UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON; D.C. 20460 

March 31 , 2015 

OFFICE OF CHEMICAL SAFETY 

AND POLLUTION PREVENTION 

MEMORANDUM 

SUBJECT: 

FROM: 

THROUGH: 

TO: 

EPA Reg. N2: 88232-EUP-R 

PC Code: UNKNOWN 

MRID: 494093-03 

Decision N2: 492314 

DP N2: 423571 

Submission N9: 958820 //!} JhJ /;j 

Kathleen Martin, MSES; Chemist / 5 

Microbial Pesticides Branch 

Biopesticides and Pollution Prevention Division 

Alan Reynolds, MS, Team Leader /I} (J 

Microbial Pesticides Branch ~ (1-----

Biopesticides and Pollution Prevention Division 

Ken Haymes, PhD, Regulatory Action Leader 

Microbial Pesticides Branch 

Biopesticides and Pollution Prevention Division 

Action Requested. Southern Gardens is requesting an experimental use permit (EUP) to test 

Defensin 2 (SoD2) and Defensin 7 (SoD?), two plant-incorporated protectants (PIPs) that may confer 

citrus plants to confer disease resistance in citrus plants. Under this EUP, study investigators expect to 

use a maximum of 75 kilograms of protein across 600 acres in Florida and Texas. The attached Data 

Evaluation Record (DER) provides the Section G review. 
Data Evaluation Record 

Reviewed by: 

Secondary Review: 

Kathleen Martin, M.S.E.S. 

Alan Reynolds, M.S. 

Study type: Section G Calculations 

Data 

Requirement: Not Applicable 

Date: March 26, 2015 

Test OPPTS 885.2300 (Microbial Test Guidelines: Analytical Methods- Plants) 

Guideline: and OPPTS 885.2500 (Microbial Pesticide Test Guidelines: Magnitude of 

Residues in Plants 

MRID Reviewed: 494093-03 (amended; see Southern Gardens 2014) 

DP Barcode: 423571 

Decision N2: 492314 

Submission: 958820 

Test Disease Resistant Citrus Expressed Proteins-Spinach 

Material: Defensin 2 and 7 (SoD2 and SoD7) 

Report Expression of Spinach Defensin 2 and 7 Proteins in Leaf Tissue of Citrus 

Title: Events SGC-11208-5, SGC-41108-7, SGC-41332-6 and SGC-11319-8 

(amended; see Southern Gardens 201 4) 

Author: James E. Strickler, Ph.D. 

Sponsor: Southern Gardens Citrus Nursery, LLC 

Testing 

Facility: Citrus growing sites in Florida and Texas 

Project ID: Laboratory (SG14-164.01); 

Sponsor Study Number (SGC-TALR 07) 

Study 

Date: June 12, 2014 

Receipt June 17, 2014 

Date: 

Confidentiality None 

Claims: 

Good 

Laboratory NA 

Practice: 

Study Summary The requested number of acres reasonable and the total amount of protein 

and Conclusion: expressed by the four events is well-described. 

Deficiencies: None 

Classification: ACCEPTABLE 
Southern Gardens is requesting an experimental use permit (EUP) to test a new 

plant-incorporated protectants (PIP's) that may confer disease resistance to citrus plants. In total, 

Southern Gardens is requesting 720 acres in Florida and Texas (600 PIP and 120 border) to test 

four insertion events (SGC-411 08-7, SGC-41332-6, SGC-11319-8, and SGC-11208-5) that produce 

two proteins (SoD2, SoD7, or SoD2, SoD7); the maximum amount of protein is expected to be no 

greater than 75 kg. The events, their expressed proteins, and type of citrus are listed in Table 1: 

T a e bl 1 E ven t s, P ro t ems . , an dT Ype o l fc1trus · 

Event Proteina Citrus Type 

SGC-411 08-7 SoD2 sweet orange 

SGC-41332-6 SoD2, SoD7 sweet orange 

SGC-11 319-8 SoD2, SoD7 lemon 

SGC-11208-5 SoD7 grapefruit 

asoD2-Defensln 2; SoD7=Defens~n 7 

TESTING LOCATIONS 

Southern Gardens plans to conduct their study in one or more locations in Hendry County, 

Florida, and Hidalgo County, Texas. Testing will be conducted at both locations to generate 

agronomic, efficacy and regulatory data and information. 

ACRES 

In total, Southern Gardens intends to plant a maximum of 720 acres at test sites in 

Florida and Texas. Of the 720 acres, 600 are expected to be PIP acres and 120 border. These are 

tabulated in Table 2: 

Table 2 Summary of Acres by Event, Type (PIP, Border), and Location 

SGC-41108-7 

Hendry County, FL 

Hidalgo County, TX 

Total PIP· 

Total Border: 

PIP 

100 

50 

150 

AMOUNT OF PROTEIN: 

Border 

20 

10 

30 

SGC-41332-6 

PIP Border 

100 20 

50 10 

150 

30 

SGC-11319-8 SGC-11208-5 TOTAL 

PIP Border PIP Border PIP Border 

100 20 100 20 400 80 

50 10 50 10 200 40 

150 150 600 

30 30 120 

To measure the amount of PIP protein in the leaf material of trees, study investigators used the 

Western Blot method which yielded concentrations of ~ 0.2 to more than 20 mg/kg for each of the four 

events. Southern Gardens considered these results to be semi-quantitative due to problems 

encountered in the quantitative analyses. They believe that the true concentrations most likely range 

from 0.1 to 25 mg/mg. Provided in Table 4 is the total amount of protein (kg) expected in the leaf 

material over the duration of the study; the assumptions used are in Table 3 and the calculation is at 

Equation 1. 

DER-2 
Table 3 

Planting 

Density 

Lea f 

Materia 

per Tree 

I 

%Active 

Ingredient 

Assumptions Use d 1n t e a cu at1on . h C I I . 

Value Source 

250 Trees/A Research Director at Southern Gardens 

20 kg, dry weight Published literature (Morgan et al 2006) 

<1 to >20 mg/kg Measured 

Equation 1 Total Amount of Protein Expected in Leaf Material Over the Duration of the Study 

(

Tre es) Amt Leaf Material 

Amt of Protein (kg)= Acres x Planting Density -- x T (kg, dry wgt) x% AI 

Acre ree 

For example, in Hendry County, FL: 

(

250 Trees) 20 kg leaf mg protein 

Amt of Protein (kg) = 100 Acres x x x% AI ( k ) Acre Tree g 

Table 4 Amount of Protein (kg) Expected in Leaf 

Mat en . a I 0 ver th e D ura f 1on o f th e St u d ly 

SGC- SGC- SGC- SGC- TOTAL 41108-7 41332-6 11319-8 11208-5 

Hendry County, FL : 10.6 0.9 5.6 0.15 17.3 

Hidalgo County, TX : 5.3 0.45 2.8 0.075 8.7 

Total 15.90 1.35 8.40 0.23 30.0 

Because of the uncertainties regarding the measured concentration of SoD2 and SoD? in the 

leaf material, Southern Gardens proposes a conservative approach, where the maximum concentration 

measured (i.e., >20 mg/kg) be rounded up to 25 mg/kg and this value used in the calculation for all four 

events. Under this scenario, the total amount of protein (kg) is expected to be 75 kg; see Table 5 for 

the amount for each event and county. 

Table 5 Amount of Protein Expected Assuming a 

Con centration o f 25 mq /k (~ f E h E or ac vent 

SGC- SGC- SGC- SGC- TOTAL 41108-7 41332-6 11319-8 11208-5 

Hendry County, FL 12.5 12.5 12.5 12.5 50.0 

Hidalgo County, TX : 6.25 6.25 6.25 6.25 25.0 

Total 18.75 18.75 18.75 18.75 75.0 

DER-3 
CONCLUSION: 

The number of PIP and border acres (600 and 120, respectively) across two counties, one each 

in Florida and Texas, is reasonable. EPA agrees with Southern Gardens' conservative approach in 

using 25 mg/kg to calculate the amount of protein in the leaf material. The problems encountered in 

quantifying the expected concentrations of SoD2 and SoD? are well-described and documented in the 

study and appear to be insurmountable. The study investigators explored alternate analytical methods 

such as using a different extraction method and re-analyzing the samples. 

Overall, the information and data provided in the submitted materials (i.e., 494093-03 and 

Southern Gardens 2014) was well-described, easy-to-follow, and nicely documented. For example, 

appropriate justification (e.g., citing published literature) was provided for all the assumptions. 

Regarding the limitations encountered in quantify the amount of protein per event, study investigators 

provided a thorough discussion on their methodology and how they attempted to ameliorate the 

problems. 

REFERENCES 

Morgan et al. 2006. Size, Biomass and Nitrogen Relationships with Sweet Orange Tree Growth. 

J. Amer. Soc. Hort. Sci. 131 (1):149-156. 

Southern Gardens. 2014. Letter from Vickie Foster, Southern Gardens Citrus to U.S. Environmental 

Protection Agency. Information Submitted in Accordance with FIFRA Section 5 and 40 CFR 172: 

Revised Section G of 88232-EUP-R (sublabel acreage added). October 3, 2014. 

DER-4