EPA BIOPESTICIDES AND POLLUTI ON PREVENTION DIVISION

COMPANY NOTICE OF FILING FOR PE STICIDE 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 p reparing the pesticide petition. In

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

maintain the outline. Please do not chan ge 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 temp late 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, a nd 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

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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 Gardens2

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 ed ited by EPA if the terminology used was unclear, the summary contained extraneous material, or the su mmary 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 in corporated into citrus trees. Spinach defensin proteins have been show n to be effective in the control of citrus greening (CG) also known as Huanglongbing (H LB). 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 laborat ories throughout the world for rapidly expressing or silencing genes in plants.

B. Product Identity/Chemistry

1. Identity of the pestic ide and corresponding residues. The pesticide and corresponding residues ar e 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 an tibacterial activ ity. Plant defensins are understood to act by binding to receptors on the membrane of plant pathogens and thereby causing the integrity of the host me mbrane to become compromised, leading to cell disruption and death.

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

residue. Preliminary research indicates that spinach defensin proteins 2, 7 and 8, expressed in genetically engine ered CTV, are anticipated to occur at very low levels in citrus tissues (reference report ID SGC-039 subm itted with this application). Western blot method is the current Analytical Enforcemen t 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 mammalia n dietary consumption of food (spinach). These proteins have no known deleterious hum an health effects or any evidence of toxicity to people, wildlife , or beneficial insects.

Spinach defensin proteins are derived from a fo od plant (spinach) that has a long history of3

safe use in the human diet. EPA has c oncluded (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 hum an digestive tract (report ID SGC-042 submitted with this application). A dditional studies (report IDs SGC-040 and SGC-041, also submitted in support of this application) on the bioinf ormatics of these spinach defensins show no relationship to any know n toxin or allergen.

The genetic material necessary for the pr oduction 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 ha ve 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) citr us 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 viruse s and plant viral proteins is co mmon 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 th e diet. Plant viruses are not pa thogenic to humans. Indeed, the human gastrointestinal tract har bors a wide variety of plant viru ses within the intestines. There are no reports of adverse effects from CTV exposures by any r oute 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 gene tic 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 de fensin proteins 2, 7 and 8 is unlikely to occur due to the anticipated lo w expression of the defe nsin 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 e xposure 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 thes e proteins from regular consumption of spinach. Moreover, such exposures are unlik ely to be problematic because of the low toxicity and the high degree of di gestibility of the proteins.4

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 grea ter than the wild type CTV which is ubiquitous in Florida citrus.

2. Non-dietary exposure

GE CTV expressing spinach defe nsin proteins 2, 7 and 8 ar e located in citrus phloem. Expressed defensins bind tightly in plant ti ssue within the plant ma trix. 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 considerati on of the cumulative exposure to GE CTV-SoD. The product has demonstrat ed low mammalian toxicity and there are currently no EPA-registered pesticide pr oducts containing defensin proteins. Furthermore, there exists a long and documente d history of safe use and consumption of the naturally occurring plant virus and spinach proteins such that bi ological 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 hum an 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 sour ce of vitamins, minerals, and antioxidants. [“Food $ense,” Utah State Univ. Cooperative Extension (Apr. 2011) (a vailable online at: http://extension.usu.edu/files/publicati ons/publication/FN_Food$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 Pr ofile,” 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 exposur e 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 allerg enic or antinutrient proteins. Finally, the prot eins will be expresse d at very low levels.

2. Infants and children

. Based on the lack of demonstr ated toxicity, absence of allergenic potential, the high de gree of digestibility of spinac h defensin proteins and their5

documented history of safe use, dietary exposur e is anticipated to be at very low levels, indicating with reasonable certai nty that no harm to infants, children or adults will result from aggregate exposure to these expresse d proteins. Exempti ng GE CTV expressing spinach defensin proteins 2, 7 and 8 from th e 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 de fensin 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 carci nogenic, therefore, no carcinogenic risk is associated wi th spinach defensin proteins.

H. Existing Tolerances

Residues of the plant incorporated-protect ants 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 fr om the requirement of a tolerance when used as a plant-incorporated protectant in citrus pl ants in accordance with the terms of Experimental Use Permit No. 88232–EUP–1. This temporary exemption from the requirement of a tole rance 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 fr om the requirement of a tolerance when used as a plant-incorporated protectant in citrus pl ants in accordance with the terms of Experimental Use Permit No. 88232–EUP–1. This temporary exemption from the requirement of a to lerance expires on April 18, 2018.

I. International Tolerances

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