EPA BIOPESTICIDES AND POLLUTION PREVENTION DIVISION 

COMPANY NOTICE OF FILING FOR PESTICIDE PETITIONS PUBLISHED IN 

THE FEDERAL REGISTER 

(DRAFT July__, 2014) 

EPA Biopesticides and Pollution Prevention Division contact: [Kenneth Haymes, 

Ph.D. 703-347-0398] 

INSTRUCTIONS: Please utilize this outline in preparing the pesticide petition. In 

cases where the outline element does not apply, please insert “NA-Remove” and 

maintain the outline. Please do not change the margins, font, or format in your 

pesticide petition. Simply replace the instructions that appear in green, i.e., “[insert 

company name],” with the information specific to your action.

SUBMISSION: E-mail the completed template to: duggard.mari@epa.gov.

TEMPLATE: 

Southern Gardens Citrus Nursery, LLC, 1820 County Road 833, Clewiston, FL 33440

PP 4F8289

 EPA has received a pesticide petition (XXXXXX) from Southern Gardens Citrus 

Nursery, LLC, 1820 County Road 833, Clewiston, FL 33440 proposing, pursuant to 

section 408(d) of the Federal Food, Drug, and Cosmetic Act (FFDCA), 21 U.S.C. 

346a(d), to amend 40 CFR part 180

 1. by establishing a tolerance for residues of NA-Remove

 2. to establish an exemption from the requirement of a tolerance for the 

3. to establish an amendment/expansion of an existing tolerance exemption for the

NA-Remove

 1. microbial pesticide Genetically Engineered Citrus tristeza virus expressing 

Spinach Defensin Proteins 2, 7 and 8 in or on Citrus Fruits (Citrus Spp., Fortunella 

Spp., Crop Group 10-10; 40 CFR Section 180.41(c)(12)(13)).

 2. biochemical pesticide NA-Remove

3. plant-pesticide NA-Remove

Pursuant to section 408(d)(2)(A)(i) of FFDCA, as amended, Southern Gardens 
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Citrus Nursery, LLC has submitted the following summary of information, data, and 

arguments in support of their pesticide petition. This summary was prepared by Southern 

Gardens Citrus Nursery, LLC and EPA has not fully evaluated the merits of the pesticide 

petition. The summary may have been edited by EPA if the terminology used was 

unclear, the summary contained extraneous material, or the summary unintentionally 

made the reader conclude that the findings reflected EPA’s position and not the position 

of the petitioner. 

I. Southern Gardens Citrus Nursery, LLC Petition Summary

PP XXXXXX

A. Product Name and Proposed Use Practices

The product, genetically engineered Citrus tristeza virus expressing defensin proteins 2, 7 

and 8 from spinach (Spinacia oleracea) (GE CTV-SoD) has been incorporated into citrus 

trees. Spinach defensin proteins have been shown to be effective in the control of citrus 

greening (CG) also known as Huanglongbing (HLB). GE CTV-SoD is inoculated into 

citrus trees already diseased with HLB, in an effort to restore tree health; or, is intended 

to prevent HLB by inoculating healthy trees with this modified CTV vector prior to 

putting them in the field. Virus-based transient-expression vectors are routine tools used in 

plant molecular biology laboratories throughout the world for rapidly expressing or 

silencing genes in plants.

B. Product Identity/Chemistry 

 1. Identity of the pesticide and corresponding residues. The pesticide and 

corresponding residues are identified as spinach defensin proteins 2, 7 and 8, expressed 

by genetically engineered Citrus tristeza virus. Defensin proteins are ubiquitous in plants 

and are known to exhibit antifungal and antibacterial activity. Plant defensins are 

understood to act by binding to receptors on the membrane of plant pathogens and 

thereby causing the integrity of the host membrane to become compromised, leading to 

cell disruption and death. 

2. Magnitude of residues at the time of harvest and method used to determine the 

residue. Preliminary research indicates that spinach defensin proteins 2, 7 and 8, 

expressed in genetically engineered CTV, are anticipated to occur at very low levels in 

citrus tissues (reference report ID SGC-039 submitted with this application). Western blot 

method is the current Analytical Enforcement Method on file with the EPA for spinach 

defensin protein (MRID 49409303). 

C. Mammalian Toxicological Profile. Spinach defensins are naturally occurring proteins 

with a long history of safe use in mammalian dietary consumption of food (spinach). 

These proteins have no known deleterious human health effects or any evidence of 

toxicity to people, wildlife, or beneficial insects. 

Spinach defensin proteins are derived from a food plant (spinach) that has a long history of 
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safe use in the human diet. EPA has concluded (Memorandum, March 19, 2015, from J. 

Facey to K. Haymes) that neither SoD2 nor SoD7 have amino acid sequence similarities to 

known allergenic or toxic proteins, and that SoD2 and SoD7 are degraded in simulated 

gastric fluid. Defensin 8 (SoD8) from spinach is similar to SoD2 and SoD7, and is likewise 

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

with this application). Additional studies (report IDs SGC-040 and SGC-041, also submitted 

in support of this application) on the bioinformatics of these spinach defensins show no 

relationship to any known toxin or allergen. 

The genetic material necessary for the production of the GE CTV-SoD proteins are 

nucleic acids (DNA) which are common to all forms of plant and animal life. There are 

no known instances in which nucleic acids have caused toxic effects as a result of 

dietary exposure. 

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

respectively, are exempted from the requirement of a tolerance (on a temporary basis) when 

expressed in modified (rDNA) citrus trees (40 CFR Part 174.535). 

Citrus tristeza virus is endemic throughout the Florida citrus 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 viral proteins is common in the diet. Plant viruses 

are not pathogenic to humans. Human exposure to a variety of natural plant viruses and plant 

viral proteins is common in the diet. Plant viruses are not pathogenic to humans. Indeed, the 

human gastrointestinal 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. 

Collectively, the available data on spinach defensin proteins and CTV, along with the 

history of safe consumption of spinach and citrus, establish the safety of the plant 

pesticides GE CTV-SoD proteins and the genetic material necessary for their production 

in all citrus fruit commodities. 

D. Aggregate Exposure

 1. Dietary exposure. 

 i. Food

Significant dietary exposure to GE CTV expressing spinach defensin proteins 2, 7 and 8 

is unlikely to occur due to the anticipated low expression of the defensin proteins from 

the vector. Dietary exposure to GE CTV is not expected to be greater than exposure to 

wild type CTV in citrus products. Dietary exposure to spinach defensins, to the degree 

they may occur via ingestion of fresh and processed citrus commodities, will be at levels 

far below existing dietary exposure to these proteins from regular consumption of 

spinach. Moreover, such exposures are unlikely to be problematic because of the low 

toxicity and the high degree of digestibility of the proteins. 

 
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ii. Drinking water 

The spinach defensin proteins are not likely to be present in drinking water because the 

proteins are only present in minute quantities within the citrus plant. GE CTV is not 

expected to be present in drinking water in amounts greater than the wild type CTV 

which is ubiquitous in Florida citrus. 

2. Non-dietary exposure 

GE CTV expressing spinach defensin proteins 2, 7 and 8 are located in citrus phloem. 

Expressed defensins bind tightly in plant tissue within the plant matrix. Due to their 

location in citrus trees, availability for any dermal or respiratory exposure is reduced. 

E. Cumulative Effects

Common modes of toxicity are not relevant to consideration of the cumulative exposure 

to GE CTV-SoD. The product has demonstrated low mammalian toxicity and there are 

currently no EPA-registered pesticide products containing defensin proteins. 

Furthermore, there exists a long and documented history of safe use and consumption of 

the naturally occurring plant virus and spinach proteins such that biological effects do not 

appear to be cumulative with any other known compounds. 

F. Safety Determination

 1. U.S. population. There is a long history of human consumption of spinach 

plants (both raw and cooked), without causing any known deleterious human health 

effects or any evidence of toxicity. On the contrary, spinach is commonly regarded as a 

“super food” that serves as an excellent source of vitamins, minerals, and antioxidants. 

[“Food $ense,” Utah State Univ. Cooperative Extension (Apr. 2011) (available online at: 

http://extension.usu.edu/files/publications/publication/FN_Food$ense_2100-08pr.pdf).] 

Recent U.S. consumption statistics indicate that, on average, 2 lbs. of spinach are 

consumed per person per year in the United States. [“Spinach Profile,” Agricultural 

Marketing Resource Center (June 2013) 

(http://www.agmrc.org/commodities__products/vegetables/spinach-profile/).] 

The U.S. human population has been exposed to CTV in citrus products for at least two 

decades since its discovery as widespread in the Florida citrus industry in the mid 1990s. 

No adverse effects from this exposure in humans have been reported. 

Non-occupational exposure such as drinking water exposure is minimal to non-existent 

since the genes are only expressed within plant tissues. GE CTV expressing spinach 

defensin proteins, does not represent a source of novel potential allergenic or antinutrient 

proteins. Finally, the proteins will be expressed at very low levels. 

 2. Infants and children. Based on the lack of demonstrated toxicity, absence of 

allergenic potential, the high degree of digestibility of spinach defensin proteins and their 
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documented history of safe use, dietary exposure is anticipated to be at very low levels, 

indicating with reasonable certainty that no harm to infants, children or adults will result 

from aggregate exposure to these expressed proteins. Exempting GE CTV expressing 

spinach defensin proteins 2, 7 and 8 from the requirement of a tolerance should pose no 

significant risk to humans or the environment.

G. Effects on the Immune and Endocrine Systems 

Given the rapid digestibility of spinach defensin proteins, no chronic effects are 

expected. GE CTV expressing spinach defensin proteins 2, 7 and 8 (or degradate 

products of these proteins) are not known, nor expected, to have any effect on the 

immune or endocrine systems. Proteins in general are not carcinogenic, therefore, no 

carcinogenic risk is associated with spinach defensin proteins.

H. Existing Tolerances

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

SoD7, respectively, are exempted from the requirement of a tolerance at 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 are temporarily exempt from the requirement of a 

tolerance when used as a plant-incorporated protectant in citrus plants in accordance with 

the terms of Experimental Use Permit No. 88232–EUP–1. This temporary exemption 

from the requirement of a tolerance expires on April 18, 2018. 

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

or on citrus food commodities are temporarily exempt from the requirement of a 

tolerance when used as a plant-incorporated protectant in citrus plants in accordance with 

the terms of Experimental Use Permit No. 88232–EUP–1. This temporary exemption 

from the requirement of a tolerance expires on April 18, 2018. 

I. International Tolerances

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