### UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON ; D.C . 20460

March 31 , 2015 O F FICE OF C H EM I CAL SAFETY A ND P O LLUT IO N PREV ENTI ON



### MEMORANDUM SUBJECT:

FROM: THROUGH : 

### TO: E PA Reg . N2: 88232- EU P-R PC Cod e : UN KN OWN M RID : 4 94 093- 03 D ecision N2 : 4923 14 DP N2: 42357 1 S ubm iss i o n N9 : 958820 

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# JhJ /;j 5 Kathleen Martin , MSES ; Chemist / Microbial Pesticides Branch Biopesticides and Pollution Prevention Division Alan Reynolds , MS , Team Leader

## /I} (J Microbial Pesticides Branch

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## (1----- Biopesticides and Pollut i on Prevention Division Ken Ha y mes , PhD , Regulatory Action Leader Microbial Pest icides Branch Biopesticides and Pollution Prevention D ivision Action Requested . Southern Gard ens 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 ma ximum of 75 kilograms of protein across 600 acres in Florida and Texas . The attached Data Evaluation Record ( DER ) provi des the Section G review .

## Data Evaluation Record Reviewed by: Se condary Review : Kat h leen Martin , M . S . E . S. Al a n Reynolds , M . S . Study type : Se ct io n G Ca lculati o n s Data Requirement: Not Applicable Date : March 26 , 2015

Test O PP TS 885.23 00 ( Mi c ro bi al Te st Guide l ines : Analytic al Methods- Plants ) Guideline: and O PPT S 885.2500 ( Mi c robi al Pest ic id e Test G uid el i nes: Magni t ude of Residues i n P lants MRID Reviewed: 494093-03 (am e nded ; see So ut hern G a rdens 2014) DP Barcode : 423571 Decision N2 : 492314 Submission : 958820 Test D is e ase R esi sta nt C it rus Expresse d Proteins-Sp i n ach Material: Defen s in 2 and 7 (So D2 and So D7) Report Expression of S pinach Defe nsi n 2 and 7 Proteins i n Leaf Tissue of Citrus

Title : Events SGC - 11208- 5 , SG C-41108-7 , SG C-41332-6 and SGC-11319-8 (am e nded ; see So ut he rn Garde ns 201 4 ) Author : Ja mes E . Strick le r , Ph . D . Sponsor: So ut he rn G ard ens Citrus N ursery , L LC

Testing Facility: C itru s g rowi ng s ites i n F lo rida and Texas Project ID : La bo ratory (S G 14- 164 .01) ; Spo n so r Study Number (S G C-TAL R 07) Study Date : J un e 1 2 , 20 14 Receipt J u ne 17 , 20 14 Date : Confidentiality N o n e Cla i ms : Good Laboratory NA Practice : Study Summary Th e req uested nu mber of acres reason a b le and the total amount of protein and Conclusion : expressed by t he f ou r events is we ll -de scribed . Deficiencies : None Classification : ACC EPTA BLESout h ern Ga rdens is requesting an experimental use permit ( E UP) to test a new plant-incorporated protectants (PIP's) that may confer d isease resistance to c itrus plants . I n total , Southern Gardens is requesting 720 acres in Florida and Texas (600 P I P 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 max imum amount of protein is expected to be no greater than 75 kg . The events , their expressed prote i ns , and type of cit rus are listed in Table 1 : T bl 1



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### t P 

### t . ven s , ro ems , an dT pe o Y

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### fc · 1trus Event Protein a Citrus Type SGC-411 08-7 SoD2 sweet orange SGC-41332-6 SoD2 , So D7 sweet orange SGC-11 319-8 SoD2 , SoD7 lemon SGC-11208-5 So D7 grapefruit a so D2 -D efens l n 2 ;**SoD 7=** **Defe ns~n**  7 TESTING LOCATIONS Southern Gardens plans to conduct their study in one or more locations in Hendry County, Flor ida , and Hida lgo County , Te xas . Testing will be conducted at both locations to generate agronomic, efficacy and regulatory data and information . AC RES In total , Southern Gardens intends to plant a maximum of 720 acres at test sites in Fl o rida and Te xas . Of the 720 acres , 600 are expected to be PIP acres and 120 border . These are t a bulated in Table 2 : Table 2 Summary of Acres by Event , Type (P I P , Border) , and Location SGC-41108-7 Hen dry County , FL Hidalgo County , TX Total P I P · Total B order: PIP 100 50 150

A MOUNT OF PROTEIN : Border 20 10 30 SGC-41332-6 PIP Border 100 20 50 10 150 30 SGC-11319-8 SGC-11208 -5 TOTAL P I P Border P I P 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 Wes tern 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-2Table 3 Planting Density f Lea Materia per Tree I %Active Ingredient Assumptions Use 1n t e a cu at1on 

### d . 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 >2 0 mg/kg Measured Equation 

### 1 Total Amount of Protein Expected in Leaf Material Over the Duration of the Study ( Tre es ) Amt LeafMaterial Amt of Protein (kg)= Acres x Planting Dens i ty -- x T (kg, dry wgt) x% AI Acre ree For ex ample , in Hendry County , FL : ( 250 Trees ) 20 kg leaf mg prote in Amt of Protein (kg) 

## = 1 00 Acres x x x% A I ( k ) Acre Tree g Table 

### 4 Amou nt of Protein (kg) Expected in Leaf Mat . 

### I 0 th D 

### f f th St d ena ver e ura 1on o e u 

### 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 .3 5 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 A mount of Protein Expected Assumi ng a Con f 25 /k f E h E ce ntration o mq**(~** 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-3CONCLUSION : The number of PIP and border acres (6 00 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 th e expected concen trations of SoD2 and SoD? are well-described and documented in the study and appear to be insurmountable . The study investigators exp l ored a lternate 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 j ustification (e .g ., cit ing 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 ):1 49 - 156 . Southern Gardens . 2014 . Letter from Vick ie 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 (s ublabel acreage added) . October 3 , 2014 .

DER-4