumatin II
Batch 5

98.0

96.15

Batch 17

96.2

95.35

Batch 31

99.4

96.15

96.2
99.4

94.85
94.55

Thaumatin protein purity (thaumatin protein as percent of total protein) was maintained when dry protein powders
were stored over prolonged periods. None of the samples of Thaumatin II showed degradation fragments oraggregation upon storage at either 4°C or at room temperature (~22°C).

C.4. Manufacturing Yield and Consistency
The manufacturing yield, consistency, and product purity were determined from multiple non-sequential
batches of Thaumatin II produced during product and process development at bench scale. Consistency is
expected to increase as the process is upgraded and optimized at pilot-scale and eventually at industrial scale.
Table C-2 summarizes multi-batch yield and purity of N. nicotiana-produced Thaumatin II.
Table C-2. Consistency of production yield and purity of Thaumatin II

ProteinYield of purified protein(mg/kg FW plant biomass)
SinglebatchesAve. ± SD

Number nonconsecutive
batchesanalyzed

Thaumatin purity(% of total protein)SinglebatchesAve. ± SD

Number nonconsecutive
batchesanalyzed

97.8 ± 1.55

4

-

1

710
240
Thaumatin II(bench-scale)

420
470

98.0
550 ± 160

6

540

96.1
97.3
99.4

610
Thaumatin II(pilot-scale)

117

-

1

98.2

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
Table C-2 lists results of analyses of individual production batches at developmental (bench) scale. The yieldof purified protein per unit of biomass processed (fresh weight; FW) was determined for each batch, as wellas the purify of the isolated protein obtained. Results from 6 independent production batches of Thaumatin
II show the recoverable yield of protein and the resultant protein purity. In addition to data from bench-scale
developmental studies, data from one pilot-scale run for Thaumatin II are included in Table C-2. Processscale-up focused on purity first, and results suggest that the purity release criterion of >95% can be achievedat larger scale.

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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA

C.9.

Bioburden and Residual Vector

The bioburden level and level of residual Agrobacterium vector was determined for N. nicotiana-produced
Thaumatin II from non-consecutive batches. Results shown in Table C-8 demonstrate that the isolates had nobioburden or residual Agrobacterium vector (inoculum), therefore meeting the Specification.
Table C-8. Multi-batch analyses of bioburden and residual Agrobacterium inoculum
Batch1

Bioburden (CFU/g Thaumatin II; average of 3 replicates per batch)2
Total aerobic microbial
content

Total combined yeast
and mold (TYMC)

Total residual
Agrobacterium(inoculum) content

Batch A000Batch B000Batch C000Batches analyzed were obtained from non-consecutive production runs
Thaumatin II solution at 1 mg/ml stock, 100 µl aliquots plated in 3 replicates on different
media. Total aerobic microbial count (TAMC) was determined in Soybean-Casein Digest
Agar (Roth #X937.1) plates incubated at 33°C for 3 days. Total combined yeast and molds
count (TYMC) was determined in Sabouraud-Glucose Agar (Roth #932.1) plates incubatedat 20-25°C for 6 days. Residual Agrobacterium inoculum was determined in LB-Bacto Agar
+ 50 µg/ml rifampicin plates incubated at 28°C for 2 days. Agrobacteria containing binary
plasmid were enumerated in plates of LB-Bacto Agar + 50 µg/ml rifampicin and 50 µg/ml
kanamycin incubated at 28°C for 2 days.

1
2

C.10. Summary
APPENDIX C summarizes the various methods used to characterize Notifier’s Thaumatin II protein expressed
recombinantly in the plant host Nicotiana benthamiana. The sweet-tasting thaumatin protein that comprise
Notifier's THAUMATIN II product is expressed with signal peptide to enhance its accumulation in plant tissue
and facilitate isolation. The precursor protein is correctly processed in planta post-translationally to produce
the final mature Thaumatin II protein.
All three methods described in GRN 738 (APPENDIX C, pp 47-51) for purifying thaumatins from edible plantbiomass (heat/acid-induced precipitation, cool ambient temperature chromatography, or tartrate-induced
crystallization) yielded highly pure product (95-98%). Cool neutral extraction followed by cation-exchange
chromatography applied to N. benthamiana-produced process stream yielded similarly pure protein (~9598%). The batch-to-batch yield in developmental studies has been generally reproducible and the process
generates proteins of consistent quality. Improvements in overall yield and quality are expected as theprocess is scaled and optimized, as suggested by results of a pilot-scale manufacturing run with Thaumatin II.
The amino acid composition, amino acid sequence, and molecular mass of the resultant protein is identicalto the corresponding thaumatins II found in Thaumatococcus daniellii, which forms the basis of currently
marketed thaumatin products (GRN 738; this Notice).
The methods used to characterize thaumatins, summarized in GRN 738 and in this section of the Notice, areindustry standard and are validated and conducted by licensed external analytical laboratories. Other release
tests (e.g. pH with various excipients) are applied to each batch of Thaumatin II (not shown here) to ensure
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THAUMATIN II SWEETENER FROM NICOTIANA BENTHAMIANA
that the product meets its specification. Purified Thaumatin II protein is stable for >6 months in storage as
dry powders and are also stable (>3 months) when mixed and stored with sucrose. The stability program isin progress and longer stable-storage times should be supported by subsequent results.
Notifier’s purification process is undergoing optimization during scale up. The residual host-derived alkaloid
impurities in Thaumatin II are consistently low and preliminary results suggest that they can be further
reduced during purification at larger (i.e. pilot) manufacturing scale, which employs more rigorous processcontrols. Even at the current level of total alkaloids, these impurities do not constitute a health risk due totheir low concentration and the low level of ingestion of thaumatin proteins in the diet. Likewise, the levelsof elemental impurities in Thaumatin II are not considered a health risk due to their low concentrations andthe low per capita consumption of thaumatin.
It is important to note that thaumatin products approved as food additives in multiple countries are not 100%pure thaumatin, and contain a number of host- and process-derived impurities as well, including extraneous
protein and heavy metals (Lim 2012; Cusack 1991; Stephen 1991; Adesina 1977; Adesina 1978). Regardless,the safety of the commercial products has been conclusively shown and, by comparison, the safety ofNotifier's final product has also been affirmed (see Section 6.1 (overall safety), Section 6.1.1 (allergenic
potential), and Section 6.2 (safety in relation to dietary intake).
The sweetness intensity, threshold of detection and other flavor characteristics, as well as the sugar-replacing
potential of Notifier’s Thaumatin II, have been defined under controlled conditions in three sensory
evaluation studies conducted to date. These studies have determined that sucrose and potentially othercaloric sweeteners of similar intensity (e.g., corn syrup) used in food and beverages could be replaced at up
to 50 wt% with Notifier’s Thaumatin II while retaining generally the same perceived level of sweetness. The
functionality of Notifier’s Thaumatin II as a high-intensity sweetener has therefore been verified.
Importantly, consumption of these proteins at the projected rates of application to achieve sweetening is
expected to be safe with respect to the intake of thaumatin itself and any residual host or process-derived
impurities remaining in the final THAUMATIN II product.

END OF NOTICE.

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