PETITION FOR TOLERANCE EXEMPTION 

Genetica lly Engineered Citrus tristeza l'irus ex1>ressing Spinach Defensin Proteins 2, 7 a nd 8 (G E CTY-SoD) 

Southern Gardens Citrus Nursery, LLC 

October 20 15 

Genetically Engineer ed Citrus tristeza virus Expressing Spinach Defensin Proteins 2, 7 

a nd 8 (GE CTV-SoD) in Citrus Fruits (Crop G r oup 10-10): Petition for Establishment of 

an Exemption from the Requirement of a Tolerance on a Temporary Basis 

Prepared by 

Vickie A. Forster, Principal 

Forster & Associates Consulting, LLC 

For, Southern Gardens Citrus Nursery, LLC 

Date 

October 28, 20 15 

Submitted by 

Southern Gardens Citrus Nursery, LLC 

1820 County Road 833 

Clewiston, FL 33440 

Volume 1 of 21 

Administr ative Materials 

Page 1 of 13 
PETITIO:'\ FOR TOLERANCE EXE IPTJ O~ 

Genetically Enginee red Citrus tristeu1 1•ims expressing Spinach Defensin Proteins 2, 7 and 8 (GE CTV-SoD) 

Southern Ga rdens Citrus 'ursery, LLC 

October 20 I 5 

Introduction 

Spinach (Spinacia oleracea) dcfcnsin genes 2, 7 and 8 have been inserted into Citrus tristeza 

virus, isolate T36 of strain T36, a decl ine isolate nati ve to Florida. The genetically engineered 

CTY is incorporated into citrus trees via inoculation: A full-genome eDNA of CTV isolate T36 in binary plasmids was used to agroinfiltratc Nicotiana benthamiana leaves. When N. 

benthamiana plants became in fected, as determined by Northern blot ana lysis and ELISA, 

expressed sap of the tobacco plants was used to bark fl ap or stem slash inoculate citrus trees. 

Citrus trees initially inoculated as descri bed above, and subsequently determined to have been 

successfu lly infected with GE CTY expressing one or two spinach defensin proteins (GE 

CTY-SoD), have served as the source material for inoculating, via budwood grafting, citrus 

trees that are fie ld tested fo r resistance or tolerance - by reducing or eliminating disease 

symptoms- to citrus greening (CG), also known as Huanglongbing (HLB). 

Spinach has a long history of safe use in the human diet. Residues of spinach 6C;.f..:.ts:n 

proteins 2 and 7, So02 and So07, respectively, are exempted from the requircmer:t '),.a 

tolerance (on a temporary basis) when expressed in modified (rONA) citrus trees (40 CFR 

Part 174.535). Defcnsin 8 (So0 8) from spinach is similar to So02 and So07, and is likewise 

expec ted to be rapidly degraded in the human digestive tract (report lD SGC-042 st..b1.1itt..:d 

with this application). Additional studies included in support of the temporary t0lcr l'lCC 

exemption (report IDs SGC-040 and SGC-04 1) on the bioinformatics of these r..i.uc h 

dcfcnsins show no relationship to any known toxin or allergen. 

Human exposure to a variety of natural pl ant viruses and plant viral proteins is commort in the 

diet. Plant viruses arc not pathogenic to humans. Human exposure to a variety of natun~ phnt 

viruses and plant vira l proteins is common in th e diet. Plant viruses are not pathogenic to 

humans. The human gastrointestinal tract harbors a wide variety of plant viruses within the 

intestines. There arc no reports of adverse effects from CTV exposures by any route of 

admi nistration (Government of Australia, Dept. Agriculture and Fisheries, 20 15). 

This petition, to exempt from the requirement of a tolerance, genetically engineered Citrus 

tristeza virus expressing spinach dcfensin proteins 2, 7 and 8 (G E CTV -SoD) in citrus fru its 

(40 CFR 180.4 l(c) ( 13)), demonstrates the safety of these proteins based on their history of 

safe usc in commonly consumed foods and via lack of toxicity or all ergenic ity of spinach 

dcfcnsin proteins to humans and livestock animals. 

This petition contains data or information regarding the elements set fo rth in Section 408(a) of 

the FFDCA; however EPA has not fu lly evaluated the sufficiency of the submitted data at this 

time or whether the data supports granting of the exemption. 

Page 2 of 13 
PETITION FOR TOLERANCE EXEMPTION 

Genetically Engineered Citrus tristeza virus expressing Spinach Defensin Proteins 2, 7 and 8 (CE CTV-SoD) 

Southern Gardens Citrus Nursery, LLC 

October 20 15 

Contents 

Title Page .................................................................................................................................. . 

Introduction ................................................................................................................................ 2 

Section A: Name, Chemical Identity and Composition of the Active Ingredient: Spinach 

Defensin Proteins ..................................................................................................... 4 

Section 8: Amount, Frequency, and Timing of Expression of the Plant-IncorporatedProtectant ................................................................................................................. 6 

Section C: Reports of Investigations Made with Respect to the Safety of Spinach 

Defensin Proteins 

i. Matnmalian Toxicology ....................................................................................... 7 

Section D: Report on the Amount of Residue Found in Food and/or Feed Commodities 

and a Description of the Analytical Method ............................................................ 9 

Section E: Practical Method for Detecting and Measuring Food Residues and Methods tc r 

Removing Residues Which May Exceed Proposed Tolerances .................... ...... I 0 

Section F: Proposed Exemption from the Requirement of a Temporary Tolerance for 

Geneticall y Engineered Citrus tristeza virus expressing Spinach Defensin 

Proteins 2, 7 and 8 in Citrus Fruits (Crop Group 10- 1 0) ..................................... .. I I 

Section G: Reasonable Grounds in Support of the Petition ..................................................... 12 

Page 3 of 13 
PETITION FOR TOLERANCE EXEM PTION 

Genetica lly Engineered Citrus tristeza virus expressing Spinach Derensin Proteins 2, 7 and 8 (GE CTV-SoD) 

Southern Gardens Citrus Nursery, LLC 

October 2015 

Section A 

Name, Chemical Identity, and Composition of the Active Ingredient: 

Genetically Engineered Citrus tristeza virus Expressing 

Spinach Defensin Proteins 2, 7 and 8 

Chemical Name: N/ A 

CAS Numbers: 103220-14-0 (Plant Defensins (as a class) 

Formula: Amino Acid Sequences of Spinach De fens in Proteins 2, 7 and 8 are 

provided with this submissio n in Report ID SGC-039 

Molecular Weight: 4227 (SoD7) Daltons to 7099 (SoDS) Daltons 

Commo n Name: Genetically Engineered Citrus tristeza virus Exp•·essing Spinach 

Defensin Proteins 2, 7 and 8 

Code Number: N/A 

Trade Name: Genetically Engineered Citrus tristeza virus Expressing Spinach 

Defensin Proteins 2, 7 and 8 

Spinach (Spinacia oleracea) defensin genes 2, 7 and 8 have been inserted into Citrus tristeza 

virus, isolate T36 of strain T36, a decline isolate native to Florida. The gene tically engineered 

CTV is incorporated in to citrus trees via inoculation: A fu ll-genome eDNA ofCTV isola te T36 in binary plasmids was used to agroinfiltrate Nicotiana benthamiana leaves. When N. 

benthamiana plants became infected, as determined by Northern blot analysis and ELISA, 

expressed sap of the tobacco plants was used to bark flap or stem slash inoculate c itrus trees. 

Citrus trees initially inoculated as described above, and subsequently determined to have been 

successfully infected with GE CTV expressing one or two spinach defensin proteins (GE 

CTV -SoD), serve as the source ma terial for inoculating, via bud wood grafting, citrus trees 

that are fie ld tested for resistance or tolerance - by reducing or eliminating disease symptoms 

-to citrus greening (CG), also known as Huanglongbing (HLB). 

A list of the data and infonnation on the product characterization submitted with this petition 

to support the requested tole rance exemptio n are provided by reference on the fo llowing page. 

Page 4 of 13 
PETITION FO R TOLERANCE. EXEMPTION 

Genetically Engineered Citrus tristeza virus expressing Spinach Defensin Proteins 2, 7 a nd 8 (GE CT V-SoD) 

Southern Ga rdens Citrus Nursery, LLC 

October 20 15 

PRODUCT ANALYSIS METHOD OF S UPPORT 

Guideline/ 

OCSPP 

885. 1100 Product 

Identity 

885. 1200 Manufacturing 

Process 

885. 1300 Formation of 

Unintentional 

Ingredients 

885. 1400 Analysis of 

Samples 

885. 1500 Certification of 

Limits 

Study Title 

"Product Characterization of Genetically Engineered Citrus 

tristeza virus Expressing Spinach Defensin Proteins 2, 7 and 8 

(GE CTV-SoD), SGC-039". Volume 2 

"Product Characterization of Genetically Engineered Citrus 

tristeza virus Expressing Spinach Defensin Proteins 2, 7 and 8 

(GE CTV-SoD), SGC-039". Volume 2 

"Product Characterization of Geneticall y Engineered Citrus 

tristeza virus Expressing Spinach Defensin Proteins 2, 7 and 8 

(GE CTV-SoD), SGC-039". Volume 2 

" Product Characterization of Genetica lly Engineered Citrus 

tristeza virus Expressing Spinach Defensin Proteins 2, 7 and 8 

(GE CTY-SoD), SGC-039". Volume 2 

Confidentia l Statements of Formula, EPA Form 8570-4 

Volume 1 

Page 5 of 13 
PETITION FOR TOLERANCE EXEM PTION 

Genetically Engineered Citrus tristezCI virus expressing Spinach Defensin Proteins 2, 7 and 8 (GE CT V-SoD) 

Southern Gardens Citrus Nursery, LLC 

October 20 IS 

Section B 

Amount, Frequency, and Timing of Expression of the Genetically Engineered Microbial 

Pesticide Expressing Spinach Defensin Proteins 2, 7 and 8 

Draft Product labeling is included in Volume I of this submission. 

Page 6 of 13 
PETITION FOR TOLERANC E EXEMPTION 

Genetically Engineered Citrus triste:a vims expressing Spinach Defensin Proteins 2, 7 and 8 (GE CTV-SoD) 

Southern Gardens Citrus 'ursery, LLC 

October 20 I 5 

Section C 

Reports of the Investigations Made with Respect to the Safety of 

Spinach Defensin Proteins 2, 7 and 8 

i. Mammalian Toxicolo 

Spinach has a long history of safe usc in the human diet. Residues of spinach de fensin 

prote ins 2 and 7, SoD2 and SoD7, respecti vely, a rc exempted from the requirement of a 

tolerance (on a temporary basis) when expressed in modified (rONA) c itrus trees (40 CFR 

Part 174.535). Defensin 8 (SoD8) from spinach is similar to SoD2 and SoD7, and is li kewise 

expected to be rapidly degraded in the human digestive tract (report JD SGC-042 submitted 

with this appli cation). Additional studies included in support of the temporary tolerance 

exemption (report IDs SGC-040 and SGC-041 ) on the bioinformatics o f these spinach 

de fcnsins show no re lationship to any known toxin or a llergen. 

Human exposure to a variety of natural plant viruses and plant vira l prote ins is common in the 

diet. Pl ant viruses arc not pathogenic to humans. Human exposure to a variety of natural plant 

viruses and plant vira l proteins is common in the die t. Plant viruses are no t pa thogenic to 

humans. The human gastrointestina l tract harbors a wide variety of plant viruses within the 

intestines. There are no reports of adverse effects from CTV exposures by any route of 

administration (Government of Austra lia, Dept. Agriculture and Fisheries, 20 15). 

A list of the data and information submitted with this petitio n to support this tole rance 

exemption is provided by reference be low. 

TOX ICOLOGY M ETHOD OF S UPPORT 

Guide line/ 

OCSPP 

885.3050 Ac ute oral toxicity -

waiver request 

885.3 100 Acute dem1al 

toxicity - waiver 

request 

885.3 150 Acute pulmonary 

toxic ity - wa iver 

request 

Study Title 

"Geneti cally Engineered Citrus tristeza virus Expressing 

Spinach Dcfcnsin Protei ns 2, 7 and 8 (GE CTV-SoD): 

Rationale for Not Conducting a G uide line Study - Acute O ra l 

Toxic ity/Pathogenicity" , SGC-043. Volume 3 

"Genetically Engineered Citrus tristeza virus Expressing 

Spinach De fens in Prote ins 2, 7 and 8 (GE CTV -SoD): 

Rationa le for Not Conducting a Guideline Study- Acute 

Dermal Toxic ity/Pathology", SGC-044. Volume 4 

"Geneti call y Engineered Citrus tristeza virus Expressing 

Spinach Dcfcnsin Proteins 2, 7 and 8 (GE CTV-SoD): 

Rationa le fo r Not Conducting a Guide line Study - Acute 

Pulmonary Toxic ity/Pathogenic ity", SGC-045. Volume 5 

Page 7 of 13 
PETITION FOR TOLERANCE EXEMPTION 

Genetically Engineered Citrus tristeza 1•irus expressing Spinach Defensin Proteins 2, 7 and 8 (GE CTV-SoD) 

Southern Gardens Citrus Nursery, LLC 

October 20 IS 

TOXICOLOGY METHOD OF SUPPORT, CON TIN UED 

Guide line/ 

OCSPP 

885.3200 Acute inj ection 

toxic ity - wai ver 

request 

885.3400 Hypersensiti vity 

inc idents - wa iver 

request 

885.3500 Cell Culturewa iver request 

Non- Stability of SoD8 in 

Guide line digestive fluids -

wa iver request 

Non- Bioinfo rmatics 

Guide line Search of All ergens 

Non- Bioinforma tics 

Guide line Search of Toxins 

Non- Evaluation of 

Guide line Stability of SoD8 

Study Title 

"Gene tically Engineered Citrus tristeza virus Expressing 

Spinach De fens in Prote ins 2, 7 and 8 (GE CTV -SoD): 

Rationa le for Not Conducting a Guide line Study - Acute 

Injection Toxicity/Pathogenic ity", SGC-046. Volume 6 

"Gene tically Engineered Citrus tristeza virus Ex pressing 

Spinach Defcnsin Prote ins 2, 7 and 8 (GE CTV -SoD): 

Rationa le for Not Conducting a Guide line Study -

Hypersensitivity Inc idents", SGC-047. Volume 7 

"Genetica lly Engineered Citrus tristeza virus Expressing 

Spinach De fens in Prote ins 2, 7 and 8 (GE CTV -SoD): 

Rationa le for Not Conducting a Guide line Study - Ce ll 

Culture", SGC-048. Volume 8 

"Genetica lly Engineered Citrus tristeza virus Expressing 

Spinach Defensin Prote in 8 (GE CTV-SoD8): Ratio na le for 

Not Conducting a Digestibility Study in Simulated Gastric 

Fluid", SGC-049. Volume 9 

"Bioinfo rmatic Ana lysis of Po tentia l Allcrgenic ity of 

Dcfensin Prote ins 2, 7 and 8 from Spinach for usc in Citrus 

Fruits". Non-Guide line. SGC-040. Volume 10 

"Bioinformati c Ana lysis of Potentia l Toxic ity of Defensin 

Pro te ins 2, 7 and 8 from Spinach for usc in Citrus Fruits". 

SGC-04 1. Volume 11 

"Evaluation of the Similarities of Spinach De fens in SoDS 

Compa red to Those of SoD2 and SoD7 Proteins to Estimate 

the Digestibility of SoD8 for the Safety Assessment of 

Geneti ca lly Engineered Crops", SGC-042. Volume 12 

Page 8 of 13 
PETIT ION FOR TOLERA NCE EXE IPTIO:\' 

Genetically Engineered Citrus triste:a virus expressing Spinach Defensin Proteins 2, 7 and 8 (GE CTV-So D) 

Southern Ga rdens Citrus Nursery, LLC 

October 20 IS 

Section 0 

Report on the Amount of Residue Found in Food a nd/or Feed Commodities and a 

Description of the Analytical Method 

Preliminary research citrus samples (fruit and leaves) from trees infected with GE CTY-SoD2 

or GE CTY-SoD7 were shipped from Southern Ga rdens Citrus Nursery in Clewiston, FL, to 

LifeSensors, Inc., Malvern, PA, to measure any detec table residues of SoD2 or SoD7 via 

western blot. Western blot method is the current Analytical Enforcement Method on file with 

the EPA for spinach defensin protein (MRID 49409303). Additional information on these 

analyses is provided in report ID SGC-039. 

The analytical enforcement method (western blot) has been reviewed previously and 

determined to be "Acceptable" by the Agency, MRID 49409303. 

RESIDUE METHOD OF SUPPORT 

Guideline/ 

OCSPP Study Title 

885.2300; Expression in "Expression of Spinach De fens in 2 and 7 Proteins in Leaf 

885.2500 Citrus LeafTissue Tissue of Citrus Events SGC- 11 208-5, SGC-4 11 08-7, SGC41332-6 and SGC- 11 319-8", SGC-008. MRID 49409303 

Page 9 of 13 
PETITIO:\' FOR TOLERAi\CE EXEMPTION 

Genetica lly Engineered Citms riste~a vims expressing Spinach Defen sin Proteins 2, 7 a nd 8 (GE CTV- oD) 

o uthern Gardens Citrus l\'ursery, LLC 

October 20 I 5 

Section E 

A Practical Method for Detecting and Measuring Food Residues and 

Methods for Removing Residues Which May Exceed Proposed Tolerances 

It is not practical, and there is no need for removal of residues of genetically engineered 

Citrus tristeza virus or residues of spinach defensin proteins 2, 7 and 8, from citrus tissues and 

commodities, as an exemption from the requirement of a tolerance i requested for these 

proteins when expressed in citrus. A tolerance is not needed to protect the public health. 

Page 10 of 13 
PETITIO~'\ FOR TOLERAI'\CE EXEM PTION 

Genetica lly Engineered Citrus triste~a 1>irus expressing Spinac h Defensin Proteins 2, 7 and 8 (GE CTV- oD) 

outhern Gardens Citrus Nursery, LLC 

October 20 15 

Section F 

Proposed Exemption from the Requirement of a Time-limited Tolerance for Spinach 

Defensin Proteins in Citrus Fruits (Crop Group 1 0-10) 

Southern Gardens Citrus Nursery, LLC, is requesting that the Agency grant, on a temporary 

basis, an exemption from the requirement of a tolerance for residues of genetically engineered 

Citrus tristeza virus expressing spinach dcfcnsin proteins 2, 7 and 8 deri ved from spinach 

(Spinacia oleracea) - GE CTY-SoD - in Citrus Fruits, Crop Group I 0- 10 (40 

CFR§ 180.4 1 (c)( 13)). 

Page It of l3 
PETIT ION FOR TOLERANCE EXEMPTION 

Genetically Engineered Citrus triste:a l'irus expressing Spinach Defensin Proteins 2, 7 and 8 (GE CTV-SoD) 

Southern Gardens Citrus Nursery, LLC 

October 20 IS 

Section G 

Reasonable Grounds in Support of the Petition 

Spinach dc fe nsin proteins arc deri ved from a food plant (spinach) that has a lo ng history of 

safe use in the human d iet. EPA has concluded (Memorandum, March 19, 2015, from J. 

Facey to K. Haymes) that ne ithe r SoD2 nor SoD7 have amino acid sequence simi larities to 

known allerge nic or toxic proteins, and that SoD2 and SoD7 are degraded in simu lated gastri c 

flu id. These prote ins have no known deleterious human health effects or any evidence of 

toxic ity to people, wildlife, or bene ficial insects. 

Citrus tristeza virus is endemic throughout the Florida c itrus growing regions. The 

engineered isolate, T36, is a ubiquitous decline isolate native to Florida. Human exposure to a 

variety of natural plant viruses and plant vira l proteins is common in the diet. Plant viruses 

are not pathogenic to huma ns. Indeed, the human gastrointestinal tract ha rbors a wide variety 

of plant viruses within the intestines. There arc no reports of adverse effects from CTV 

exposures by any route o f administration. 

The genetic material necessary for the production of the GE CTV-SoD proteins a re nucle ic 

acids (DNA) which are common to a ll forms of plant and anima l life. There are no known 

instances in wh ich nucleic acids have caused toxic effects as a result of dietary exposure. 

Significa nt die tary exposure to GE CTV expressing spinach de fensin pro teins 2, 7 and 8 is 

unl ikcly to occur due to the anticipa ted low expression of the de fcn sin prote ins from the 

vector. Dietary exposure to GE CTV is not expected to be g reate r than exposure to wild type 

CTV in c itrus products. Di etary exposure to spinach de fensins, to the degree they may occur 

via ingestion of fre sh and processed citrus commodities, wi ll be at levels far below existing 

dietary exposure to these prote ins from regu lar consumption of spinach. (Recent U.S. 

consumption da ta indicates a n average of 2 lbs. of spinach are consumed pe r person per year 

in the United States ("Spinach Profile", Agricultural Marketing Resource Center (June 20 13)). 

Moreover, such exposures are unlikely to be problema tic because o f the low toxic ity and the 

high degree of di gcstibil ity of the proteins. 

Non-occupationa l exposure to GE CTV -SoD via a route other than consumption o f fresh 

citrus and/or processed citrus commodities, such as drinking water, will be minimal to nonexistent since these proteins are expressed only with in plant tissues. 

Based on data and information submitted with th is petition, we ha ve conc luded tha t there is a 

reasonable certa inty that no harm will result to the United States population, including infants 

and children, from aggregate exposure to residues of these proteins. Exempting genetically 

eng ineered Citrus tristeza virus expressing spinach de fens in prote ins 2, 7 and 8 (GE CTVSoD) from th e requirement of a tolerance should pose no significa nt risk to humans or the 

environment. 

Page 12 of l3 
PETITION FOR TOLERANCE EXEM PTION 

Genetically Engineered Citrus tristeza virus expressing Spinach Defensin Proteins 2, 7 and 8 (GE CTV-SoD) 

Southern Ga rdens Citrus Nursery, LLC 

October 20 IS 

Residues of the plant incorporated-protectants spinach defensins 2 and 7, SoD2 and SoD7, 

respectively, a re exempted from the requ irement of a tolerance a t 40 CFR § 174.535 : 

§ 174.535 Spinach Defensin proteins; temporary exemption from the requirement 

of a tolerance. 

(a) Residues of the defensin protein SoD2 derived from spinach (Spinacia oleracea L.) in or 

on citrus food commodities arc temporarily exempt from the requ irement of a tole rance when 

used as a plant-incorporated protectant in c itrus plants in accordance with the tem1s of 

Experimental Use Permit No. 88232- EUP- 1. This temporary exemption from the 

requirement of a tolerance expires on Apri l 18, 20 18. 

(b) Residues of the defensin prote in SoD7 derived from spinach (Spinacia oleracea L.) in or 

on c itrus food commodities are temporarily exempt from the requirement of a tolerance when 

used as a plant-incorporated protectant in c itrus plants in accordance with the terms of 

Experimenta l Usc Pe rmit No. 88232- EUP- 1. This temporary exemption from the 

requirement of a tolerance expires on Apri I 18, 20 18. 

Page 13 of 13