2,4

- D Summary of Analytical Chemistry and Residue Data DP#: 400085



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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



### MEMORANDUM

Date: July 11, 2013

SUBJECT: 2,4-D. Summary of Analytical Chemistry and Residue Data for Use of2,4

- D Choline inion Herbicide Tolerant Soybeans Containing the Aryloxyalkanoate Dioxygenase

- 12 (AAD-12) Gene.

PC Codes: 051505, 03000 I Decision No.: 447951 DP Barcode: D400085; 

### '111$71 Registration No

. : 62719-AUU Regulatory Action: Section 3 Petition No.: NA Risk Assessment Type: NA TXRNo.:NA Case No

. : NA CAS No.: 1048373

- 72

- 3

, 94-75

- 7 40 CFR: 40 CFR §180

. 142 MRID No

. : (See Table Below)



### FROM:

THROUGH:

TO : Danette Drew,

# Chemist~ Risk Assessment Branch V Healt h Effects Division (7509P) 

# /fk ; Ideliz Negron

- Encarnacion, Chem 'f

. ·s r 

## j**~/ /)**  

# J**~ichael** 

### Metzger, Branch Chief 

## h 

# td'-M..

_ . · ' Rtsk Assessment Branch V Nil " 

### Health Effe cts Division (7509P)

Michael Walsh, RM23 Herbicide Branch Registration Division ( 7505P)

MRID Summary Table MRIDNo. Study Type Comments 48449801 860

. 1300 Nature of the Residues in Plants

- AAD-12 New DER; 4844 9801

. der.doc Soybean 48449802 860.1500 F ield Trial! Residue Decline- Soybean New DER: 48449802. derl.doc 860.1520 Processed Food and Feed

- Soybe an New DER; 48449802

. der2

. doc 491 22 501 860

. 1 3 80 Stora g e Stability

- Soybean N ew D E R; 4 91 22 501



### Page 1 of232,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 2 of 23 Executive Summary

2,4-Dichlorophenoxyacetic acid (2,4-D) is an alkyl chlorophenoxy herbicide used to control a variety of broadleaf weeds. The proposed us e is for 2,4-D choline on soybean containing the inserted aryloxyalkanoate dioxygenase-12 (AAD-12) gene. This soybean is identified as containing DAS-68416-4. Expression of the AAD-12 protein encoded by the AAD-12 gene results in a trait that increases the herbicid e tolerance of soybean to 2,4-D via increased metabolism through a pathway involving the metabolite 2,4-dichlorophenol (2,4-DCP).

Dow AgroSciences is proposing that the existin g tolerance (0.02 ppm) for residues of 2,4-D on soybean seed are adequate to cover the use of 2,4-D choline on AAD-12 soybean. A livestock feeding/grazing restriction for this use is proposed ; therefore, the current tolerances for residues of 2,4-D on soybean forage and hay are not being reevaluated.

The end-use product relevant for this peti tion request is GF-2654 TS (Reg. no. 62719-AUU), containing 56.3% 2,4-D choline salt (38.4% of 2,4-D, or 3.8 lb ae/ gal of 2,4-D). GF-2654 TS is proposed for pre-plant, pre-emergence, or post-em ergence (when soybean is no later than the R2, or full flowering, growth stage) application to soybean at a maxi mum single application rate of 2.1 pints per acre (0.95 lb ae/A). For post-em ergence applications, a maximum of two applications are permitted with a minimum of 12 days between applications. The maximum seasonal application rate is 6.3 pi nts of GF-2654 TS per acre per s eason (3 lbs ae/A/season). The directions indicate no more th an one pre-emergence applica tion and no more than two postemergence applications per use season. Aerial application and chemiga tion is prohibited. The proposed preharvest interval is 30 days. Grazing of treated soybean by livestock is prohibited as is harvesting for forage or hay.

An acceptable metabolism study using AAD-12 soybean was submitted to elucid ate the nature of the residue in plants. The predominant residues in soybean forage and hay were 2,4-D, 2,4-DCP, Metabolite A (acetyl-glucose-DCP conjugate) and Metabolite B (X11963417; Glucose conjugate of 2,4-DCP). In soybean seed the primary id entified residues were 2,4-D, Metabolite B and Metabolite C (2,4-DCP disaccharide). The study indicates that the primary pathway of metabolism of 2,4-D in AAD-12 soybean proceeds ra pidly via dissociation to the acid. After cleavage of the acid side chai n, the resulting metabolite 2,4-DCP is rapidly conjugated with glucose. Very low levels of 2,4-DCP compared to conjugated 2,4-DCP i ndicate that conjugation is rapid and is a preferential route of metabolis m. Metabolism proceeds through incorporation of the radiolabeled carbon into na tural plant constituents. Prev ious plant metabolism studies conducted on AAD-1 corn also concluded that metabolism of 2,4-D DMA proceeded to 2,4- DCP, glucose conjugates of 2,4-DCP, and to inco rporation into natural plant constituents. Overall, the 14 

### C-residues in the AAD-12 soybeans were simi lar to those observed on transgenic corn and conventional crops. EPA has determined the residue of concern for plant tolerance enforcement is parent 2,4-D only. The residues of concern for risk assessment are 2,4-D and 2,4- DCP.

The requirements for livestock metabolism are fulfilled. Adequate goat and hen metabolism studies are available, and the metabolism of 2,4- D is similar in both sp ecies. Based on the available data, on 9/3/03, the Agency determined th at the residue of concern in livestock for the tolerance expression and for risk assessment is 2,4-D, free and conjugated, determined as the acid (W. Hazel and L. Taylor, 12/3/03, D293119, TXR No. 0052264). The existing tolerances2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 3 of 23 for residues of 2,4-D on livestock commodities are adequate to cover the use on AAD-12 soybeans, provided the grazing (soybean) and harv esting (forage and hay) restrictions are maintained on the label.

Adequate methods are available for data collect ion. In addition, the 10/97 edition of FDA PAM Volume I, Appendix I indicates that 2,4-D is partially recovere d (50-80%) using Multiresidue Methods Section 402 E1 and 402 E2. Analytical standards for 2,4-D are currently available in the EPA National Pesticide Standards Repository.

In the crop field trials, samples were stored frozen (~-20 °C) fr om harvest to extraction for 2,4-D and 2,4-DCP for up 15.2 months for forage, 15.1 m onths for hay, and 13.3 months for seed. Storage stability data are available for 2,4-D and 2,4-DCP indicating that residues are stable under frozen storage conditions for at least 27 months in soybean raw agricultural commodities (seed, hay, forage). The available storage stabi lity data are adequate to support sample storage conditions and durations in the soybean field trials.

In the processing study, samples were stored frozen ( ≤ -20 °C) from harvest/processing to extraction for 2,4-D and 2,4-DCP for 11.7-12.4 mont hs for soybean seed, and 9.1-10.3 months for the soybean processed commodities (aspirated grain fractions (AGF), meal, hulls, and oil). Storage stability data are available to s upport the storage durati ons for the processed commodities soybean hulls, meal, AGF, and oil.

Magnitude of the residue data for AAD-12 soybean have been submitted to support the proposed use, including twenty-four crop trials that were conducted in North American Free Trade Agreement (NAFTA) Growing Zones 2, 4, and 5 during the 2009 growing season. Three broadcast applications of the 3.79 lb ae/gal WP formulation of 2,4-D DMA were made as one preemergence application and two postemergen ce foliar applications at a total seasonal application rate of 2.97-3.13 lb ae/A soybean containing the AAD-12 gene. These data support of the use of 2,4-D choline on AAD-12 soybean as 2,4-D salts readily di ssociate into 2,4-D parent and the cation. Based on the study results, residue s (and per trial average residues) of 2,4- D and 2,4-DCP, respectively, may be up to 4.01 (3.93) and 25.2 (22.4) ppm in early-harvest forage; 18.4 (18.3) and 37.0 (32.1) ppm in typical-harvest forage ; 21.8 (20.3) and 190 (190) ppm in early-harvest hay; 133 (132) and 60.4 (58.8) ppm in typical-harves t hay; and 0.054 (0.047) and <0.01 (<0.01) ppm in soybean seed (51-103 day PHI for seed). The field trial data are acceptable for regulatory purposes. A revised la bel should be submitted which specifies a minimum PHI of 50 days for soybean. The pr oposed label currently specifies a 30 day PHI which does not reflect the minimum PHI (51 days) used for soybean seed in the submitted crop field trials.

A processed food and feed study for soybean has been submitted. Following three foliar broadcast applications to AAD-12 soybean of the 3.79 lb ae/ gal WP formulation of 2,4-D DMA made as one preemergence application and two postemergence foliar applications at a total seasonal application rate of 6.0 lb ae/A. Residues of 2,4-D were below the LOQ (<0.01 ppm) in all samples of soybean seed RAC, meal, hulls, an d alkali-refined oil; therefore, no processing factors were calculated for these commodities. Th e processing data indicate that residues of 2,4- D concentrate in AGF (average processing factor of >1.4x). The processing data indicate that residues of 2,4-DCP also concentrate in AGF (ave rage processing factor of 6.9x), as well as in meal (average processing factor of 1.3x), but do not concentrate in alkali -refined oil or hulls2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 4 of 23 (average processing factors of <0.5x and < 0.7x, respectively). The processing data are acceptable for regulatory purposes.

The requirements for confined/field rotational crop st udies are satisfied. No plant- back interval (PBI) or rotational crop tolerances are required to support the new use.

The maximum soybean seed residues in the subm itted field trial study do not exceed the current tolerance of 0.02 ppm for residues of 2,4-D on soybean seed and is adequate to cover the use of 2,4-D choline on AAD-12 soybean. However, in order to harmonize with the Codex maximum residue levels (MRLs) for soybean seed the current U.S. tolera nce for soybean seed (0.02 ppm) should be decreased to 0.01 ppm. This revised to lerance level will also support the proposed 2,4- D choline use on soybean. The established tolera nce for AGF will cover residues of 2,4-D in/on soybean AGF. A livestock feeding/grazing restricti on for this use is propo sed; therefore, the current tolerances for residues of 2,4-D on soybean forage and hay are not being reevaluated at this time.

Recommended Tolerances

Table 1. Toleran ce Summary for 2,4-D
Commodity Established/Proposed Tolerance (ppm) Recommended Tolerance (ppm) Comments Correct Commodity Definition Soybean, seed 0.02 0.01



### Regulatory Recommendations

Provided the registrant revise s the proposed label for GF- 2654-TS (56.3% 2,4-D choline; Reg. no. 62719-AUU) end use products as described be low, and pending submission of adequate storage stability data described below, there are no residue chemistry deficiencies that would preclude the requested registration for the us e of 2,4-D choline on AAD-12 soybean. HED has determined that the residue of concern for toleran ce expression is 2,4-D. The existing tolerances for 2,4-D are adequate to support the proposed ne w use. However, HED recommends that the existing tolerance for soybean seed be decr eased to 0.01 ppm to allow harmonization with Codex. The tolerance of 0.01 ppm on soybean se ed will also support the proposed 2,4-D choline use on soybeans. Tolerances for 2,4-D re sidues are listed in 40 CFR §180.142.

A human health risk assessment is forthcoming.

Residue Chemistry Deficiencies

The following label modifications are needed pr ior to the registration of GF-2654-TS (Reg. no. 62719-AUU) for use on soybeans:

860.1200 Directions for Use

 A revised label should be submitted which speci fies a minimum preharvest interval of 50 days for soybean. The proposed label curren tly specifies a 30 day PHI which does not reflect the minimum PHI (51 days) used fo r soybean seed in the crop field trials.2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 5 of 23 Background

2,4-Dichlorophenoxyacetic acid (2,4-D) is an alkyl chlorophenoxy herbicide used to control a variety of broadleaf weeds. The proposed us e is for 2,4-D choline on soybean containing the inserted aryloxyalkanoate dioxygenase-12 (AAD-12) gene. This soybean is identified as containing DAS-68416-4. Expression of the AAD-12 protein encoded by the AAD-12 gene results in a trait that increases the herbicid e tolerance of soybean to 2,4-D via increased metabolism through a pathway involving the metabolite 2,4-dichlorophenol (2,4-DCP).

The chemical structure and nomenclature of 2,4-D, 2,4-D DMA, 2,4-D choline and 2,4-DCP are presented in Table 2. Physicochemical propert ies of 2,4-D are summarized in Table 2. The dissociation constant for 2,4-D c holine is listed in Table 3.

2,4-D choline is expected to read ily dissociate into parent 2,4-D and choline cation when mixed with water. There is no toxicological concern fo r choline. 2,4-D is th e dissociation product of concern for the formulation asso ciated with this action.

Table 2. Test Compound Nomenclature. 2,4-Dichlorophenoxyacetic acid 

### ( 2,4-D)

Common name 2,4-D acid IUPAC name (2,4-dichlorophenoxy)acetic acid CAS name 2-(2,4-dichlorophenoxy)acetic acid
CAS # 94-75-7
PC Code 030001 End-use product/(EP) Not applicable 2,4-D dimethylamine salt (DMA)

IUPAC name (2,4-dichlorophenoxy)ace tic acid - dimethylamine (1:1) CAS name 2-(2,4-dichlorophenoxy)acetic ac id compound with N-methylethanamine (1:1)
CAS # 2008-39-1
PC Code 030019 End-use product/(EP) 3.79 lb ae /gal SL/L formulation (Weedar ® 64 Herbicide) 2,4-D choline

IUPAC name 2-hydroxy-N,N,N-trimethyleth anaminium (2,4-dichlorophenoxy)acetate2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 6 of 23 Table 2. Test Compound Nomenclature. CAS name Ethanaminium, 2-hydroxy-N,N,N-trim ethyl-, 2-(2,4-dichlorophenoxy)acetic acid hydroxide (1:1:1)
CAS # 1048373-72-3
PC Code 051505 2,4-dichlorophenol (2,4-DCP)

2,4-dichlorophenol

Table 3. Physicochemical Properties of the T echnical Grade Test Co mpound: 2,4-D acid.
Parameter Valu e Reference Melting point/range 138-141 °C DP#309452, T. Jimerson, 10/13/04 pH Not available Density Specific gravity = 1.416 at 25 °C Water solubility ( __°C) 569 mg/L at 20 °C Solvent solubility (mg/L at __°C) g/100 g at 25 °C acetone 85.0 benzene 1.07 diethyl ether 220 ethanol 130.0 isopropanol 31.6 toluene 0.067 xylene 0.58 Vapor pressure at __°C 1.4 x 10 -7 mm Hg at 25 °C Dissociation constant (pK a ) Not available Octanol/water partition coefficient Log(K OW ) In 0.001 M solution: 2.14 at pH 5 0.177 at pH 7 0.102 at pH 9 UV/visible absorption spectrum Not available

Table 4. Physicochemical Properties of 99.3% purified active ingredient (PAI) 2,4-D Choline*.
Parameter Valu e Reference Dissociation constant (pK) pK = 1.002 ± 0.177 at 25 °C [99.3% PAI; OECD 112; conductometric] MRID 48260701



### 860.1200 Directions for Use

The end-use product relevant for this petiti on request is GF-2654 TS, containing 56.3% 2,4-D choline salt (38.4% of 2,4-D, or 3.8 lb ae/gal of 2,4-D). GF-2654 TS is proposed for pre-plant, pre-emergence, or post-emergence (when soybean is no later than the R2, or full flowering, growth stage) applicatio n to soybean at a maximum single app lication rate of 2.1 pints per acre (0.95 lb ae/A). For post-emergence applications , a maximum of two applications are permitted with a minimum of 12 days betwee n applications. The maximum s easonal application rate is 6.3 pints of GF-2654 TS per acre per season (3 lbs ae/A/ season). The directions indicate no more than one pre-emergence application and no more than two post-emergence applications per use season. Aerial application and chemigation is prohi bited. The proposed prehar vest interval is 30 days for soybean. Grazing of treated soybean is prohibited as is harvesting for forage or hay.2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 7 of 23 Table 5. Summary of Directions for Us e of 2,4-D Choline on AAD-12 Soybean

Applic. Timing, Type, and Equip. Formulation Applic. Rate (lb ae/A) Max. No. Applic. per

Season Max. Seasonal Applic.

Rate (lb ae/A) PHI (days) Use Directions and Limitations

Broadcast preplant (burndown)
preemergence 3.8 lb 2,4- D/gal (GF-2654 TS) 0.95 3 3 30 days Preplant: Apply any time prior to and up through soybean planting Preemergence: Apply anytime after planting but before soybean emerges

Broadcast postemergence Apply when weeds are small and soybean growth rate is no later than R2 (full flowering stage). Make one to two a pplications with a minimum of 12 days between applications.



### HED Conclusion : The use directions provided in the peti tion are adequate to allow evaluation of the residue data. However, a revised label should be submitted which specifies a minimum preharvest interval of 50 days for soybean. Th e proposed label currentl y specifies a 30 day PHI which does not reflect the PHI (51- 103 days) used in the soybean crop field trials. No plantback intervals following 2,4-D application are needed for rotational crops.

860.1300 Nature of the Residue – Plants

48449801.der



### Adequate plant metabolism data are availabl e reflecting the applic ation of [phenyl-U- 14 

### C]-2,4- dichlorophenoxy acetic acid dimethylammonium salt (2,4-D DMA) to 2,4-D-tolerant soybean. The submitted AAD-12 soybean metabolism study is acceptable.

The radiolabeled test substance was formulated as a soluble concentr ate liquid (SL/L) and applied to 2,4-D-tolerant soyb eans grown outdoors in a planting box or pots as a preemergence soil application at 1.04 -1.11 lb ae/A (1.166-1.248 kg ae/ha) followe d by two foliar applications at 0.98-1.11 lb ae/A/application (1.103- 1.243 kg ae/ha/application), for a total application rate of 3.13-3.19 lb ae/A (3.512-3.578 kg ae/ha). The preemergence application was made on the day of planting, and foliar applications were made at the V4 and R2 growth stages at retreatment intervals (RTIs) of 34 and 21 days to soybean grown in the planting box and 22 and 20 days to soybean grown in pots. Samples of forage and hay were collected from the planting box 7 days after the second and third applic ations, respectively (days after treatment (DAT) 2 and 3), 7- DAT2 for forage and 7-DAT3 for hay. Because the 7-DAT3 hay sample was not dried, another sample of hay was collected 22 days after the third application (22-DAT3) and dried. Samples of seed were collected from the planting box and pots at 62 and 41 da ys after the third application (62-DAT3 and 41-DAT3), respectively.

Total radioactive residues (TRR) determined by combustion/LSC (liquid scintillation counter) were 50.2 ppm in 7-DAT2 forage, 35.6 ppm in 7-DAT3 hay, 37.0 ppm in 22-DAT3 hay, 0.415 ppm in 62-DAT3 seed, and 0.372 ppm in 41-DAT3 seed.

Residues were identified and quantitated in soybean forage, hay, and seed extracts and hydrolysates by high performance liquid chromatogr aphy (HPLC); seed extracts were purified2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 8 of 23 by solid phase extraction (SPE) prior to analysis. Hexane extracts from 41 -DAT3 seed were also subjected to thin layer chromatography (TLC) fo r residue characterizatio n/identification. In addition, identification of 2,4-D and 2,4-DCP was confirmed, and the glucose conjugates of metabolite 2,4-DCP, including Metabolites A (acetyl-glucose conjugate), B (X11963417; glucose conjugate of 2,4-DCP), and C (disaccharid e conjugate), were identified in forage and hay extracts/hydrolysates following isolation by SPE and analysis by liquid chromatography with tandem mass spectrometry (LC/MS/MS). Stru ctures of the metabolites may be found in Attachment 1 to this memorandum.

The parent, 2,4-D, was the major residue identif ied in forage (85.8% TRR) and hay (59.4-64.3% TRR), while 2,4-DCP was present at 7.1% TRR in forage and 1.4-3.6% TRR in hay. Metabolite A accounted for 10.4% TRR in forage and 12.6-13.4% TRR in hay. Metabolites B and C were present at 0.8% and 0.1% TRR (0.381 and 0.055 ppm) , respectively, in forage and 5.4-7.0% and 1.1-2.7% TRR (1.92-2.57 and 0.395-1.00 ppm), respectively, in hay. Remaining radioactivity, designated “other”, accounted for 5.1% TRR in fora ge and 2.5% and 22.3% TRR, respectively in 7- and 22-DAT3 hay.

In 41-DAT3 seed, following accelerated solven t extraction (ASE) with methanol and methanol/water, and base hydrolysis of the nonextractable residues, 2,4-D accounted for 1.4% TRR, free 2,4-DCP comprised only 0.1% of the TRR, and the 2,4-DCP conjugates Metabolites B and C accounted for 3.0% and 4.6% TRR, respectivel y. TLC of the hexane extracts (10% TRR) indicated that >90% of the radioactivity was distributed in natural constituents. These components were attributed to triglycerides and unsaturated fatty acids. “Other” radioactivity accounted for 56.9% TRR (0.211 ppm) and was f ound to be multicomponent and polar. Precipitates following base hydrolys is of the nonextractable residue s accounted for 13.4% TRR.

Based on the results of the metabolism study wi th AAD-12 soybean (Event 416), the petitioner proposed that the metabolism of 2,4-D DMA proceed s rapidly via dissociation to the acid: After cleavage of the acid side chai n, the resulting dichlorophenol ( 2,4-DCP) was rapidly conjugated with acetylated glucose and other glucose conjug ates. Low levels of free 2,4-DCP compared to conjugated 2,4-DCP indicated that the conjuga tion was rapid and a pr eferential route of metabolism. In seed, bound residues were produ ced that liberated la rge numbers of polar metabolites upon acid and base hydrolysis. The petitioner noted that since 2,4-D has been demonstrated to degrade to CO 2 

### in soil, it was likely that any 2,4-D applied to the soil was converted to CO 2 

### and became incorporated into natura l plant constituents. Hydrolysis experiments conducted with extracted soybean lipi ds and bound residues indi cated that this was a likely route of metabolism.2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 9 of 23 Table 6. Summary of Characterization and Identification of Radioactive Residues in AAD-12 Soybean (event 416) Forage and Hay Following Application of [ 14 C]2,4-D DMA at 2.14 lb ae/A (2.401 kg ae/ha) for forage and 3.19 lb ae/A (3.578 kg ae/ha) for hay. 1

Metabolite Fraction 7-DAT2 Fo rage 7-DAT3 Hay 22-DAT3 Hay TRR = 50.227 ppm TRR = 35.565 ppm TRR = 37.008 ppm %TRR ppm %TRR ppm %TRR ppm 2,4-D 85.8 43.12 64.3 22.885 59.4 21.98 2,4-DCP 7.1 3.558 3.6 1.295 1.4 0.518
Metabolite A 10.4 5.223 12.6 4.474 13.4 4.969 Metabolite B (X11963417) 0.8 0. 381 5.4 1.919 7.0 2.573
Metabolite C 0.1 0.055 1.1 0.395 2.7 1.000
Other 5.1 2.557 2.5 0.901 22.3 8.256
Total identified 104.2 52.33 87.1 30.97 83.9 31.04
Total characterized 5.1 2. 557 2.5 0.901 22.3 8.256
Total extractable 2 110.3 55.39 93.8 33.36 108.7 40.24 Unextractable (PES) 3 2.8 1.393 4.2 1.478 8.2 3.027 Accountability 4 113 97.9 117

1 Metabolite fractions were obtained from the summed AS E extracts (40-110 °C) as calculated by the petitioner.
2 Calculated by the study reviewer by summing ppm in ASE extracts (40-110 °C).
3 Residues remaining after exhaustive extractions.
4 Accountability = (Total ex tractable + Total unextractable)/(TRR from combus tion analysis; see TAB LE C.2.1) * 100. TRR were calculated by summing extractable and nonextractable radioactivity.

Table 7. Summary of Characterization and Identification of Radioactive Residues in AAD-12 Soybean (event 416) Seed Following Application of [ 14 C]2,4-D DMA at 3.133 lb ae/A (3.512 kg ae/ha). 1

Metabolite Fraction 41-DAT3 Seed TRR = 0.372 ppm %TRR ppm 2,4-D 1.4 0.005 2,4-DCP 0.1 <0.001 Metabolite B (X11963417) 3.0 0.011
Metabolite C 4.6 0.018
Other 56.9 0.211 Hexane (triglycerides/f atty acids 10.0 0.037 Precipitates from base hydrolysis 13.4 0.050
Total identified 9.1 0.035
Total characterized 80.3 0.298
Total extractable 2 90.9 0.338 Unextractable (PES) 3 2.6 0.010 Accountability 4 93.5

1 Metabolite fractions were calculated by the study reviewer by summing results from ASE extraction (40-70 °C) for combined methanol 40 °C, combined methanol/water 70 °C, and base hydrolysis of nonextractable residues.
2 Calculated by the study reviewer by summing ppm in ASE extracts (40-70 °C) and ba se hydrolysis of nonextractable residues.
3 Residues remaining after exhaustive extractions.
4 Accountability = (Total ex tractable + Total unextractable)/(TRR from combus tion analysis; see TAB LE C.2.1) * 100. TRR were calculated by summing extractable and nonextractable radioactivity.



### HED Conclusion : The submitted soybean metabolism study is acceptable. The predominant residues in soybean forage and hay were 2,4- D, 2,4-DCP, Metabolite A (acetyl-glucose-DCP conjugate) and Metabolite B (X11963417; Glucose c onjugate of 2,4-DCP). In soybean seed the primary identified residues were 2,4-D, Metabol ite B and Metabolite C (2,4-DCP disaccharide).2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 10 of 23 The study indicates that the primary pathwa y of metabolism of 2,4-D in AAD-12 soybean proceeds rapidly via dissociation to the acid. Afte r cleavage of the acid side chain, the resulting metabolite 2,4-DCP is rapidly conjugated with glucose. Very low levels of 2,4-DCP compared to conjugated 2,4-DCP indicate th at conjugation is rapid and is a preferential route of metabolism. Metabolism proceeds through incorporat ion of the radiolabeled carbon into natural plant constituents. Previous plant metabolis m studies conducted on AAD-1 corn also concluded that metabolism of 2,4-D DMA proceeded to 2,4- DCP, glucose conjugates of 2,4-DCP, and to incorporation into natural plant constituents. Overall, the 14 

### C-residues in the AAD-12 soybeans were similar to those observed on transgenic co rn and conventional crops. EPA has determined the residue of concern for plant tolerance enfo rcement is parent 2,4-D only. The residues of concern for risk assessment are 2,4-D and 2,4-DCP.

860.1300 Nature of the Residue - Livestock

The requirements for livestock metabolism are fulfilled. Adequate goat and hen metabolism studies were previously submitted, and the metabolism of 2,4-D is similar in both species. Based on the available data, on 9/3/03, the Agency determined that the re sidue of concern in livestock for the tolerance expression and for risk assessm ent is 2,4-D, free and conjugated, determined as the acid (W. Hazel and L. Tayl or, 12/3/03, D293119, TXR No. 0052264).

860.1340 Residue Analytical Methods

Data Collection Methods

Magnitude of Residue Study Samples were analyzed for residues of 2,4-D and its metabolite 2,4-DCP by a high performance liquid chromatography method with tandem mass spectrometry detection (LC/MS/MS). A complete description of the met hod was included in the submission.

Briefly, samples were extracted with metha nol:1.0 N NaOH (90:10, v:v) and centrifuged. An aliquot of the extract was comb ined with a mixed stable isot ope internal standard of 2,4-D( 13 

### C 6 

### ) or 2,4-DCP( 13 

### C 6 

### ) and evaporated to near dryness. Th e sample was mixed with 2 N HCl (at 90 °C for 1 hour). After cooling, the extract was diluted with me thanol, filtered, and analyzed by on-line solid-phase extraction (SPE ; residues eluted with water:me thanol containing 0.1% formic acid (70:30, v:v)) and LC/MS/MS in the negative ion mode.

The reported LOQ (equivalent to the lower leve l of method validation, or LLMV) was 0.01 ppm, and the LOD was 0.003 ppm for 2,4-D and 2,4-DCP in all matrices. The method was validated concurrently with analysis of the field trial samples.

Concurrent method recovery data are presente d in Table 8. The LC/MS/MS method used to analyze soybean samples for residues of 2,4-D and 2,4-DCP is adequate for data collection based on acceptable concurrent method recove ry data. Concurrent recoveri es were generally within the acceptable range of 70-120% in soybean fortifie d with 2,4-D and 2,4-DCP each at 0.01-100 ppm in forage, 0.01-200 ppm in hay, and 0.01-1 ppm in s eed. The fortification levels were adequate to bracket residues found in treat ed samples. Apparent residu es of 2,4-D were below the LOQ (<0.01 ppm) in/on all samples of untreated soybe an forage; apparent residues of 2,4-D were observed in/on nine samples of untreated hay at 0.011-0.034 ppm and four samples of untreated2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 11 of 23 seed at 0.014-0.312 ppm. Apparent residues of 2,4-DCP were below the LOQ (<0.01 ppm) in/on all samples of untreated soybean seed; appare nt residues of 2,4-DCP were observed in/on 21 samples of untreated forage at 0.010-0.058 ppm and 44 samples of untreated hay at 0.010-0.214 ppm. The petitioner stated that the residue s of 2,4-D and 2,4-DCP found in the untreated samples did not appear to be the result of c ontamination during anal ysis; instead, it appears contamination may have taken place at some earlier stage, perhaps from spray drift and/or from sample collection and handling in the field a nd/or during sample prep aration. Concurrent recoveries were corrected for apparent for residu es in controls; residues in treated samples were not corrected for residues in controls.

Table 8. Summary of Concu rrent Recoveries of 2,4-D and 2,4-DCP from Soybean. Matrix Spike level (ppm) Sample size (n) Recoveries (%) 1 Mean ± std dev 2 (%) 2,4-D Forage 0.01 10 81, 84, 86, 88, 89, 91, 93, 96, 99, 101 91 ± 6 1 4 91, 92, 93, 98 94 ± 3 10 2 99, 101 100 100 4 77, 76, 87, 89 82 ± 7 Hay 0.01 10 87, 90, 90, 92, 92, 94, 98, 98, 98, 98 94 ± 4 1 2 93, 97 95 100 8 76, 78, 82, 86, 89, 90, 91, 91 85 ± 6 200 6 76, 81, 85, 85, 86, 89 84 ± 5 Seed 0.01 8 79, 81, 83, 83, 85, 87, 91, 102 86 ± 7 1 8 79, 81, 86, 88, 94, 96, 96, 97 90 ± 7 2,4-DCP Forage 0.01 10 69, 83, 93, 100, 101, 101, 102, 106, 106, 110 97 ± 12 1 4 87, 90, 96, 99 93 ± 5 10 2 94, 96 95 100 4 76, 79, 81, 83 80 ± 3 Hay 0.01 10 89, 89, 90, 93, 94, 96, 98, 99, 104, 109 96 ± 7 1 2 93, 103 98 100 8 70, 71, 71, 72, 73, 75, 79, 83 74 ± 5 200 6 75, 77, 78, 78, 79, 86 79 ± 4 Seed 0.01 8 79, 90, 91, 93, 94, 98, 105, 109 95 ± 9 1 8 81, 82, 86, 92, 94, 97, 97, 103 92 ± 8

1 Recoveries were corrected for appare nt residues in the untreated samples.
2 Standard deviation is not applicab le for sample sizes n < 3 samples.



### Processing Study Samples of soybean and processed commodities we re analyzed for residues of 2,4-D and its metabolite 2,4-DCP using the analytical method desc ribed above for the field trail (magnitude of the residue) samples.

The reported LOQ (equivalent to the LLMV) was 0.01 ppm, and the LOD was 0.003 ppm for 2,4-D and 2,4-DCP in all matrices. The method was validated concurrently with analysis of the processing study samples.

The LC/MS/MS method used to analyze soybean processing samples for residues of 2,4-D and 2,4-DCP was adequate for data collection based on acceptable concurrent method recovery data. Concurrent method recoveries were within th e acceptable range of 70-120% for soybean and2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 12 of 23 processed commodities fortifie d with 2,4-D and 2,4-DCP each at 0.01 and 1 ppm for seed, AGF, hulls, and meal and at 0.01 and 0.1 ppm for refine d oil (see Table 9). The fortification levels were adequate to bracket residues found in treat ed samples. Apparent residues of 2,4-D were observed in one sample of untr eated AGF at 0.013 ppm; apparent residues of 2,4-D and 2,4-DCP were below the LOQ in/on all remaining untreat ed samples of soybean seed, AGF, meal, hulls, and refined oil. Concurrent rec overies were corrected for apparent residues in controls; residues in treated samples were not correct ed for residues in controls.

Table 9. Summary of Concurrent Recoveries of 2,4-D from Soybean Processed Commodities. Matrix Spike level (ppm) Sample size (n) Recoveries (%) 1 Mean ± std dev 2 (%) 2,4-D Soybean seed (RAC) 0.01 8 79, 81, 83, 83, 85, 87, 91, 102 86 ± 7 1 8 79, 81, 86, 88, 94, 96, 96, 97 90 ± 7 Aspirated grain fractions 0.01 2 96, 102 99 1 2 93, 95 94 Meal 0.01 2 89, 106 98 1 2 95, 97 96 Hulls 0.01 2 89, 101 95 1 2 96, 98 97 Refined oil 0.01 2 91, 95 93 0.1 2 96, 99 98 2,4-DCP Soybean seed (RAC) 0.01 8 79, 90, 91, 93, 94, 98, 105, 109 95 ± 9 1 8 81, 82, 86, 92, 94, 97, 97, 103 92 ± 8 Aspirated grain fractions 0.01 2 70, 73 72 1 2 100, 104 102 Meal 0.01 2 98, 105 102 1 2 99, 107 103 Hulls 0.01 2 85, 94 90 1 2 91, 100 96 Refined oil 0.01 2 89, 92 91 0.1 2 89, 95 92

1 Recoveries were corrected for detectable residues in the untreated samples, if present.
2 Standard deviation is not applicable for sample sizes n < 3 samples.



### Enforcement methods There are adequate methods for the enforcement of plant commodity tolerances. An adequate proposed GC/ECD enforcement method for plan ts (designated as EN -CAS Method No. ENC2/93) has been submitted to FDA for inclusion in PAM II.

HED Conclusions : Sufficient data have been submitted to support the data collection methods. The methods from the soybean field trial and pro cessing studies have been adequately validated for data collection purposes based on acceptabl e concurrent fortific ation recovery data.

860.1360 Multiresidue Methods2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 13 of 23 The 10/97 edition of FDA PAM Volume I, Appendix I indicates that 2,4-D is partially recovered (50-80%) using Multiresidue Me thods Section 402 E1 and 402 E2. Conjugated metabolites would not be determined by these multiresidue methods.

HED Conclusion: Multi-residue method tes ting requirements have been adequately addressed.

860.1380 Storage Stability

48449802.de1 48449802.de2 49122501.der


### In the crop field trials, samples were stored frozen (~-20 °C) fr om harvest to extraction for 2,4-D and 2,4-DCP for up 15.2 months for forage, 15.1 m onths for hay, and 13.3 months for seed. The summary of storage conditions and durations for soybean is presented in Table 10.

Adequate storage stability data are available for parent 2,4-D indicating th at residues are stable under frozen storage conditions for at least 12 mo nths in/on a variety of field crop commodities including corn grain, forage, and fodder; sorghu m grain; wheat grain, fo rage, and straw; rice grain; grass forage and hay.

In addition, storage stability data are availa ble indicating residues of 2,4-D and 2,4-DCP are stable for up to 27.8 months in soybean raw agricultural commodities (RACs; seed, hay, forage) stored frozen in High-Density Po lyethylene (HDPE) containers (as used the field trial studies). The available storage stability da ta for are adequate to support sample storage conditions and durations in the soybean field trials.

In the processing study, samples were stored frozen ( ≤ -20 °C) from harvest/processing to extraction for 2,4-D and 2,4-DCP for 11.7-12.4 mont hs for soybean seed, and 9.1-10.3 months for the soybean processed commodities (aspirated grain fractions (AGF), meal, hulls, and oil). The summary of storage conditions and durations for soybean is presented in Table 11.

Storage stability data are available for parent 2,4-D indicating that re sidues are stable under frozen storage conditions for at least 12 months in the processed commodities of various crops (corn starch, flour, and oil; wheat flour; sugarcan e, sugar, molasses, and bagasse; and rice bran and hulls) and for at least 27 months in the soyb ean RACs (seed, forage, hay). These 2,4-D data are adequate to support the storage durations ( up to 10.3 months) in the soybean processing study for the processed commodities (AGF, hulls, meal, and refined oil).

There are no data available examining the st ability of the metabolite 2,4-DCP in soybean processed commodities. However, given the stability of 2,4-DCP in all the soybean RACs (seed, forage, hay) for at least 27 months, and the compositional similarity of these RACs to the processed commodities of AGF, hulls, and meal , the existing stability data for 2,4-DCP is adequate to support the storage du rations (up to 10.3 months) in the soybean processing study for AGF, hulls, and meal. The available storage stability data for corn oil, indicating that residues of 2,4-DCP are stable in oil for up to 13 months, are adequate to support the storage duration (up to 9.9 months) of soybean oil in the processing study.2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 14 of 23 Table 10. Summary of Storage Conditions for Soybean

. Matrix Storage Temperature (°C) Actual Storage Duration 1 Interval of Demonstrated Storage Stability (for various RACs) Forage Frozen at field sites

Approximately -20 at analytical lab 417-461 days (13.7-15.2 months) Adequate storage stability data are available for 2,4-D indicating that residues are stable under frozen storage conditions for at least 12 months in/on corn grain, forage, and fodder; sorghum grain; whea t grain, forage, and straw; rice grain; grass forage and hay; and soybean seed 2

, and for up to 13 months on transgenic (AAD-1) corn grain, stover and forage. 3

Storage stability data are available indicating that 2,4-DCP residues are stable for up to 13 months on transgenic (AAD1) corn stover, up to 5 mont hs on AAD-1 corn forage, and up to 2 months on AAD-1 corn grain. 3

Storage stability data are available indicating that residues of both 2,4-D and 2,4-DCP are st able in AAD-1 soybean raw commodities (seed, forage, hay) for up to 27.8 months. 4 Hay 415-460 days (13.7-15.1 months) Seed 327-405 days (10.8-13.3 months)

1 Duration from harvest to extraction; interval between extractio n and analysis was not provided. Hay sample duration is from collection after drying to extraction; samples were allowed to dry for 0-12 days.
2 Residue Chemistry Chapter of the 2,4-D RED; 10/12/04.
3 MRID 49063401.
4 MRID 49122501.

Table 11. Summary of Storage Condition s for Soybean Processed Commodities

. Matrix Storage Temperature (°C) Actual Storage Duration 1 Interval of Demonstrated Storage Stability (for various processed commodities) Soybean seed (RAC) Frozen at field sites

< -12 at processing facility

Approximately -20

at analytical lab 357-376 days (11.7-12.4 months) Adequa te storage stability data are available for 2,4-D indicating that residues are stable under frozen storage conditions for at least 12 months in/on soybean seed; corn starch, flour, and oil; wheat flour; sugarcane, sugar, molasses, and bagasse; and rice bran and hulls 2 and for up to 13 months on transgenic (AAD-1) corn grain, starch, flour and oil. 3

Storage stability data are available indicating that 2,4-DCP residues are stable for up 13 months in AAD-1 corn starch and corn oil, and up to 2 months in AAD-1 corn grain and flour. 3

Storage stability data are available demonstrating residues of 2,4-D and 2,4- DCP are stable in AAD-1 soybean raw commodities (seed, forage, hay) for up to 27.8 months. 4 Aspirated grain fractions 276 days (9.1 months) Hulls 287-312 days (9.4-10.3 months) Meal 282-304 days (9.3-10.0 months) Refined oil 280-302 days (9.2-9.9 months)

1 Duration from harvest to extraction; interval between extract ion and analysis was not provide d. Samples were processed within 65-102 days of harvest.
2 Residue Chemistry Chapter of the 2,4-D RED; 10/12/04.
3 MRID 49063401.
4 MRID 49122501.



### HED Conclusions : Storage stability data are available indicating residues of 2,4-D and 2,4-DCP are stable for up to 27.8 months in soybean raw agricultural commodities (RACs; seed, hay, forage). The available storage stability data are adequate to support sample storage conditions2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 15 of 23 and durations in the soybean field trials. Stor age stability data are available to support the storage durations (up to 10.3 months) for the pr ocessed commodities soybean hulls, meal, AGF, and refined oil.

860.1400 Water, Fish, and Irrigated Crops
There are no proposed uses that are relevant to this guideline topic.

860.1460 Food Handling
There are no proposed uses that are relevant to this guideline topic.

860.1480 Meat, Milk, Poultry, and Eggs

Soybean seed, meal, hulls, and aspirated grai n fractions (AGF) are the potential livestock feedstuffs associated with the proposed use of 2,4-D choline on AAD-12 soybean. Because the proposed label restricts grazing a nd the harvesting of forage and hay, those are not considered feedstuffs associated with the proposed use. S oybean meal and AGF are included in the livestock dietary burden determin ation (Table 12).

The tolerances for residues of 2,4-D in livestock feedstuffs range from 0.02 ppm in soybean seeds to 360 ppm in grass forage. Based upon th e established tolerances for 2,4-D residues in livestock feedstuffs, the maximum reasonabl y balanced diets (MRBD) for livestock are calculated below (Table 11). The MRBD for beef is 55.2 ppm and for dairy cattle livestock is 649 ppm; the majority of which is derived from th e tolerance level for grass forage and hay. The MRBD for poultry is 1.5 ppm and fo r swine is 0.9 ppm, based prim arily on the 2.0 ppm tolerance on cereal grains.

Table 12. Maximum Reasonably Balanced Diets: Livestock Dietary Burden for 2,4-D % diet Dietary contribution (ppm) Feedstuff Type Tolerance (ppm) % dry
matter Beef Dairy Poultry Swine Beef Dairy Poultry Swine

Grass Forage R 360 25 - 45 - - - 648 - - Grass Hay R 300 88 15 - - - 51.14 - - -

Aspir. Grain frac. CC 40 85 5 - - - 2.35 - - -

Barley Grain CC 2 88 50 45 75 20 1.14 1.02 1.5 0.4

Millet Grain CC 2 88 25 - - 20 0.57 - - 0.4 Sorghum

grain CC 0.2 86 - - - 45 - - - 0.09 Soybean

meal PC 0.02 92 5 10 5 - 0.001 0.002 0.001 -

Pea seed PC 0.05 90 - - 20 15 - - 0.01 0.0075

Totals - - - 100 100 100 100 55.2 649 1.5 0.9



### HED Conclusions : Soybean meal and AGF, when included as part of the MRDB, does not have a significant impact on the overall dietary burden. The addition of any of the relevant soybean2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 16 of 23 feedstuffs (seed, meal, hulls) would not impact the MRDB, which is largely driven by high tolerance level residues on grass forage and hay ( cattle) and cereal grains (poultry and swine). The previously submitted cattle feeding study is adequate. The existing livestock commodity tolerances are adequate to cove r the use on feedstuffs of AAD- 12 soybeans, provided the grazing and harvesting (forage and hay) restri ctions are maintained on the label.

860.1500 Crop Field Trials

48449802.de1


### Dow AgroSciences LLC has submitted field tria l data for 2,4-D on soybean containing the inserted aryloxyalkanoate dioxygenase-12 ( AAD-12) gene (seed line Event DAS-68416-4). Expression of the AAD-12 protein encoded by the AAD-12 gene results in a trait that increases the herbicide tolerance of soybean to 2,4- D via increased metabolism through a pathway involving the metabolite 2,4-dichlorophenol (2,4-DCP ). Twenty-four field trials were conducted in the United States and Canada during the 2009 growing season in the North American Free Trade Agreement (NAFTA) Growing Zones 2 (GA a nd AL; 3 trials), 4 (AR and MS; 4 trials), and 5 (IL, IN, MO, MI, OH, NE, IA, MN, and ON; 17 trials).

Each trial consisted of one untre ated and one treated plot. The plots were planted with soybeans containing the AAD-12 gene (Event DAS-68416-4 seed line). Each treated plot received three broadcast applications of a 3.79 lb ae (acid equivalents)/gal so luble concentrate liquid (SL/L) formulation of 2,4-D dimethylam ine salt (2,4-D DMA; Weedar ® 

### 64 Herbicide) at 0.95-1.10 lb ae/A/application, for a tota l seasonal application ra te of 2.97-3.13 lb ae/A. 2,4-D was applied as one preemergence application and two foliar appl ications, made ~12 days before the soybeans reached the R2 growth stage (ful l flowering stage), and at the ~R 2 growth stage. Retreatment intervals (RTIs) were 4-64 days between the firs t and second applications and 7-21 days between the second and third applications . Applications were made using ground equipment in spray volumes of 19.9-32.3 gal/A and included a non-ioni c surfactant (NIS) at all trial sites.

Soybean forage and hay were harvested at two preharvest intervals (P HIs), reflecting early harvest (targeted 7-day PHI following the second a pplication) and typical harvest (targeted 7-day PHI following the final application); samples of hay were allowed to dry in the field for 0-12 days prior to collection. Seed wa s harvested at maturity following three applications. PHIs were 6-8 days for forage and hay following two or three applications, and 51-103 days for seed. Additional samples of forage a nd hay were collected from one trial each conducted in GA and MI at PHIs of 0-1, 3-4, 7-8, 10-11, and 14-15 days ; additional seed samples for residue decline were collected at PHIs of 37- 56, 44-63, 51-70, 58-77, and 65-84 days.

Following two to three broadcast applications of the 3.79 lb ae/ gal SL/L formulation of 2,4-D DMA made as one preemergence application and one or two postemergence foliar applications at total seasonal applic ation rates of 1.97-2.10 lb ae/A for early -harvest forage and hay, and 2.97- 3.13 lb ae/A for typical harvest of all RACs, resi dues of 2,4-D (and per trial averages) were as follows: (1) <0.01-4.014 (<0.01-3.930) ppm in/on early-harvest forage (6- to 8-day PHI following application 2), <0.01-18.42 4 (<0.01-18.282) ppm in/on typical -harvest forage (6- to 8- day PHI), (2) <0.01-21.855 (<0.01-20.281) ppm in/ on early-harvest hay (6- to 8-day PHI following application 2), <0.01- 60.413 (<0.01-58.783) ppm in/on typi cal-harvest hay (6- to 8- day PHI), and (3) <0.01 (<0.01) ppm in/ on seed (51- to 103-day PHI).2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 17 of 23 Additionally, residues of 2,4-DC P (and per trial average re sidues) were <0.01-25.195 (<0.01- 22.451) ppm in/on early-harvest forage, <0.01- 36.966 (<0.01-32.080) ppm in/ on typical-harvest forage, <0.01-190.589 (<0.01-190.347) ppm in /on early-harvest hay, <0.01-132.961 (<0.01- 131.509) ppm in/on typical-harvest hay, a nd <0.01-0.054 (<0.01-0.047) ppm in/on seed.

The submitted residue decline data indicate that residues of 2,4-D generally decreased in/on forage and hay with increasing PHI. Residues of 2,4-D in/on seed were <LOQ in both decline studies; therefore, resi due decline could not be assessed. Residues of 2,4-DCP in forage, hay, and seed generally decreased or did not increase with increasing PHI.

Table 13. Summary of Residue Data fr om Soybean Field Trials with 2,4-D. Commodity Total Applic.

Rate (lb ae/A) PHI (days) Residue Levels 1 (ppm) n Sample Min. Sample Max. LAFT 2 HAFT 2 Median Mean Std. Dev.

2,4-D Soybean, early forage 1.97-2.10 6-8 24 <0.01 4.014 <0.01 3.930 0.272 0.696 1.146

Soybean, forage 2.97 - 3.13 6-8 24 <0.01 18.424 <0.01 18.282 0.202 1.420 3.900

Soybean, early hay 1.97-2.10 6-8 24 <0.01 21.885 <0.01 20.281 0.754 2.552 4.758

Soybean, hay 2.97 - 3.13 6-8 24 <0.01 60.413 <0.01 58.783 0.613 3.769 11.903 Soybean, seed 2.97 - 3.13 51-103 24 <0.01 <0.01 <0.01 0.01 0.01 0.01 N/A

2,4-DCP Soybean, early forage 1.97-2.10 6-8 24 <0.01 25.195 <0.01 22.451 10.772 9.521 7.264

Soybean, forage 2.97 - 3.13 6-8 24 <0.01 36.966 <0.01 32.080 14.729 13.329 9.783

Soybean, early hay 1.97-2.10 6-8 24 <0.01 190.589 <0.01 190.347 32.338 40.018 48.419

Soybean, hay 2.97 - 3.13 6-8 24 <0.01 132.961 <0.01 131.509 36.701 40.509 35.918 Soybean, seed 2.97 - 3.13 51-103 24 <0.01 0.054 <0.01 0.047 0.014 0.019 0.012

1 Except for sample min/max, values reflect per trial averages; n = no. of field trials. For ca lculation of median, mean, and standard deviation, the LOQ (0.01 ppm) wa s used for any results reported as ND (0 .003 ppm) or between the LOD and LOQ in Table 13. N/A = Not applicable.
2 LAFT = lowest average field trial; HAFT = highest average field trial.



### HED Conclusions : Pending submission of a revised la bel reflecting a PHI of 50 days for soybean, the submitted field trial data are adeq uate and support of the use of 2,4-D choline on AAD-12 soybean. The field trials reflected two to three broadcast applications of the 3.79 lb ae/gal SL/L formulation of 2,4-D DMA made as one preemergence application and one or two postemergence foliar applications to soybeans containing the AAD-12 gene at total seasonal application rates of 1.97-2.10 lb ae/A for early-harvest forage and hay and 2.97-3.13 lb ae/A for typical harvest.

Based on the study results, residues of 2,4-D (and per trial averages) may be up to 4.01 (3.93) ppm in/on early-harvest forage (6- to 8-day PHI following applic ation #2), up to18.4 (18.3) ppm in/on typical-harvest forage (6- to 8-day PHI), up to 21.8 (20.3) ppm in/on early-harvest hay (6- to 8-day PHI following applicati on #2), up to 60.4 (58.8) ppm in/on typical-harvest hay (6- to 8-2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 18 of 23 day PHI), and <0.01 (<0.01) ppm in/ on seed (51- to 103-day PHI).

Additionally, residues of 2,4-DCP (and per trial average residu es) were up to 25.2 (22.4) ppm in/on early-harvest forage, up to 40.0 (32.1) pp m in/on typical-harvest forage, up to 190 (190) ppm in/on early-harvest hay, up to133 (132) ppm in/on typica l-harvest hay, and up to 0.054 (0.047) ppm in/on seed.

The submitted residue decline data indicate that residues of 2,4-D generally decreased in/on forage and hay with increasing PHI. Residues of 2,4-D in/on seed were <LOQ in both studies; therefore, residue declin e could not be assessed. Residues of 2,4-DCP in forage, hay, and seed generally decreased or did not in crease with increasing PHI.

The trial numbers and geographical locations ar e adequate; application and harvest conditions reflect the proposed label except that the label specifies a PHI of 30 days for soybean and the minimum PHI in the study was 51 days for soybean seed. An acceptable method was used for quantitation of 2,4-D and 2,4-DCP.

Under the conditions and parameters used in the study, the field tria l residue data are classified as scientifically acceptable pe nding submission of a revised la bel which specifies a minimum PHI of 50 days for soybean.

The existing tolerances for 2,4-D use on soybean are adequate to cover the use of 2,4-D choline on AAD-12 soybean.

860.1520 Processed Food and Feed

48449802.de2


### Dow AgroSciences LLC has submitted a proces sing study for 2,4-D on soybean containing the inserted aryloxyalkanoate dioxygenase-12 ( AAD-12) gene (seed line Event DAS-68416-4). Expression of the AAD-12 protein encoded by the AAD-12 gene results in a trait that increases the herbicide tolerance of soybeans to 2,4- D via increased metabolism through a pathway involving the metabolite 2,4-dichlorophenol (2,4-D CP). Two trials were conducted in GA and NE during the 2009 growing season.
In both trials, soybean seed was harvested 66 days (GA-01) or 90 days (NE-16) following the last of three broadcas t applications of a 3.79 lb ae/gal soluble concentrate liquid (SL/L) formulation of 2,4-D dimethylam ine salt (2,4-D DMA; Weedar ® 

### 64 Herbicide) at 1.93-2.02 lb ae/A/application, for a to tal seasonal application rate of 5.97-6.00 lb ae/A. 2,4-D was applied as one preemergence application and two foliar appli cations. Retreatment intervals (RTIs) were 4- 33 days between the first and second applica tions and 13 days between the second and third applications. Applications were made usi ng ground equipment in spray volumes of 21.9-32.36 gal/A and included a non-ionic surfac tant (NIS) at all trial sites. The petitioner noted that the application rate used in the study was only 2x th at used in the corres ponding crop field trials (refer to the 48449802.de1) due to concerns over the potential for crop injury

Samples of soybean seed were processed into as pirated grain fractions (AGF), meal, hulls, and alkali-refined oil by GLP Technologies (Navasota, TX) using procedures simulating commercial processing.2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 19 of 23 Residues of 2,4-D were below the LOQ (<0.01 ppm) in/on all samples of soybean seed RAC, meal, hulls, and alkali-refined oil; therefore, no processing factors were calculated. Residues of 2,4-D were 0.012-0.016 ppm in AGF. The processi ng data indicate that residues of 2,4-D concentrate in AGF (average processing factor of >1.4x). Average residues of 2,4-DCP were 0.012-0.109 ppm in/on seed prior to AGF ge neration (RAC), 0.074-0.831 ppm in AGF, 0.015- 0.155 ppm in meal, <0.01-0.050 in hulls, and below the LOQ (<0.01 ppm) in alkali-refined oil. The processing data indicate th at residues of 2,4-DCP concentrat e in AGF (average processing factor of 6.9x) and meal (average processing fa ctor of 1.3x) but do not concentrate in alkalirefined oil or hulls (average processing factors of <0.5x and <0.7x, respectively).

The processing factors calculated in this st udy were less than the maximum theoretical concentration factors of 11.3x for soybean hulls, 2.2x for meal, and 12.0x for oil (based separation of components; OPPTS 860.1520, Table 3).

A summary of residue data from the soybean pr ocessing study is presen ted in Table 14.

Table 14. Residue Data from Soyb ean Processing St udy with 2,4-D. RAC Processed Commodity Total Rate (lb ae/A) PHI (days) Residues (ppm) 1 [Average] Processing Factor 2
2,4-D 2,4-DCP 2,4-D 2,4-DCP

Sycamore, GA; 2009 (GA-01) Soybean RAC 3 : Seed (prior to AGF generation) 6.00 66 ND, ND, ND [<0.01] 0.121, 0.107, 0.099 [0.109] -- --

Seed (after AGF generation) ND, ND, ND [<0.01] 0.088, 0.101, 0.082 [0.090] -- --

AGF 0.016 0.831 >1.6x 7.6x Meal ND, ND [<0.01] 0.158, 0.152 [0.155] NC 4 1.4x

Hulls ND, ND [<0.01] 0.053, 0.046 [0.050] NC 0.5x

Alkali-refined oil ND, ND [<0.01] ND, ND [<0.01] NC <0.09x

York, NE; 2009 (NE-16) Soybean RAC 3 : Seed (prior to AGF generation) 5.97 90 ND, ND, ND [<0.01] 0.011, 0.013, 0.011 [0.012] -- --

Seed (after AGF generation) ND, ND, ND [<0.01] 0.011, (0.009), 0.012 [<0.011] -- --

AGF 0.012 0.074 >1.2x 6.2x Meal ND, ND [<0.01] 0.015, 0.015 [0.015] NC 1.3x

Hulls ND, ND [<0.01] (0.004), ND [<0.01] NC <0.8x

Alkali-refined oil ND, ND [<0.01] ND, ND [<0.01] NC <0.8x2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 20 of 23 Table 14. Residue Data from Soyb ean Processing St udy with 2,4-D. RAC Processed Commodity Total Rate (lb ae/A) PHI (days) Residues (ppm) 1 [Average] Processing Factor 2
2,4-D 2,4-DCP 2,4-D 2,4-DCP

Average Soybean RAC 3 : Seed (prior to AGF generation) 5.97-6.00 66-90 -- --

AGF >1.4x 6.9x
Meal NC 1.3x
Hulls NC <0.7x
Alkali-refined oil NC <0.5x

1 ND = Nondetectable (<LOD). The LOD was 0.003 ppm for all analytes in all ma trices; the LOQ was 0.01 ppm for all analytes. Residues between the LOD and LOQ are reported in parentheses. When calculating per trial averages, the LOQ was used for values reported as ND or between the LOD and LOQ.
2 Processing Factor = [Measured residue for analyte in the pro cessed fraction] / [Measured residue for analyte in the RAC].
3 The residues for RAC: Seed (prior to AGF generati on) were used for calculating processing factors.
4 NC = Not calculated. The processing fa ctor could not be calculated because resi dues were below the LOQ in the RAC and the processed fraction.



### HED Conclusions: The submitted soybean processing study is acceptable. Processing factors for 2,4-D could not be calculated for alkali-refined oil, meal, and hulls because residues were below the LOQ in/on all samples of soybean seed and pr ocessed commodities. The data indicated that residues of 2,4-D may concentr ate in AGF (average proces sing factor of >1.4x). The current tolerance for residues of 2,4-D on AGF (40 ppm) is adequate to support the proposed use of 2,4-D choline on soybeans.

Residues of 2,4-DCP may concentrate in AGF and meal (average processing factors of 6.9x and 1.3x, respectively) but do not con centrate in alkali-refined oil a nd hulls (processing factors of <1x).

The observed concentration factor s were less than the theoretical concentration factors. An acceptable method was used for the determinatio n of residues of 2,4-D and 2,4-DCP in soybean seed and processed commodities.

860.1650 Submittal of Analytic al Reference Standards

Analytical standards for 2,4-D are currently avai lable in the EPA National Pesticide Standards Repository (personal communication with Theres a Cole of the US EPA National Pesticide Standard Repository, 08/31/2011).

860.1850/860.1900 Confined/Field Accumula tion in Rotational Crops

A confined rotational crop study has been submitted and reviewed, and is acceptable.

No residues of concern or 2,4-D metabolites were detected at  0.01 ppm in any rotated crop matrix from either the 30- or 139-day plant-back intervals (PBIs). The data indicate that 14 

### Cresidues in rotated crops are due pr imarily to the incorporation of radioactivity into natural plant constituents. Limited field studies and rotationa l crop tolerances are not required for residues of 2,4-D. No plant-back restriction following appl ication of 2,4-D is required (D207980, D. Miller, 11/30/95).2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 21 of 23 HED conclusions: The requirements for confined/field ro tational crop studies are satisfied. No PBI or rotational crop tolerances are required to support the new use.

860.1550 Proposed Tolerances

Tolerances for 2,4-D residues are listed in 40 CFR §180.142. HED has determined that the residue of concern for tolerance expression is 2, 4-D. The maximum soybean seed residues in the submitted field trial data do not exceed the cu rrent tolerance of 0.02 pp m for residues of 2,4-D on soybean seed. However, revisions to the curr ent tolerance are recommended for international harmonization. In order to harmonize with th e Codex maximum residue levels (MRLs) for soybean seed the existing U.S. tolerance for s oybean seed (0.02 ppm) should be decreased to 0.01 ppm. This revision will also support the pro posed soybean use. A tolerance is established for AGF that will cover residues in/on soybean AGF . A livestock feeding/grazing restriction for this use is proposed; ther efore, the current tolerances for re sidues of 2,4-D on soybean forage and hay are not being reevalua ted at this time.

References

2,4-D. Summary of Analytical Chemistry and Re sidue Data for Use of 2,4-D Choline in/on Herbicide Tolerant Field Corn Containing the Aryloxyalkanoate Dioxygenase-1 (ADD-1) Gene. D384441. Alexandra LaMay, 10/27/11.

2,4-D. HED’s Revised Human Health Risk A ssessment for the Reregistration Eligibility Decision (RED) Revised to Reflect Public Comments , Timothy Dole, 5/12/2005.

2,4-D. PC Code No. 030001, Case No. 0073. DP Barcode D309450. Reregistration Eligibility Decision Revised Residue Ch emistry Considerations. 10/12/2004.

2,4-D: Residue Analytical Method for Livestock Commodities. D339654. Toiya Goodlow, 1/14/2009.

2,4-D. (030001) Magnitude of the Residue (soybeans) and Confined Rotational Field Trials (lettuce, wheat, radish). GDLNs 171-4(k ), 165-1. DP Barcode: D207980; CBRS No. 14458; MRID Nos.: 433563-01, 433563-02 and 433560-02; Case No. 0073. David J. Miller, 11/30/1995.

2, 4-D. Preliminary Data Provided to Support the Stability of 2, 4-D and 2, 4-DCP for Transgenic Soybean and Corn Commodities Held in Frozen Storage . D394948. Peter Savoia. 10/27/2011.2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 22 of 23 Attachment 1: Chemical Structures

Name Structure Common name: 2,4-D cholineIUPAC Name: 2-hydroxy-N,N,Ntrimethylethanaminium (2,4- dichlorophenoxy)acetate CAS Name: Ethanamini um, 2-hydroxy-N,N,Ntrimethyl-, 2-(2,4-dichlorophenoxy)acetic acid hydroxide (1:1:1) CAS No.: 1048373-72-3

Common name: 2,4-D acid IUPAC name: (2,4-dichlorophenoxy)acetic acid CAS name: 2-(2,4-dichlorophenoxy)acetic acid CAS No.: 94-75-7

Choline cation

Common name: 2,4-DCP IUPAC name: 2,4-Dichlorophenol CAS No.: 120-83-2

Common name: 2,4-D DMA 2,4-D dimethylamine salt (DMA) IUPAC name: (2,4-dichlorophenoxy)acetic acid - dimethylamine (1:1) CAS name: 2-(2,4-dichlorophenoxy)acetic acid compound with N-methylethanamine (1:1) CAS No.: 2008-39-12,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085



### Page 23 of 23 Name Structure Metabolite A; Acetyl-glucose-DCP conjugate; Peak D

Metabolite B (X11963417); Glucose conjugate of 2,4-DCP; Peak A

2,4-dichlorophenyl β -D-glucopyranoside

Metabolite C; 2,4-DCP disaccharide Peak E2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.3/0CSPP 860

. 1 380 /0ECD IIA 6.1.1 and IliA 8.1.1 Storage Stability

- Soybean



### Reviewed and Approved by

# ~ -s

_ _ 

### Date: 7/8/13**=-a _e_** **tt _e-=** **o=-r_e_w ,-=c~h** **=e= m~is** **:;;;; ;;,** **, HE** **~:::::: D~RA:--""'B~** **v= ==** **==- --** 

Thi s DER was originally prepared b y Summitec Corporation (9724 Kingston Pike, Suite 602, Knoxville

, TN 37922; submitted 6/13/13) an d subcon tr actor CSS -D ynamac Corporation (10301 Democracy La n e , Suite 300

, Fa i rfax, VA 22030). T he DER has been reviewed by the Health Effects Division (HED) and re vised to r eflect cu rr ent Office of Pest i c id e Programs (OPP) policies.



### STUDY REPORT:



### 49122501, Li, Q. and Wendelburg, B.M. (2013) Frozen Storage Stability of2,4-D and 2,4-DCP in Soybean- Amended Report. Lab Study Number: 101561. Unpublished study prepared b y Dow AgroSciences LLC

. 52 pages.

Note that MRID 49122501 (amended report) rep l aces MRID 490 75 901 (not r eviewed).

EXECUTIVE SUMMARY:



### Dow AgroSciences LLC has submitted the results of a storage stability study with 2

, 4

- D and 2,4

- DP in transgenic soybean. Samples of untreated soybean forage, hay, and seed were fortified with 2,4-D and 2,4

- DCP at 0.10 ppm each. Fortified samples were stored frozen (approximately -2 0 °C) and analyzed at intervals of approximately 0, 1, 2

, 5, 8, 25, and 28 months.

Samples were analyzed for residues of2

, 4-D and 2,4-DCP using a high performance liquid chroma t ography method with tandem mass spectrometry detection (LC / MS / MS)

. The method

was adequate for data collection based on acceptable concurrent method recoveries

. The limits of quantitation (LOQs) were 0

. 01 ppm for each analyte

.

Inte r im results of the storage stability study through 8 months we r e previously submitted and reviewed under DP# 394948 (P

. Savoia

, 10 / 27111)

. The final storage stabilit y results indicate that residues of 2,4-D and 2,4

- DCP are relatively stable inion soybean forage

, hay, and seed stored frozen for up to 2 7. 8 months.

STUDY fW 

### AIVER ACCEPTABILITY /DEFICIENCIES /C LARIFICATIONS:



### The storage stability data are classified as scientifically acceptable

. The acceptability of this study for regulatory purposes is addressed in the forthcoming U.S

. EPA Residue Chemistry Summary Document [DP# 400085]

.

COMPLIANCE:



### Signed and dated Good Laboratory Practice (GLP), Qualit y A ssur an ce, and Data Confidentiality
statements were provided

. No deviations from regulatory requirements we re reported which would ha v e an im pact on t he validity ofthe study

.

DP # 4118 73 /MRID N o

. 49 1 2 2 501 Page 1 of 82,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.3/OCSPP 860.1380/OECD IIA 6.1.1 and IIIA 8.1.1 Storage Stability – Soybean

DP# 411873/MRID No. 49122501 Page 2 of 8 

### A. BACKGROUND INFORMATION

2,4-Dichlorophenoxyacetic acid (2,4-D) is an alkyl chlorophenoxy herbicide used to control a variety of broadleaf weeds. It is a longstandi ng active ingredient regist ered for a variety of food/feed uses. It may be applied as broadcast, banded, or directed (spr ay or wiper) treatments during dormancy or preplant, preharvest, pr eemergence, emergence, postemergence, or postharvest using ground or aeria l equipment. Permanent tolerances for 2,4-D are established under 40 CFR 180.142 for a wide variety of field, fruit, and vegetable crops which include aquatic sites, as well as numerous livestock commodities.

The chemical structure and nomenclature of 2,4-D and 2,4-DCP, and the physicochemical properties of the technical gr ade of 2,4-D are presented in Tables A.1 and A.2.

TABLE A.1. Test Compound Nomenclature. Compound

O Cl Cl

OH

O

Common name 2,4-D; 2,4-D acid IUPAC name (2,4-dichlorophenoxy)acetic acid CAS name 2-(2,4-dichlorophenoxy)acetic acid CAS registry number 94-75-7 End-use product/(EP) Not appl icable to this submission Compound

Cl

Cl OH

Common name 2,4-DCP Chemical name 2,4-dichlorophenol CAS registry number 120-83-2

TABLE A.2. Physicochemical Properties of the Technical Grade Test Compound: 2,4-D.
Parameter Valu e Reference Melting point/range 138-141 °C DP# 309452, T. Jimerson, 10/13/04 pH Not available Density 1.416 at 25 °C (specific gravity) Water solubility 569 mg/L at 20 °C2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.3/OCSPP 860.1380/OECD IIA 6.1.1 and IIIA 8.1.1 Storage Stability – Soybean

DP# 411873/MRID No. 49122501 Page 3 of 8

TABLE A.2. Physicochemical Properties of the Technical Grade Test Compound: 2,4-D.
Parameter Valu e Reference Solvent solubility g/100 g at 25 °C acetone 85.0 benzene 1.07 diethyl ether 220 ethanol 130.0 isopropanol 31.6 toluene 0.067 xylene 0.58 Vapor pressure 1.4 x 10 -7 mm Hg at 25 °C Dissociation constant (pK a ) Not available Octanol/water partition coefficient Log(K OW ) In 0.001 M solution: 2.14 at pH 5 0.177 at pH 7 0.102 at pH 9 UV/visible absorption spectrum Not available



### B. EXPERIMENTAL DESIGN

B.1. Sample Handling and Preparation

Samples of untreated transgenic soybean raw agricultural commodities (RACs) forage, hay, and seed from a previous field trial st udy (Study ARA-09-15-11, 2011, MRID 48449802; DP# 400085) were placed into high-density polyethylene (HDPE) containers and fortified with 2,4-D and 2,4-DCP at 0.10 ppm each. MRID 48449802 indica ted that samples from that study were ground and blended. For each commodity and samp ling interval, three sub-samples per analyte were designated for storage samples; in additi on, one untreated control sample and two control samples to be used for concurrent recoveries were included in the sample set for each commodity and each analysis sample set. Fortified and unf ortified samples were then stored frozen at approximately

 20 °C and analyzed at intervals of approximately 0, 1, 2, 5, 8, 25, and 28 months. For fortification of all stored samples as well as fresh fortification samples, the mixed 2,4-D and 2,4-DCP standards were prepared in me thanol. Sample sets were prepared and fortified on three separate dates to allow for th e simultaneous extraction a nd analysis of the 0- and 8-month, 2- and 25-month, and 5- and 28-month storage interval samples. The fortification and analysis dates are pres ented in the table below.

TABLE B.1. Dates of Fortification and Extraction.

Sampling Event Fortification Date Analysis Date* Actual Storage Interval Days Months
0 month 4/8/2011 4/8/2011 0 0
1 month 8/3/2010 9/3/2010 31 1.0
2 month 7/12/2012 9/10/2012 60 2.0
5 month 7/12/2012 11/27/2012 138 4.5
8 month 8/3/2010 4/8/2011 248 8.1
25 month 8/3/2010 9/10/2012 769 25.3
28 month 8/3/2010 11/27/2012 847 27.82,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.3/OCSPP 860.1380/OECD IIA 6.1.1 and IIIA 8.1.1 Storage Stability – Soybean

DP# 411873/MRID No. 49122501 Page 4 of 8 *Analysis date is also the date of extraction.


### [We note that although the study includes a 0-day sampling interval, no samples with storage intervals ≥ 1 month were actually analyzed on the day of fortification. The petitioner is reminded that the reason for including a 0-day sampling interv al in a storage stability study is to establish the residue levels present at the time sa mples are placed into storage (see OCSPP 860.1380(d)(6)(i))].

B.2. Analytical Methodology

Samples of soybean forage, hay, and seed were analyzed for residues of 2,4-D or 2,4-DCP by the Dow AgroSciences LLC, Regulatory Scien ces and Government Affairs Laboratory (Indianapolis, IN) using an LC/MS/MS method. A summary of the method was included in the submission, along with instrument parameters.

Briefly, samples were extracted with methanol: 1.0 N sodium hydroxide (90:10, v:v). The extract was isolated by centrifugation and a mixed internal standard of 13 

### C 6 

### -2,4-D and 13 

### C 6 

### -2,4-DCP was added. The mixture was concentrated, acidif ied with 2 N hydrochloric acid, and heated at 90 °C for one hour. Upon cooling, the extract was diluted with meth anol and filtered (if necessary) prior to determination by in-line solid phase (C18) extraction and LC/MS/MS analysis. The ion transitions used for quantitation were 219.0/161.0 m/z for 2,4-D and 161.0/125.0 m/z for 2,4-DCP.

The LOQ is 0.01 ppm for each analyte. The c oncurrent recoveries for each analyte were conducted at the same level as th e fortified samples (0.10 ppm).

C. RESULTS AND DISCUSSION

Concurrent recovery data for th e storage stability study are repo rted in Table C.1. The data indicate that the LC/M S/MS method was adequate for the de termination of residues of 2,4-D and 2,4-DCP in soybean forage, hay, and seed. Concu rrent recoveries were within the acceptable range of 70-120%. It was reported that 2,4-DCP was consistently observed at the LOD and 2,4- D was occasionally observed at the LOD in/on unfortified samples; the actual results for unfortified samples were not reported in MRID 49122501. Residue results in the concurrent recovery and stored samples were reportedly corrected by th e petitioner for any apparent residues in unfortified samples (as found in study MRID 48449802). Adequate sample chromatograms and calculations were provided.

The results of the storage stabili ty study are presented in Table C .2. Graphs of residue stability of 2,4-D and 2,4-DCP in soybean commodities ar e presented in Figures C.1 and C.2. The storage stability results indicat e that residues of 2,4-D and 2,4-DC P are relatively stable in/on soybean forage, hay, and seed stored frozen for up to 27.8 months.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.3/OCSPP 860.1380/OECD IIA 6.1.1 and IIIA 8.1.1 Storage Stability – Soybean

DP# 411873/MRID No. 49122501 Page 5 of 8

TABLE C.1. Summary of Concurrent Recoveries of 2,4-D and 2,4- DCP from Soybean Commodities. Matrix Analyte Spike Level (ppm) Storage Interval 1 (months) Sample Size (n) Recoveries 2 (%) Mean Recovery 3 (%) Soybean forage 2,4-D 0.10 0, 8.1 2 93, 99 96 1.0 2 96, 100 98 2.0, 25.3 2 93, 95 94 4.5, 27.8 2 93, 97 95 2,4-DCP 0.10 0, 8.1 2 86, 90 88 1.0 2 78, 92 85 2.0, 25.3 2 88, 95 92 4.5, 27.8 2 93, 94 94 Soybean hay 2,4-D 0.10 0, 8.1 2 87, 91 89 1.0 2 94, 97 96 2.0, 25.3 2 93, 102 98 4.5, 27.8 2 92, 93 93 2,4-DCP 0.10 0, 8.1 2 82, 89 86 1.0 2 87, 88 88 2.0, 25.3 2 102, 102 102 4.5, 27.8 2 97, 99 98 Soybean seed 2,4-D 0.10 0, 8.1 2 91, 95 93 1.0 2 96, 106 101 2.0, 25.3 2 93, 96 95 4.5, 27.8 2 94, 95 95 2,4-DCP 0.10 0, 8.1 2 90, 96 93 1.0 2 94, 95 95 2.0, 25.3 2 95, 103 99 4.5, 27.8 2 97, 102 100

1 Samples for the 0-month storage interval were fortified conc urrently and analyzed with sa mples for the 8.1-month storage interval; samples for the 2.0-month storage interval were analyz ed with samples for the 25.3-month storage interval; and sample s for the 4.5-month storage interval were analyzed w ith samples for the 27.8-month storage interval.
2 Recoveries were corrected by the petitioner for apparent residues in controls (see MRID 48449802) .
3 Calculated by the study reviewer.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.3/OCSPP 860.1380/OECD IIA 6.1.1 and IIIA 8.1.1 Storage Stability – Soybean

DP# 411873/MRID No. 49122501 Page 6 of 8 

### FIGURE C.1. Graph of Stability of 2, 4-D Residues in Soybean RACs at ~

 20 °C.

60 70 80 90 100 110 120

0 5 10 15 20 25 30CorrectedRecovery(%)

Storage Interval (months) 

### 2,4

‐ D Stability in Soybean Commodities

Forage
Hay

Seed



### FIGURE C.2. Graph of Stability of 2, 4-DCP Residues in Soybean RACs at ~

 20 °C.

60 70 80 90 100 110 120

0 5 10 15 20 25 30CorrectedRecovery(%)

Storage Interval (Months) 

### 2,4

‐ DCP Stability in Soybean Commodities

Forage
Hay

Seed2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.3/OCSPP 860.1380/OECD IIA 6.1.1 and IIIA 8.1.1 Storage Stability – Soybean

DP# 411873/MRID No. 49122501 Page 7 of 8

TABLE C.2. Stability of 2,4-D and 2,4-DCP Residues in Soybean Commodities Following Storage at

~  20 °C. Commodity Spike Level (ppm) Storage Interval (months) Recovered Residues (ppm) 1 Mean Recovered Residues 2 (ppm) Mean Recovery 2 (%) Corrected Recovery 3 (%) 2,4-D Soybean forage 0.10 0 0.100, 0.091, 0.096 0.096 96 100 1.0 0.094, 0.101, 0.096 0.097 97 99 2.0 0.094, 0.095, 0.094 0.094 94 101 4.5 0.093, 0.097, 0.095 0.095 95 100 8.1 0.090, 0.092, 0.096 0.093 93 97 25.3 0.097, 0.091, 0.092 0.093 93 99 27.8 0.093, 0.092, 0.097 0.094 94 99 Soybean hay 0.10 0 0.094, 0.094, 0.095 0.094 94 106 1.0 0.104, 0.099, 0.093 0.099 99 104 2.0 0.093, 0.098, 0.094 0.095 95 98 4.5 0.088, 0.095, 0.093 0.092 92 100 8.1 0.090, 0.088, 0.088 0.089 89 100 25.3 0.090, 0.089, 0.091 0.090 90 92 27.8 0.091, 0.092, 0.086 0.090 90 97 Soybean seed 0.10 0 0.095, 0.101, 0.100 0.099 99 106 1.0 0.096, 0.097, 0.102 0.098 98 98 2.0 0.090, 0.088, 0.084 0.087 87 92 4.5 0.092, 0.089, 0.088 0.090 90 95 8.1 0.092, 0.099, 0.093 0.095 95 102 25.3 0.089, 0.089, 0.086 0.088 88 93 27.8 0.093, 0.096, 0.092 0.094 94 99 2,4-DCP Soybean forage 0.10 0 0.080, 0.087, 0.086 0.084 84 96 1.0 0.086, 0.085, 0.078 0.083 83 98 2.0 0.093, 0.092, 0.088 0.091 91 99 4.5 0.089, 0.095, 0.094 0.093 93 100 8.1 0.079, 0.080, 0.093 0.084 84 96 25.3 0.085, 0.076, 0.076 0.079 79 86 27.8 0.083, 0.088, 0.079 0.083 83 89 Soybean hay 0.10 0 0.090, 0.094, 0.097 0.094 94 109 1.0 0.108, 0.095, 0.085 0.096 96 110 2.0 0.099, 0.095, 0.098 0.097 97 96 4.5 0.101, 0.105, 0.103 0.103 103 105 8.1 0.092, 0.081, 0.128 4 0.087 87 101 25.3 0.087, 0.103, 0.090 0.093 93 92 27.8 0.082, 0.087, 0.086 0.085 85 872,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.3/OCSPP 860.1380/OECD IIA 6.1.1 and IIIA 8.1.1 Storage Stability – Soybean

DP# 411873/MRID No. 49122501 Page 8 of 8

TABLE C.2. Stability of 2,4-D and 2,4-DCP Residues in Soybean Commodities Following Storage at

~  20 °C. Commodity Spike Level (ppm) Storage Interval (months) Recovered Residues (ppm) 1 Mean Recovered Residues 2 (ppm) Mean Recovery 2 (%) Corrected Recovery 3 (%) Soybean seed 0.10 0 0.096, 0.100, 0.097 0.098 98 105 1.0 0.091, 0.094, 0.092 0.092 92 98 2.0 0.097, 0.093, 0.096 0.095 95 96 4.5 0.089, 0.094, 0.096 0.093 93 94 8.1 0.087, 0.088, 0.088 0.088 88 94 25.3 0.088, 0.087, 0.092 0.089 89 89 27.8 0.097, 0.092, 0.094 0.094 94 94

1 Uncorrected recoveries as reported by the petitioner.
2 Mean recovered residues and recovery calculated by the study reviewer.
3 Recoveries were corrected by the petitioner for apparent residues in controls and for the average concurrent recoveries (see Table C.1).
4 This value was considered an outlier compared to the othe r two recoveries and was not used in determining the average recovered residues.



### D. CONCLUSION

The storage stability data are adequate and de monstrate that residues of 2,4-D and 2,4-DCP are relatively stable in/on soybean forage, hay, and s eed stored frozen for up to about 28 months.

E. REFERENCES

DP#: 394948 Subject: 2,4-D. Preliminary Data Provided to Support the Stability of 2,4-D and 2,4-DCP for Transgenic Soybean and Corn Commodities Held in Frozen Storage. From: P. Savoia To: M. Walsh Date: 10/27/2011 MRID: 48613901

F. DOCUMENT TRACKING

DP#: 411873 PC Code: 030001

Template Version June 2005.2,4

- D / PC Code 030001/Dow AgroSc i ences LLC DACO 7.4.1 17.4.2 / 0PPTS 860.1500 / 0ECD IIA 6.3.1, 6.3.2, 6

. 3.3 and IliA 8

. 3.1, 8.3.2, 8.3

. 3 Crop Field Trial/ Residue Decline

- 2

, 4

- D-Tolerant Soybean (AAD

- 12, Event 68416

- 4)



### Reviewed and Approved by 

# ---- Danette Drew, Chemist, HED RABV Date: 3/4112

Note: This DER was originally prepared under contract by Versar
, Inc

. (6850 Versar Center

, Springfield, VA 22151 ; submitted 1 / 24/12)

. The DER has been reviewed by t h e Health Effects Division (HED) and revised to reflec t current Office of Pesticide Programs (OPP) policies.



### STUDY REPORTS:

48449802 Culligan, J. (2011) Magnitude ofthe Residue of2,4

- D inion Herbicide Tolerant Soybeans Containing the Aryloxyalkanoate Dioxygenase-12 (AAD

- 12) Gene

. Project Study Number: ARA-09-15

- 11; Dow AgroSciences Study No. 090053. Unpublished study prepared by Ag Research Associates, LLC. 338 p.



### EXECUTIVE SUMMARY:

Dow AgroSciences LLC has submitted field trial data for 2,4

- D on soybean containing the inserted aryloxyalkanoate dioxygenase-12 (AAD

- 12) gene (seed line Event DAS

- 68416-4)

. Expre ssion of the AAD -12 protein encoded by the AA D

- 12 gene results in a trait that increases the herbicide tolerance of soybean to 2

, 4-D via increased metabolism through a pathway involving the metabolite 2,4-dichlorophenol (2,4

- DCP)

. Twenty

- four field trials were conducted in the United States and Canada during the 2009 growing season in the North American Free Trade Agreement (NAFTA) Growing Zones 2 (GA and AL; 3 trials)

, 4 (AR and MS; 4 trials), and 5 (IL, IN, MO

, MI, OH, NE, lA, MN, and ON; 17 trials)

.

Each trial coqsisted of one untreated and one treated plot. The plots were planted with soybeans containing the AAD

- 12 gene (Event DAS

- 68416

- 4 seed line)

. Each treated plot received three broadcast appl i cations of a 3.79lb ae (acid equivalents) / gal soluble concentrate liquid (SLIL) formulation of2

, 4-D dimethylamine salt (2,4-D DMA ; Weedar ® 64 Herbicide) at 0.95

- 1.10 lb ae/A/application, for a total seasonal application rate of2

. 97

- 3.13 lb ae / A. 2,4-D was applied a s

one preemergence application and t wo foliar applicat ions

, made**~** 

### 12 days before the soybeans reached the R2 growth stage (full flowering stage)

, and at the**~R2** 

### growth stage. Retreatment intervals (R Tis) were 4-64 days between the first and second applications and 7-21 days between the second and third appl i cations

. Applications were made using ground equipment in spray volumes of 19.9-32

. 3 gal / A and included a non-ionic surfactant (NIS) at all tria l sites.

Soybean forage and hay were harvested at two preharvest intervals (PHis), reflecting ear l y harvest (targeted 7

- day PHI following the second application) and typical har ve st ( t ar geted 7-day PHI following the final application); samples of hay were allowed to dry in the fi eld for 0-12 days prior to collection

. Seed was harvested at maturity following three app lications

. PHis were 6-8 days for forage and hay follow ing two or three applicat ions

, and 51-103 days for s eed. Additiona l samples of forage and hay were collected from one trial each conducted in GA and MI at PHis of0-1

, 3 -4

, 7-8, 10-11

, and 14-15 days ; ad dition a l seed samples fo r residue decline

were collected at PHis of 3 7-56, 44

- 63

, 51-70

, 58-77, and 65 -84 days.

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. 4 84 4 9802.del Page I of212,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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### Samples were analyzed for residues of 2,4-D and metabolite 2,4-DCP using a high performance liquid chromatography method with tandem mass spectrometry detection (LC/MS/MS). The limit of quantitation (LOQ, equivalent to the lo west level of method validation; LLMV) was 0.01 ppm each for 2,4-D and 2,4-DCP in all matrices. The method was adequate for data validation based on acceptable concurrent recovery data. The fortification levels used in concurrent method recovery were adequate to bracket expected residue levels. Concurrent recoveries were corrected for apparent residues in controls; residues in treated samples were not corrected for residues in controls.

Samples were stored frozen (~-20 °C) from harv est to extraction for 2,4-D and 2,4-DCP for up to 461 days (15.2 months) for forage, 460 days (15.1 months) for hay, and 405 days (13.3 months) for seed. The interval between extraction and analysis was not reporte d. Adequate storage stability data are available fo r 2,4-D indicating that residues are stable und er frozen storage conditions for at least 12 months in/on a variet y of field crop commodities including corn grain, forage, and fodder; sorghum grain; wheat grain, forage, and straw; rice grain; grass forage and hay; and soybean seed. Interim storage stab ility data (DP #394948) ar e available indicating residues of 2,4-DCP are stable for up to 8 mont hs in soybean commodities. The available storage stability data for 2,4-D are adequate to support sample storage conditions and durations; however, confirmatory storage st ability data are required for 2,4- DCP reflecting frozen storage for up to 13.3-15.2 months in/on soybean forage, hay, and seed and should reflect storage in High-Density Polyethylene (HDPE) contai ners (as used in this study).

Following two to three broadcast applications of the 3.79 lb ae/ gal SL/L formulation of 2,4-D DMA made as one preemergence application and one or two postemergence foliar applications at total seasonal applic ation rates of 1.97-2.10 lb ae/A for ear ly-harvest forage and hay and 2.97- 3.13 lb ae/A for typical harvest of all RACs, resi dues of 2,4-D (and per trial averages) were as follows: (1) <0.01-4.014 (<0.01-3.930) ppm in/on early-harvest forage (6- to 8-day PHI following application 2), <0.01-18.42 4 (<0.01-18.282) ppm in/on typical -harvest forage (6- to 8- day PHI), (2) <0.01-21.855 (<0.01-20.281) ppm in/ on early-harvest hay (6- to 8-day PHI following application 2), <0.01- 60.413 (<0.01-58.783) ppm in/on typi cal-harvest hay (6- to 8- day PHI), and (3) <0.01 (<0.01) ppm in/ on seed (51- to 103-day PHI).

Additionally, residues of 2,4-DC P (and per trial average re sidues) were <0.01-25.195 (<0.01- 22.451) ppm in/on early-harvest forage, <0.01- 36.966 (<0.01-32.080) ppm in/ on typical-harvest forage, <0.01-190.589 (<0.01-190.347) ppm in /on early-harvest hay, <0.01-132.961 (<0.01- 131.509) ppm in/on typical-harvest hay, a nd <0.01-0.054 (<0.01-0.047) ppm in/on seed.

The submitted residue decline data indicate that residues of 2,4-D generally decreased in/on forage and hay with increasing PHI. Residues of 2,4-D in/on seed were <LOQ in both decline studies; therefore, resi due decline could not be assessed. Residues of 2,4-DCP in forage, hay, and seed generally decreased or did not increase with increasing PHI.

STUDY/WAIVER ACCEPTABILITY/ DEFICIENCIES/CLARIFICATIONS :2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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### Under the conditions and parameters used in the study, the field tria l residue data are tentatively classified as scientifically acceptable pending subm ission of storage stability data demonstrating the stability of 2,4-DCP in/on soybean forage, hay, and seed stored frozen for up to 13.3-15.2 months. The acceptability of this study for regulatory purposes is addressed in the forthcoming U.S. EPA Residue Chemistry Summary Document, DP# 400085.

COMPLIANCE :
Signed and dated Good Laboratory Practice (GLP), Quality Assurance, a nd Data Confidentiality statements were provided. No deviations fr om regulatory requirement s were reported which would have an impact on the validity of the study.

A. BACKGROUND INFORMATION

2,4-Dichlorophenoxyacetic acid (2,4-D) is an alkyl chlorophenoxy herbicide used to control a variety of broadleaf weeds. These data we re submitted to support the proposed use for 2,4-D choline application to soybean s containing the inserted aryl oxyalkanoate dioxygenase-12 (AAD12) gene. This soybean is identified as c ontaining DAS-68416-4. Expression of the AAD-12 protein encoded by the AAD-12 gene results in a trait that increas es the herbicide tolerance of soybean to 2,4-D via increased metabolism through a pathway involving the metabolite 2,4- dichlorophenol (2,4-DCP).

The chemical structure and nomenclature of 2,4-D, 2,4-D DMA, and 2,4-D choline are summarized in Table A.1, and the physicochemical properties are summarized in Table A.2.

TABLE A.1. Test Compound Nomenclature. 2,4-D

Common name 2,4-D acid IUPAC name (2,4-dichlorophenoxy)acetic acid CAS name 2-(2,4-dichlorophenoxy)acetic acid
CAS # 94-75-7
PC Code 030001 End-use product/(EP) Weedar® 642,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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TABLE A.1. Test Compound Nomenclature. 2,4-D dimethylamine salt (DMA)

IUPAC name (2,4-dichlorophenoxy)ace tic acid - dimethylamine (1:1) CAS name 2-(2,4-dichlorophenoxy)acetic ac id compound with N-methylethanamine (1:1)
CAS # 2008-39-1
PC Code 030019 End-use product/(EP) 3.79 lb ae /gal SL/L formulation (Weedar ® 64 Herbicide) 2,4-D choline

IUPAC name 2-hydroxy-N,N,N-trimethyleth anaminium (2,4-dichlorophenoxy)acetate CAS name Ethanaminium, 2-hydroxy-N,N,N-trim ethyl-, 2-(2,4-dichlorophenoxy)acetic acid hydroxide (1:1:1)
CAS # 1048373-72-3
PC Code 051505 2,4-DCP

2,4-dichlorophenol

TABLE A.2. Physicochemical Properties of the T echnical Grade Test Co mpound: 2,4-D acid.
Parameter Valu e Reference Melting point/range 138-141 °C DP#309452, T. Jimerson, 10/13/04 pH Not available Density Specific gravity = 1.416 at 25 °C Water solubility ( __°C) 569 mg/L at 20 °C Solvent solubility (mg/L at __°C) g/100 g at 25 °C acetone 85.0 benzene 1.07 diethyl ether 220 ethanol 130.0 isopropanol 31.6 toluene 0.067 xylene 0.58 Vapor pressure at __°C 1.4 x 10 -7 mm Hg at 25 °C Dissociation constant (pK a ) Not available2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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TABLE A.2. Physicochemical Properties of the T echnical Grade Test Co mpound: 2,4-D acid.
Parameter Valu e Reference Octanol/water partition coefficient Log(K OW ) In 0.001 M solution: 2.14 at pH 5 0.177 at pH 7 0.102 at pH 9 UV/visible absorption spectrum Not available



### B. EXPERIMENTAL DESIGN

B.1. Study Site Information

Twenty-four soybean field trials were conducte d in the United States and Canada during the 2009 growing season in NAFTA Zones 2 (GA and AL; 3 trials), 4 (AR and MS; 4 trials), and 5 (IL, IN, MO, MI, OH, NE, IA, MN, and ON; 17 trials).

Each trial consisted of one untre ated and one treated plot. The treated plots were planted with soybeans containing the AAD-12 gene (Event DAS- 68416-4). Each treated plot received three broadcast applications of a 3.79 lb ae/g al SL/L formulation of 2,4-D DMA (Weedar ® 

### 64 Herbicide) at 0.95-1.10 lb ae/A/application, for a to tal seasonal applicatio n rate of 2.97-3.13 lb ae/A. 2,4-D was applied as one preemergence a pplication and two foliar applications, made ~12 days before the soybeans reached the R2 growth st age, and at the ~R2 growth stage. RTIs were 4-64 days between the first and second applicatio ns and 7-21 days between the second and third applications. Applications were made usi ng ground equipment in spray volumes of 19.9-32.3 gal/A and included an NI S at all trial sites.

Soybean forage and hay were harvested at two PH Is, reflecting early harvest (targeted 7-day PHI following application 2) and typi cal harvest (targeted 7-day PHI following the final application); samples of hay were allowed to dry in the fiel d for 0-12 days prior to collection. Seed was harvested at maturity following three applicatio ns. PHIs were 6-8 days for forage and hay following two or three applications, and 51-103 days for seed. Additional samples of forage and hay were collected from one trial each conducte d in GA and MI at PHIs of 0-1, 3-4, 7-8, 10-11, and 14-15 days; additional seed samples for residue decline were collected at PHIs of 37-56, 44- 63, 51-70, 58-77, and 65-84 days.

Crops were grown and maintained using standard agricultural practices to yield commercially representative soybeans. Any maintenance pesticides or ferti lizers used were reported. For each field trial, the petitioner presented the aver age minimum and maximum monthly temperatures and the total monthly rainfall for the study period, as well as the historical averages. The actual temperature recordings were gene rally within normal historical ranges for the study period, with the following exceptions: temperatures during Trials IL-08, IL-09, IN -10, IA-18, NE-17, MO19, MO-22 (during October), and ON-24 were coolerthan normal; conditions at trials IL-08, IL09, IN-10, IA-18, MO-19, and MO- 22 (during October; seed harv ested 10/13) were wetter than normal; and conditions at trials NE-17 and ON- 24 were drier than normal. The petitioner reported that a hail storm early in the trial cause d significant stand loss in trial AR-04; however,2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

DP Barcode D400085/MRID No. 48449802.de1 Page 6 of 21 

### sufficient sample matrix was produced for the study. Trial IA-20 also received hail on July 24 (4 days after application 2), but signi ficant damage was not reported. In addition, an early frost was reported in trial ON-24 (seed harvested 10/28). Irrigation was used to supplement rainfall in seven trials. The petitioner noted that variations of the weat her conditions from historical averages did not have an adverse effect on any of the soybean crops.

Trial conditions are presented in Table B.1.1. Th e study use pattern is presented in Table B.1.2, and the crop varieties grown ar e identified in Table C.3.

TABLE B.1.1 Trial Site Conditions

. Trial Identification:

City, State; Year

(Trial ID No.) Soil characteristics Type %OM pH CEC meq/g Sycamore, GA; 2009 (GA-01) Tifton loamy sand Not Provided Chula, GA; 2009 (GA-02) Tifton loamy sand Columbia, AL; 2009 (AL-03) Paxville loam Lonoke, AR; 2009 (AR-04) Silt loam Stuttgart, AR; 2009 (AR-05) Dewitt silt loam Hazen, AR; 2009 (AR-06) Stuttgart silt loam Greenville, MS; 2009 (MS-07) Sharkey clay Wyoming, IL; 2009 (IL-08) Plano silt loam Carlyle, IL; 2009 (IL-09) Cisne complex silt loam Rockville, IN; 2009 (IN-10) Ragsdale silt loam Jasper, MO; 2009 (MO-11) Newtonia-Eldorado silt loam Rushville, IL; 2009 (IL-12) Rozetta silt loam Deerfield, MI; 2009 (MI-13) Lenawee silty clay loam Metamora, OH; 2009 (OH-14) Gilford fine sandy loam, Mermill loam Shipshewana, IN; 2009 (IN-15) Homer sandy loam, Sebewa loam York, NE; 2009 (NE-16) Hastings silt loam Stromburg, NE; 2009 (NE-17) Hastings silt loam Richland, IA; 2009 (IA-18) Taintor silty clay loam La Plata, MO; 2009 (MO-19) Silt loam Spillville, MO; 2009 (IA-20) Sandy loam Cherry Grove, MN; 2009 (MN-21) Loam Dudley, MO; 2009 (MO-22) Calhoun silt loam Branchton, ON; 2009 (ON-23) Silt loam Dutton, ON; 2009 (ON-24) Silt loam

TABLE B.1.2. Stud y Use Pattern.
Location City, State; Year (Trial ID No.) EP 1 Application Tank Mix/ Adjuvants 3 Method/Timing Volume (gal/A) Rate (lb ae/A) RTI 2 (days) Total Rate (lb ae/A)

Sycamore, GA; 2009 (GA-01) 3.79 lb ae/gal SL/L 1. Broadcast/Preemergen ce 21.7 0.99 -- 2.99 NIS
2. Foliar broadcast/ R1 21.6 1.00 4
3. Foliar broadcast/ R2 21.5 1.00 132,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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TABLE B.1.2. Stud y Use Pattern.
Location City, State; Year (Trial ID No.) EP 1 Application Tank Mix/ Adjuvants 3 Method/Timing Volume (gal/A) Rate (lb ae/A) RTI 2 (days) Total Rate (lb ae/A)

Chula, GA; 2009 (GA-02) 3.79 lb ae/gal SL/L 1. Broadcast/Preemergen ce 23.3 1.00 -- 3.00 NIS
2. Foliar broadcast/ R1 21.8 1.00 4
3. Foliar broadcast/ R2 21.7 1.00 13 Columbia, AL; 2009 (AL-03) 3.79 lb ae/gal SL/L 1. Broadcast/Preemergen ce 21.5 1.0 -- 3.00 NIS
2. Foliar broadcast/ R1 21.0 1.0 34
3. Foliar broadcast/ R2 21.0 1.0 11 Lonoke, AR; 2009 (AR-04) 33.79 lb ae/gal SL/L 1. Broadcast/ BBCH 00 21.0 1.02 -- 3.02 NIS
2. Foliar broadcast/ R1 20.4 0.999 50
3. Foliar broadcast/ R2 20.8 1.0 12 Stuttgart, AR; 2009 (AR-05) 3.79 lb ae/gal SL/L 1. Broadcast/ BBCH 00 19.9 0.996 -- 2.99 NIS
2. Foliar broadcast/ R1 20.1 1.0 30
3. Foliar broadcast/ R2 20.5 0.989 12 Hazen, AR; 2009 (AR-06) 3.79 lb ae/gal SL/L 1. Broadcast/ BBCH 00 19.9 0.98 -- 3.03 NIS
2. Foliar broadcast/ R1 20.8 1.02 29
3. Foliar broadcast/ R2 21.1 1.03 7 Greenville, MS; 2009 (MS-07) 3.79 lb ae/gal SL/L 1. Broadcast/Preemergen ce 20.6 0.994 -- 2.97 NIS
2. Foliar broadcast/ R1 20.5 0.989 51
3. Foliar broadcast/ R2 20.9 0.990 12 Wyoming, IL; 2009 (IL-08) 3.79 lb ae/gal SL/L 1. Broadcast/Preemergen ce 21.9 0.98 -- 2.98 NIS
2. Foliar broadcast/ BBCH 16-60 25.8 1.02 40

3. Foliar broadcast/ BBCH 60-65 24.4 0.98 12

Carlyle, IL; 2009 (IL09) 3.79 lb ae/gal SL/L 1. Broadcast/ Preemergence 31.8 1.02 -- 3.01 NIS
2. Foliar broadcast/ V4 20.8 1.0 29
3. Foliar broadcast/ R2 20.3 0.99 12 Rockville, IN; 2009 (IN-10) 3.79 lb ae/gal SL/L 1. Broadcast/ BBCH 03-06 23.15 1.022 -- 3.09 NIS
2. Foliar broadcast/ BBCH 14-15 21.34 1.023 35

3. Foliar broadcast/ BBCH 65-66 22.07 1.044 13

Jasper, MO; 2009 (MO-11) 3.79 lb ae/gal SL/L 1. Broadcast/ BBCH 00 20.3 1.00 -- 2.99 NIS
2. Foliar broadcast/ R1 20.3 0.995 27
3. Foliar broadcast/ R2 20.3 0.995 13 Rushville, IL; 2009 (IL-12) 3.79 lb ae/gal SL/L 1. Broadcast/ BBCH 00 20.4 1.01 -- 3.02 NIS
2. Foliar broadcast/ R1 20.6 1.00 25
3. Foliar broadcast/ R2 20.5 1.01 122,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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TABLE B.1.2. Stud y Use Pattern.
Location City, State; Year (Trial ID No.) EP 1 Application Tank Mix/ Adjuvants 3 Method/Timing Volume (gal/A) Rate (lb ae/A) RTI 2 (days) Total Rate (lb ae/A)

Deerfield, MI; 2009 (MI-13) 3.79 lb ae/gal SL/L 1. Broadcast/ BBCH 00 23.0 1.0 -- 3.00 NIS
2. Foliar broadcast/ BBCH 48 22.6 1.0 50
3. Foliar broadcast/ BBCH 55 22.9 1.0 11 Metamora, OH; 2009 (OH-14) 3.79 lb ae/gal SL/L 1. Broadcast/ BBCH 00 23.1 1.0 -- 3.00 NIS
2. Foliar broadcast/ BBCH 29-30 23.1 1.0 53

3. Foliar broadcast/ BBCH 35 23.1 1.0 11 Shipshewana, IN; 2009 (IN-15) 3.79 lb ae/gal SL/L 1. Broadcast/ BBCH 00 22.6 1.0 -- 3.00 NIS
2. Foliar broadcast/ BBCH 51-58 23.3 1.0 51

3. Foliar broadcast/ BBCH 65 22.9 1.0 13York, NE; 2009 (NE16) 3.79 lb ae/gal SL/L 1. Broadcast/ BBCH 00 25.16 0.97 -- 2.98 NIS
2. Foliar broadcast/ V6 31.76 0.9987 34
3. Foliar broadcast/ R2 32.27 1.0147 13 Stromburg, NE; 2009 (NE-17) 3.79 lb ae/gal SL/L 1. Broadcast/ BBCH 00 20.6 1.01 -- 3.02 NIS
2. Foliar broadcast/ R1 20.9 1.01 42
3. Foliar broadcast/ R3 20.31 1.0 12 Richland, IA; 2009 (IA-18) 3.79 lb ae/gal SL/L 1. Broadcast/ BBCH 05 22.1 1.01 -- 2.99 NIS

2. Foliar broadcast/ BBCH 17 20.9 1.01 35
3. Foliar broadcast/ BBCH 65 20.4 1.02 11 La Plata, MO; 2009 (MO-19) 3.79 lb ae/gal SL/L 1. Broadcast/ BBCH 00 20.9 1.02 -- 3.04 NIS
2. Foliar broadcast/ BBCH 16 20.4 1.00 27
3. Foliar broadcast/ BBCH 65 20.7 1.02 11 Spillville, MO; 2009 (IA-20) 3.79 lb ae/gal SL/L 1. Broadcast/ Preemergence 25.7 1.1 -- 3.10 NIS
2. Foliar broadcast/ V9 21.3 1.0 45
3. Foliar broadcast/ BBCH 69 22.3 1.0 21 Cherry Grove, MN; 2009 (MN-21) 3.79 lb ae/gal SL/L 1. Broadcast/ Preemergence 25.9 1.0 -- 2.99 NIS
2. Foliar broadcast/ V6 21.0 0.99 45
3. Foliar broadcast/ R2 22.87 1.0 11 Dudley, MO; 2009 (MO-22) 3.79 lb ae/gal SL/L 1. Broadcast/ Preemergence 19.91 0.996 -- 3.00 NIS
2. Foliar broadcast/ BBCH 17 19.90 0.995 23
3. Foliar broadcast/ BBCH 63 20.11 1.006 12 Branchton, ON; 2009 (ON-23) 3.79 lb ae/gal SL/L 1. Broadcast/ Preemergence 23.3 1.073 -- 3.13 NIS
2. Foliar broadcast/ R1 21.93 1.010 64
3. Foliar broadcast/ R2-R3 22.65 1.043 11 Dutton, ON; 2009 (ON-24) 3.79 lb ae/gal SL/L 1. Broadcast/ Preemergence 20.68 0.95 -- 3.02 NIS
2. Foliar broadcast/ R1 21.93 1.016 57
3. Foliar broadcast/ R2-R3 22.72 1.052 12

1 EP = End-use Product; Weedar 64, a 3.79 lb ae/gal SL/L formulation of 2,4-D DMA.
2 Retreatment Interval
3 NIS = Non-ionic surfactant; added to all spray mixtures at 0.25% v/v.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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TABLE B.1.3. Trial Numbers and Geographical Locations

.

NAFTA Growing Zones Soybean
Submitted Requested 1

Canada U.S.
1 -- -- --
1A -- -- --
2 3 -- 2/2
3 -- -- --
4 4 -- 3/2
5 17 11 15/11
5A -- -- --
5B -- 1 --
6 -- -- --
7 -- -- --
7A -- -- --
8 -- -- --
9 -- -- --
10 -- -- --
11 -- -- --
12 -- -- --
13 -- -- --
14 -- -- --
15 -- -- --
16 -- -- --
17 -- -- --
18 -- -- --
19 -- -- --
20 -- -- --
21 -- -- --
Total 24 12 20/15

1 For U.S., as per OCSPP 860.1500, Tables 1 and 5 for soybean; the second number reflects a 25% reduction in the number of trials allowed for the crop as a representative commodity in support of a crop group/subgroup tolerance or when application results in no quantifiable residues. For Canada, as per Regula tory Directive - Dir98-02, Section 9 - Crop Field Trials, Append ix III, Table 2 for soybean.



### B.2. Sample Handling and Preparation

Single untreated and duplicate trea ted samples were collected from each trial. Samples of forage, hay, and seed were collec ted and placed in frozen storage at the field sites within 4 hours of collection, or chilled within 4 hours of collection using a cool er with blue, dry, or wet ice, after which samples were placed in a freezer with in 8 hours of collection. The hay samples were allowed to dry in the field to a moisture conten t of ~<20% for 0-12 days prior to collection. The samples were shipped frozen via freezer truck w ithin 1-134 days of harvest to the analytical laboratory, Dow AgroSciences (Indianapolis, IN) fo r residue analysis. Samples were maintained2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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### frozen (approximately -20 °C) at the analytical laboratory. In pr eparation for analysis, samples were ground in the presence of dry ice.

B.3. Analytical Methodology

Samples were analyzed for residues of 2,4- D and its metabolite 2,4-DCP by LC/MS/MS. A complete description of the met hod was included in the submission.

Briefly, samples were extracted with metha nol:1.0 N NaOH (90:10, v:v) and centrifuged. An aliquot of the extract was comb ined with a mixed stable isot ope internal standard of 2,4-D( 13 

### C 6 

### ) or 2,4-DCP( 13 

### C 6 

### ) and evaporated to near dryness. Th e sample was mixed with 2 N HCl (at 90 °C for 1 hour). After cooling, the extract was diluted with me thanol, filtered, and analyzed by on-line solid-phase extraction (SPE ; residues eluted with water:me thanol containing 0.1% formic acid (70:30, v:v)) and LC/MS/MS in the negative ion mode.

The reported LOQ (equivalent to the LLMV) was 0.01 ppm, and the LOD was 0.003 ppm for 2,4-D and 2,4-DCP in all matrices. The method was validated concurrently with analysis of the field trial samples.

C. RESULTS AND DISCUSSION

Sample storage conditions and durations for so ybean are summarized in Table C.2. Samples were stored frozen (~-20 °C) from harvest to extraction for 2,4-D and 2,4-DCP for up to 461 days (15.2 months) for forage, 460 days (15.1 m onths) for hay, and 405 days (13.3 months) for seed. The interval between extraction and analys is was not reported. Stability of 2,4-D has been demonstrated in a variety of field crop commodities for at least 12 months, including in/on corn grain, forage, and fodder; sorghum grain; wheat gr ain, forage, and straw; ri ce grain; grass forage and hay; and soybean seed (Residue Chemistr y Chapter of the 2,4-D RED; 10/12/04). Interim storage stability data are available (DP #394948) indicating residues of 2,4-DCP are stable for up to 8 months in soybean commodities. The availa ble storage stability data for 2,4-D are adequate to support sample storage conditi ons and durations; however, confir matory storage stability data are required for the metabolite 2,4-DCP reflec ting storage for up to 13.3-15.2 months in/on soybean forage, hay, and seed in HDPE containers (samples from this study were stored in HDPE containers).

Concurrent method recovery data are presented in Table C.1. The LC/MS/MS method used to analyze soybean samples for residues of 2,4-D and 2,4-DCP was adequate for data collection based on acceptable concurrent me thod recovery data. Concurrent recoveries were generally within the acceptable range of 70-120% in s oybean fortified with 2,4-D and 2,4-DCP each at 0.01-100 ppm in forage, 0.01-200 ppm in hay, and 0.01-1 ppm in seed. The fortification levels were adequate to bracket residues found in treat ed samples. Apparent residues of 2,4-D were below the LOQ (<0.01 ppm) in/on all samples of unt reated soybean forage; apparent residues of 2,4-D were observed in/on nine samples of unt reated hay at 0.011-0.034 ppm and four samples of untreated seed at 0.014-0.312 ppm. Apparent residues of 2,4-DCP were below the LOQ2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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### (<0.01 ppm) in/on all samples of untreated soybean seed; apparent resi dues of 2,4-DCP were observed in/on 21 samples of untreated forage at 0.010-0.058 ppm and 44 samples of untreated hay at 0.010-0.214 ppm. The petitioner stated th at the residues of 2,4-D and 2,4-DCP found in the untreated samples did not appear to be the re sult of contamination during analysis; instead, it appears contamination may have taken place at some earlier stage, perhaps from spray drift and/or from sample collection and handling in the field and/or during sample preparation. Concurrent recoveries were corrected for apparent for residues in controls; residues in treated samples were not corrected for residues in controls.

Residue data from the soybean trials are reporte d in Table C.3. A summary of residue data for soybean is presented in Table C.4. Following two to three broadcast app lications of the 3.79 lb ae/gal SL/L formulation of 2,4-D DMA made as one preemergence application and one or two postemergence foliar applications at total seasonal application rates of 1.97-2.10 lb ae/A for early-harvest forage and hay and 2.97-3.13 lb ae/A for typical harvest, residues of 2,4-D (and per trial averages) were <0.01-4.014 (<0.01-3.930) ppm in /on early-harvest forage (6- to 8-day PHI following application 2), <0.01-18.42 4 (<0.01-18.282) ppm in/on typical -harvest forage (6- to 8- day PHI), <0.01-21.855 (<0.01-20.281) ppm in/on early -harvest hay (6- to 8-day PHI following application 2), <0.01-60.413 (<0.01-58.783) ppm in/on typical-harvest hay (6- to 8-day PHI), and <0.01 (<0.01) ppm in/on seed (51- to 103-da y PHI). Residues of 2,4-DCP (and per trial average residues) were <0.01-25.195 (<0.01-22.451) ppm in/on early-harvest forage, <0.01- 36.966 (<0.01-32.080) ppm in/on typical-harve st forage, <0.01-190.589 (<0.01-190.347) ppm in/on early-harvest hay, <0.01-132.961 (<0.01-1 31.509) ppm in/on typical-harvest hay, and <0.01-0.054 (<0.01-0.047) ppm in/on seed.

The submitted residue decline data indicate that residues of 2,4-D generally decreased in/on forage and hay with increasing PHI. Residues of 2,4-D in/on seed were <LOQ in both studies; therefore, residue declin e could not be assessed. Residues of 2,4-DCP in forage, hay, and seed generally decreased or did not in crease with increasing PHI.

TABLE C.1. Summary of Concu rrent Recoveries of 2,4-D and 2,4-DCP from Soybean. Matrix Spike level (ppm) Sample size (n) Recoveries (%) 1 Mean ± std dev 2 (%) 2,4-D Forage 0.01 10 81, 84, 86, 88, 89, 91, 93, 96, 99, 101 91 ± 6 1 4 91, 92, 93, 98 94 ± 3 10 2 99, 101 100 100 4 77, 76, 87, 89 82 ± 7 Hay 0.01 10 87, 90, 90, 92, 92, 94, 98, 98, 98, 98 94 ± 4 1 2 93, 97 95 100 8 76, 78, 82, 86, 89, 90, 91, 91 85 ± 6 200 6 76, 81, 85, 85, 86, 89 84 ± 5 Seed 0.01 8 79, 81, 83, 83, 85, 87, 91, 102 86 ± 7 1 8 79, 81, 86, 88, 94, 96, 96, 97 90 ± 72,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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TABLE C.1. Summary of Concu rrent Recoveries of 2,4-D and 2,4-DCP from Soybean. Matrix Spike level (ppm) Sample size (n) Recoveries (%) 1 Mean ± std dev 2 (%) 2,4-DCP Forage 0.01 10 69, 83, 93, 100, 101, 101, 102, 106, 106, 110 97 ± 12 1 4 87, 90, 96, 99 93 ± 5 10 2 94, 96 95 100 4 76, 79, 81, 83 80 ± 3 Hay 0.01 10 89, 89, 90, 93, 94, 96, 98, 99, 104, 109 96 ± 7 1 2 93, 103 98 100 8 70, 71, 71, 72, 73, 75, 79, 83 74 ± 5 200 6 75, 77, 78, 78, 79, 86 79 ± 4 Seed 0.01 8 79, 90, 91, 93, 94, 98, 105, 109 95 ± 9 1 8 81, 82, 86, 92, 94, 97, 97, 103 92 ± 8

1 Recoveries were corrected for appare nt residues in the untreated samples.
2 Standard deviation is not applicab le for sample sizes n < 3 samples.

TABLE C.2. Summary of Storage Conditions for Soybean

. Matrix Storage Temperature (°C) Actual Storage Duration 1 Interval of Demonstrated Storage Stability

Forage Frozen at field sites

Approximately -20 at analytical lab 417-461 days (13.7-15.2 months) Adequate storage stability data are available for 2,4-D indicating that residues are stable under frozen storage conditions for at least 12 months in/on corn grain, forage, and fodder; sorghum grain; whea t grain, forage, and straw; rice grain; grass forage and hay; and soybean seed. 2 Interim storage stability data are avai lable demonstrating residues of 2,4-DCP are stable in soybean commodities for up to 8 months 3

. Hay 415-460 days (13.7-15.1 months) Seed 327-405 days (10.8-13.3 months)

1 Duration from harvest to extraction; interval between extractio n and analysis was not provided. Hay sample duration is from collection after drying to extraction; samples were allowed to dry for 0-12 days.
2 Residue Chemistry Chapter of the 2,4-D RED; 10/12/04.
3 DP #394948 of interim storage stability data review.

TABLE C.3. Residue Data from Crop Field Trials with 2,4-D.
Trial ID: City, State; Year

(Trial ID No.) Zone Soybean variety Commodity

or Matrix Total Rate (lb ae/A) PHI 1 (days) Residues 2 , ppm [Average]

2,4-D 2,4-DCP

Forage Sycamore, GA; 2009 (GA-01) 2 ADD-12 Event DAS-68416-4 Forage 1.99 7 (Early) 0.090, 0.090 [0.090] 6.987, 5.625 [6.306] 2.99 7 (Typical) 0.421, 0.575 [0.498] 16.525, 18.083 [17.304]2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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TABLE C.3. Residue Data from Crop Field Trials with 2,4-D.
Trial ID: City, State; Year

(Trial ID No.) Zone Soybean variety Commodity

or Matrix Total Rate (lb ae/A) PHI 1 (days) Residues 2 , ppm [Average]

2,4-D 2,4-DCP

Chula, GA; 2009 (GA02) 2 ADD-12 Event DAS-68416-4 Forage 2.00 7 (Early) 0.802, 0.909 [0.855] 21.267, 23.815 [22.541]
3.00 0 21.647, 26.976 [24.312] 36.796, 35.793 [36.294]
3 5.227, 5.007 [5.117] 33.533, 32.009 [32.771]

7 (Typical) 2.777, 2.070 [2.423] 31.385, 23.503 [27.444]
10 0.066, 0.064 [0.065] 20.091, 21.183 [20.637]
14 0.070, 0.033 [0.052] 19.350, 17.575 [18.462]
Columbia, AL; 2009 (AL-03) 2 ADD-12 Event DAS-68416-4 Forage 2.00 7 (Early) 0.074, 0.090 [0.082] 5.822, 5.418 [5.620] 3.00 7 (Typical) 0.244, 0.805 [0.525] 12.484, 24.860 [18.672] Lonoke, AR; 2009 (AR-04) 4 ADD-12 Event DAS-68416-4 Forage 2.02 7 (Early) ND, 0.033 [<0.022] ND, 0.021 [<0.016] 3.02 7 (Typical) ND, ND [<0.01] (0.005), ND [<0.01] Stuttgart, AR; 2009 (AR-05) 4 ADD-12 Event DAS-68416-4 Forage 2.00 7 (Early) ND, (0.004) [<0.01] 0.010, 0.107 [0.058] 2.99 7 (Typical) (0.003), (0.007) [<0.01] 0.012, (0.006) [<0.011]
Hazen, AR; 2009 (AR06) 4 ADD-12 Event DAS-68416-4 Forage 2.00 7 (Early) (0.006), 0.012 [<0.011] (0.008), 0.010 [<0.01] 3.03 6 (Typical) ND, ND [<0.01] (0.004), (0.005) [<0.01] Greenville, MS; 2009 (MS-07) 4 ADD-12 Event DAS-68416-4 Forage 1.98 7 (Early) (0.007), (0.005) [<0.01] (0.006), (0.006) [<0.01] 2.97 7 (Typical) (0.008), (0.004) [<0.01] (0.007), (0.007) [<0.01] Wyoming, IL; 2009 (IL-08) 5 ADD-12 Event DAS-68416-4 Forage 2.00 7 (Early) 0.294, 0.336 [0.315] 9.934, 9.170 [9.552] 2.98 7 (Typical) 0.747, 0.676 [0.712] 15.129, 12.776 [13.952]Carlyle, IL; 2009 (IL09) 5 ADD-12 Event DAS-68416-4 Forage 2.02 7 (Early) 0.348, 0.240 [0.294] 17.654, 25.195 [21.424] 3.01 7 (Typical) 0.474, 0.594 [0.534] 27.195, 36.966 [32.080] Rockville, IN; 2009 (IN-10) 5 ADD-12 Event DAS-68416-4 Forage 2.05 7 (Early) 0.714, 0.523 [0.619] 11.247, 10.653 [10.950] 3.09 7 (Typical) 0.089, 0.138 [0.114] 8.287, 13.202 [10.744]2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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TABLE C.3. Residue Data from Crop Field Trials with 2,4-D.
Trial ID: City, State; Year

(Trial ID No.) Zone Soybean variety Commodity

or Matrix Total Rate (lb ae/A) PHI 1 (days) Residues 2 , ppm [Average]

2,4-D 2,4-DCP

Jasper, MO; 2009 (MO-11) 5 ADD-12 Event DAS-68416-4 Forage 2.00 7 (Early) ND, ND [<0.01] (0.004), (0.003) [<0.01] 2.99 7 (Typical) ND, ND [<0.01] ND, 0.025 [<0.018] Rushville, IL; 2009 (IL-12) 5 ADD-12 Event DAS-68416-4 Forage 2.01 7 (Early) ND, ND [<0.01] ND, 0.014 [<0.012] 3.02 7 (Typical) ND, ND [<0.01] 0.012, 0.037 [0.024] Deerfield, MI; 2009 (MI-13) 5 ADD-12 Event DAS-68416-4 Forage 2.00 7 (Early) 0.868, 0.845 [0.857] 12.097, 13.373 [12.735] 3.00 1 22.007, 19.402 [20.705] 17.980, 16.492 [17.236] 4 1.512, 1.610 [1.561] 18.796, 19.304 [19.050]
8 3 (Typical) 1.012, 1.063 [1.038] 18.690, 20.777 [19.734] 11 0.182, 0.173 [0.178] 15.817, 15.871 [15.844] 15 0.129, 0.192 [0.161] 18.151, 19.412 [18.781] Metamora, OH; 2009 (OH-14) 5 ADD-12 Event DAS-68416-4 Forage 2.00 7 (Early) 0.452, 0.383 [0.417] 13.650, 15.497 [14.573] 3.00 7 (Typical) 0.166, 0.163 [0.165] 11.764, 11.671 [11.718] Shipshewana, IN; 2009 (IN-15) 5 ADD-12 Event DAS-68416-4 Forage 2.00 7 (Early) 0.949, 1.332 [1.141] 15.063, 14.273 [14.668] 3.00 7 (Typical) 0.337, 0.313 [0.325] 19.685, 16.819 [18.252]York, NE; 2009 (NE16) 5 ADD-12 Event DAS-68416-4 Forage 1.97 8 (Early) 0.156, 0.145 [0.151] 5.737, 4.794 [5.265] 2.98 7 (Typical) 0.135, 0.178 [0.156] 6.771, 7.952 [7.361] Stromburg, NE; 2009 (NE-17) 5 ADD-12 Event DAS-68416-4 Forage 2.02 7 (Early) 0.319, 0.490 [0.405] 10.516, 11.219 [10.867] 3.02 6 (Typical) 0.222, 0.254 [0.238] 12.469, 19.203 [15.836] Richland, IA; 2009 (IA-18) 5 ADD-12 Event DAS-68416-4 Forage 2.00 7 (Early) 3.845, 4.014 [3.930] 14.955, 19.919 [17.437] 2.99 6 (Typical) 18.424, 18.140 [18.282] 26.933, 28.791 [27.862] La Plata, MO; 2009 (MO-19) 5 ADD-12 Event DAS-68416-4 Forage 2.02 6 (Early) 2.707, 3.521 [3.114] 19.874, 20.347 [20.111] 3.04 8 (Typical) 0.823, 1.098 [0.960] 24.899, 24.312 [24.606]2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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TABLE C.3. Residue Data from Crop Field Trials with 2,4-D.
Trial ID: City, State; Year

(Trial ID No.) Zone Soybean variety Commodity

or Matrix Total Rate (lb ae/A) PHI 1 (days) Residues 2 , ppm [Average]

2,4-D 2,4-DCP

Spillville, MO; 2009 (IA-20) 5 ADD-12 Event DAS-68416-4 Forage 2.10 7 (Early) 0.043, 0.093 [0.068] 5.593, 7.381 [6.487] 3.10 7 (Typical) 0.048, 0.147 [0.098] 8.074, 9.289 [8.681]
Cherry Grove, MN; 2009 (MN-21) 5 ADD-12 Event DAS-68416-4 Forage 1.99 7 (Early) 0.131, 0.211 [0.171] 10.609, 11.159 [10.884]
2.99 6 (Typical) 7.312, 7.432 [7.372] 21.595, 20.312 [20.954]
Dudley, MO; 2009 (MO-22) 5 ADD-12 Event DAS-68416-4 Forage 1.99 7 (Early) 3.704, 3.454 [3.579] 18.975, 15.772 [17.373] 3.00 8 (Typical) 0.037 4 , 0.041 [0.039] 13.028 4 , 16.147 [14.588] Branchton, ON; 2009 (ON-23) 5 ADD-12 Event DAS-68416-4 Forage 2.08 8 (Early) 0.228, 0.281 [0.255] 9.782, 12.051 [10.916] 3.13 8 (Typical) 0.122, 0.089 [0.106] 16.671, 13.795 [15.153] Dutton, ON; 2009 (ON-24) 5 ADD-12 Event DAS-68416-4 Forage 1.97 8 (Early) 0.278, 0.301 [0.289] 9.791, 11.561 [10.676] 3.02 8 (Typical) 0.388, 0.477 [0.432] 14.066, 15.676 [14.871] Hay Sycamore, GA; 2009 (GA-01) 2 ADD-12 Event DAS-68416-4 Hay 1.99 7 (Early) [4] 0.128, 0.187 [0.158] 5.625, 10.937 [8.281] 2.99 7 (Typical) [4] 0.601, 0.670 [0.635] 27.290, 26.485 [26.887]Chula, GA; 2009 (GA02) 2 ADD-12 Event DAS-68416-4 Hay 2.00 7 (Early) [4] 0.970, 3.836 [2.403] 37.498, 66.225 [51.861]
3.00 0 [3] 84.428, 121.063 [102.745] 72.861, 81.561 [77.211]
3 [4] 25.350, 25.827 [25.589] 114.315, 116.490 [115.403] 7 (Typical) [4] 9.365, 7.805 [8.585] 77.664, 79.250 [78.457]
10 [5] 0.109, 0.106 [0.108] 10.940, 15.554 [13.247]
14 [5] 0.119, 0.071 [0.095] 43.517, 39.450 [41.484] Columbia, AL; 2009 (AL-03) 2 ADD-12 Event DAS-68416-4 Hay 2.00 7 (Early) [4] 0.556, 0.499 [0.528] 21.869, 19.994 [20.931] 3.00 7 (Typical) [5] 1.055, 0.993 [1.024] 107.363, 42.111 [74.737] Lonoke, AR; 2009 (AR-04) 4 ADD-12 Event DAS-68416-4 Hay 2.02 7 (Early) [3] ND, ND [<0.01] (0.006), (0.006) [<0.01] 3.02 7 (Typical) [6] (0.004), ND [<0.01] (0.008), (0.007) [<0.01]2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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TABLE C.3. Residue Data from Crop Field Trials with 2,4-D.
Trial ID: City, State; Year

(Trial ID No.) Zone Soybean variety Commodity

or Matrix Total Rate (lb ae/A) PHI 1 (days) Residues 2 , ppm [Average]

2,4-D 2,4-DCP

Stuttgart, AR; 2009 (AR-05) 4 ADD-12 Event DAS-68416-4 Hay 2.00 7 (Early) [2] (0.005), (0.008) [<0.01] 0.016, 0.011 [0.013] 2.99 7 (Typical) [3] 0.025, 0.012 [0.018] 0.033, (0.010) [<0.021]Hazen, AR; 2009 (AR06) 4 ADD-12 Event DAS-68416-4 Hay 2.00 7 (Early) [2] 0.012, 0.010 [0.011] 0.014, 0.011 [0.013] 3.03 6 (Typical) [2] (0.007), (0.008) [<0.01] 0.013, 0.012 [0.013] Greenville, MS; 2009 (MS-07) 4 ADD-12 Event DAS-68416-4 Hay 1.98 7 (Early) [2] (0.007), 0.011 [<0.011] (0.009), (0.008) [<0.01] 2.97 7 (Typical) [3] ND, (0.007) [<0.01] (0.007), (0.006) [<0.01] Wyoming, IL; 2009 (IL-08) 5 ADD-12 Event DAS-68416-4 Hay 2.00 7 (Early) [5] 1.602, 1.579 [1.590] 57.502, 52.297 [54.900] 2.98 7 (Typical) [4] 4.164, 4.502 [4.333] 55.594, 55.219 [55.407]Carlyle, IL; 2009 (IL09) 5 ADD-12 Event DAS-68416-4 Hay 2.02 7 (Early) [5] 0.959, 1.286 [1.122] 99.752, 98.058 [98.905] 3.01 7 (Typical) [6] 2.036, 2.592 [2.314] 130.056, 132.961 [131.509] Rockville, IN; 2009 (IN-10) 5 ADD-12 Event DAS-68416-4 Hay 2.05 7 (Early) [2] 1.530, 1.204 [1.367] 46.764, 29.591 [38.177] 3.09 7 (Typical) [5] 0.604, 0.577 [0.590] 38.941, 40.037 [39.489] Jasper, MO; 2009 (MO-11) 5 ADD-12 Event DAS-68416-4 Hay 2.00 7 (Early) [3] ND [<0.01] (0.004) [<0.01]

2.99 7 (Typical) [4] ND, ND [<0.01] ND, ND [<0.01]

Rushville, IL; 2009 (IL-12) 5 ADD-12 Event DAS-68416-4 Hay 2.01 7 (Early) [2] ND, ND [<0.01] 0.011, (0.005) [<0.011] 3.02 7 (Typical) [4] ND, ND [<0.01] (0.007), (0.009) [<0.01] Deerfield, MI; 2009 (MI-13) 5 ADD-12 Event DAS-68416-4 Hay 2.00 7 (Early) [0] 1.075, 2.469 [1.772] 6.009, 15.295 [10.652]
3.00 1 [7] 22.493, 26.216 [24.355] 26.785, 19.445 [23.115]
4 [7] 1.014, 1.092 [1.053] 7.344, 5.969 [6.657]
8 3 (Typical) [0] 0.852, 0.713 [0.783] 38.127, 36.868 [37.497]

11 [0] 0.489, 0.624 [0.556] 41.613, 31.225 [36.419]
15 [7] 0.308, 0.304 [0.306] 13.684, 10.877 [12.281]2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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TABLE C.3. Residue Data from Crop Field Trials with 2,4-D.
Trial ID: City, State; Year

(Trial ID No.) Zone Soybean variety Commodity

or Matrix Total Rate (lb ae/A) PHI 1 (days) Residues 2 , ppm [Average]

2,4-D 2,4-DCP

Metamora, OH; 2009 (OH-14) 5 ADD-12 Event DAS-68416-4 Hay 2.00 7 (Early) [6] 1.669, 2.218 [1.944] 5.220, 5.249 [5.234] 3.00 7 (Typical) [8] 0.411 5 31.810 5

Shipshewana, IN; 2009 (IN-15) 5 ADD-12 Event DAS-68416-4 Hay 2.00 7 (Early) [6] 2.619, 7.224 [4.921] 13.890, 13.001 [13.445] 3.00 7 (Typical) [4] 0.765, 0.912 [0.839] 32.510, 46.304 [39.407]York, NE; 2009 (NE16) 5 ADD-12 Event DAS-68416-4 Hay 1.97 8 (Early) [6] 0.503, 0.636 [0.570] 27.033, 30.909 [28.971] 2.98 7 (Typical) [5] 0.524, 0.550 [0.537] 28.710, 29.985 [29.347] Stromburg, NE; 2009 (NE-17) 5 ADD-12 Event DAS-68416-4 Hay 2.02 8 (Early) [3] 0.625, 0.674 [0.649] 39.784, 38.792 [39.288] 3.02 6 (Typical) [5] 0.595, 0.801 [0.698] 40.012, 61.762 [50.887] Richland, IA; 2009 (IA-18) 5 ADD-12 Event DAS-68416-4 Hay 2.00 7 (Early) [4] 7.000, 9.415 [8.207] 64.422, 101.888 [83.155] 2.99 6 (Typical) [5] 60.413, 57.154 [58.783] 95.647, 94.852 [95.250] La Plata, MO; 2009 (MO-19) 5 ADD-12 Event DAS-68416-4 Hay 2.02 6 (Early) [2] 11.240, 13.390 [12.315] 190.589, 190.105 [190.347] 3.04 8 (Typical) [3] 3.020, 3.338 [3.179] 106.061, 102.542 [104.301] Spillville, MO; 2009 (IA-20) 5 ADD-12 Event DAS-68416-4 Hay 2.10 7 (Early) [2] 0.623, 0.695 [0.659] 38.524, 32.883 [35.704] 3.10 7 (Typical) [2] 0.085, 0.122 [0.103] 24.873, 25.743 [25.308] Cherry Grove, MN; 2009 (MN-21) 5 ADD-12 Event DAS-68416-4 Hay 1.99 7 (Early) [2] 0.700, 0.568 [0.634] 48.349, 49.917 [49.133] 2.99 6 (Typical) [5] 4.462, 6.177 [5.320] 13.070, 16.489 [14.780] Dudley, MO; 2009 (MO-22) 5 ADD-12 Event DAS-68416-4 Hay 1.99 7 (Early) [12] 18.677, 21.885 [20.281] 147.994, 147.785 [147.890] 3.00 8 (Typical) [5] 0.095, 0.160 [0.128] 47.578, 45.814 [46.696] Branchton, ON; 2009 (ON-23) 5 ADD-12 Event DAS-68416-4 Hay 2.08 8 (Early) [8] 0.894, 0.802 [0.848] 47.035, 32.177 [39.606] 3.13 8 (Typical) [9] 0.172, 0.182 [0.177] 38.811, 32.996 [35.904] Dutton, ON; 2009 (ON-24) 5 ADD-12 Event DAS-68416-4 Hay 1.97 8 (Early) [8] 1.082, 1.359 [1.220] 37.742, 50.033 [43.887] 3.02 8 (Typical) [8] 1.444, 2.477 [1.960] 49.278, 59.682 [54.480]2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

DP Barcode D400085/MRID No. 48449802.de1 Page 18 of 21

TABLE C.3. Residue Data from Crop Field Trials with 2,4-D.
Trial ID: City, State; Year

(Trial ID No.) Zone Soybean variety Commodity

or Matrix Total Rate (lb ae/A) PHI 1 (days) Residues 2 , ppm [Average]

2,4-D 2,4-DCP

Seed Sycamore, GA; 2009 (GA-01) 2 ADD-12 Event DAS-68416-4 Seed 2.99 66 ND, ND [<0.01] 0.033, 0.041 [0.037]

Chula, GA; 2009 (GA02) 2 ADD-12 Event DAS-68416-4 Seed 3.00 37 ND, ND [<0.01] 0.036, 0.037 [0.037] 44 ND, (0.006) [<0.01] 0.034, 0.030 [0.032]
51 (Target) ND, ND [<0.01] 0.034, 0.034 [0.034] 58 ND, ND [<0.01] 0.035, 0.031 [0.033] 65 ND, ND [<0.01] 0.033, 0.031 [0.032] Columbia, AL; 2009 (AL-03) 2 ADD-12 Event DAS-68416-4 Seed 3.00 59 ND, ND [<0.01] 0.039, 0.031 [0.035] Lonoke, AR; 2009 (AR-04) 4 ADD-12 Event DAS-68416-4 Seed 3.02 91 ND, ND [<0.01] ND, ND [<0.01]

Stuttgart, AR; 2009 (AR-05) 4 ADD-12 Event DAS-68416-4 Seed 2.99 77 ND, ND [<0.01] ND, ND [<0.01]

Hazen, AR; 2009 (AR06) 4 ADD-12 Event DAS-68416-4 Seed 3.03 83 ND, ND [<0.01] ND, ND [<0.01]

Greenville, MS; 2009 (MS-07) 4 ADD-12 Event DAS-68416-4 Seed 2.97 77 ND, ND [<0.01] ND, ND [<0.01]

Wyoming, IL; 2009 (IL-08) 5 ADD-12 Event DAS-68416-4 Seed 2.98 102 ND, ND [<0.01] 0.020, 0.022 [0.021]Carlyle, IL; 2009 (IL09) 5 ADD-12 Event DAS-68416-4 Seed 3.01 90 ND, ND [<0.01] (0.008), (0.008) [<0.01] Rockville, IN; 2009 (IN-10) 5 ADD-12 Event DAS-68416-4 Seed 3.09 96 ND, ND [<0.01] (0.003), (0.005) [<0.01] Jasper, MO; 2009 (MO-11) 5 ADD-12 Event DAS-68416-4 Seed 2.99 101 ND, ND [<0.01] 0.013, 0.015 [0.014] Rushville, IL; 2009 (IL-12) 5 ADD-12 Event DAS-68416-4 Seed 3.02 102 ND, ND [<0.01] 0.013, 0.015 [0.014] Deerfield, MI; 2009 (MI-13) 5 ADD-12 Event DAS-68416-4 Seed 3.00 56 ND, ND [<0.01] 0.046, 0.053 [0.049] 63 ND, ND [<0.01] 0.064, 0.049 [0.057]
70 (Target) ND, ND [<0.01] 0.047, 0.048 [0.047] 77 ND, ND [<0.01] 0.041, 0.040 [0.041] 84 ND, ND [<0.01] 0.052, 0.034 [0.043]2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

DP Barcode D400085/MRID No. 48449802.de1 Page 19 of 21

TABLE C.3. Residue Data from Crop Field Trials with 2,4-D.
Trial ID: City, State; Year

(Trial ID No.) Zone Soybean variety Commodity

or Matrix Total Rate (lb ae/A) PHI 1 (days) Residues 2 , ppm [Average]

2,4-D 2,4-DCP

Metamora, OH; 2009 (OH-14) 5 ADD-12 Event DAS-68416-4 Seed 3.00 71 ND, ND [<0.01] 0.021, 0.020 [0.020] Shipshewana, IN; 2009 (IN-15) 5 ADD-12 Event DAS-68416-4 Seed 3.00 70 ND, ND [<0.01] 0.054, 0.033 [0.044]York, NE; 2009 (NE16) 5 ADD-12 Event DAS-68416-4 Seed 2.98 90 ND, ND [<0.01] (0.004), (0.005) [<0.01] Stromburg, NE; 2009 (NE-17) 5 ADD-12 Event DAS-68416-4 Seed 3.02 70 ND, ND [<0.01] 0.012, (0.008) [<0.011] Richland, IA; 2009 (IA-18) 5 ADD-12 Event DAS-68416-4 Seed 2.99 87 ND, ND [<0.01] (0.006), 0.017 [<0.014] La Plata, MO; 2009 (MO-19) 5 ADD-12 Event DAS-68416-4 Seed 3.04 85 ND, ND [<0.01] (0.004), (0.004) [<0.01] Spillville, MO; 2009 (IA-20) 5 ADD-12 Event DAS-68416-4 Seed 3.10 94 ND, ND [<0.01] 0.021, 0.028 [0.025] Cherry Grove, MN; 2009 (MN-21) 5 ADD-12 Event DAS-68416-4 Seed 2.99 103 ND, ND [<0.01] 0.015, 0.017 [0.016] Dudley, MO; 2009 (MO-22) 5 ADD-12 Event DAS-68416-4 Seed 3.00 70 ND, ND [<0.01] (0.005), (0.004) [<0.01] Branchton, ON; 2009 (ON-23) 5 ADD-12 Event DAS-68416-4 Seed 3.13 92 ND, ND [<0.01] 0.020, 0.018 [0.019] Dutton, ON; 2009 (ON-24) 5 ADD-12 Event DAS-68416-4 Seed 3.02 71 ND, ND [<0.01] 0.021, 0.030 [0.025]

1 For “Early” harvest, samples were collected following applica tion 2; for “Typical” harvest, sa mples were collected following final application. The number of days hay was allowed to dry prior to coll ection is presented in parentheses.
2 ND = Nondetectable (<LOD). The LOD was 0.003 ppm for all analytes in all ma trices; the LOQ was 0.01 ppm for all analytes. Residues between the LOD and LOQ are reported in parentheses. When calculating per trial averages, the LOQ was used for values reported as ND or between the LOD and LOQ.
3 We note that the PHI reported here was confirmed based on application and harvest dates, and differs from that presented in Table 1 of MRID 48449802.
4 The petitioner reported that the results indicate that the cont rol and treated values were switched. The appropriate value f or the treated sample is reported.
5 The petitioner reported an apparent error in sample identification and stated that these results are for the only sample considered to be a representative treated sample for this trial.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

DP Barcode D400085/MRID No. 48449802.de1 Page 20 of 21 TABLE C.4. Summary of Residue Data fr om Soybean Field Trials with 2,4-D. Commodity Total Applic.

Rate (lb ae/A) PHI (days) Residue Levels 1 (ppm) n Sample Min. Sample Max. LAFT 2 HAFT 2 Median Mean Std. Dev.

2,4-D Soybean, early forage 1.97-2.10 6-8 24 <0.01 4.014 <0.01 3.930 0.272 0.696 1.146

Soybean, forage 2.97 - 3.13 6-8 24 <0.01 18.424 <0.01 18.282 0.202 1.420 3.900

Soybean, early hay 1.97-2.10 6-8 24 <0.01 21.885 <0.01 20.281 0.754 2.552 4.758

Soybean, hay 2.97 - 3.13 6-8 24 <0.01 60.413 <0.01 58.783 0.613 3.769 11.903 Soybean, seed 2.97 - 3.13 51-103 24 <0.01 <0.01 <0.01 0.01 0.01 0.01 N/A

2,4-DCP Soybean, early forage 1.97-2.10 6-8 24 <0.01 25.195 <0.01 22.451 10.772 9.521 7.264

Soybean, forage 2.97 - 3.13 6-8 24 <0.01 36.966 <0.01 32.080 14.729 13.329 9.783

Soybean, early hay 1.97-2.10 6-8 24 <0.01 190.589 <0.01 190.347 32.338 40.018 48.419

Soybean, hay 2.97 - 3.13 6-8 24 <0.01 132.961 <0.01 131.509 36.701 40.509 35.918 Soybean, seed 2.97 - 3.13 51-103 24 <0.01 0.054 <0.01 0.047 0.014 0.019 0.012

1 Except for sample min/max, values reflect per trial averages; n = no. of field trials. For ca lculation of median, mean, and standard deviation, the LOQ (0.01 ppm) wa s used for any results reported as ND (0 .003 ppm) or between the LOD and LOQ in Table C.3. N/A = Not applicable.
2 LAFT = lowest average field trial; HAFT = highest average field trial.



### D. CONCLUSION

Pending submission of additional storage stability data for 2,4-DCP, the submitted soybean trial data are adequate. Following two to three br oadcast applications of the 3.79 lb ae/gal SL/L formulation of 2,4-D DMA made as one pr eemergence application and one or two postemergence foliar applicati ons to soybeans containing th e AAD-12 gene (Event DAS-68416- 4) at total seasonal applicati on rates of 1.97-2.10 lb ae/A for early -harvest forage and hay and 2.97-3.13 lb ae/A for typical harvest, residues of 2,4-D (and per trial av erages) were <0.01-4.014 (<0.01-3.930) ppm in/on early-harvest forage (6 - to 8-day PHI following application 2), <0.01- 18.424 (<0.01-18.282) ppm in/on typi cal-harvest forage (6- to 8-day PHI), <0.01-21.855 (<0.01- 20.281) ppm in/on early-harvest hay (6- to 8- day PHI following application 2), <0.01-60.413 (<0.01-58.783) ppm in/on typical-harvest hay (6 - to 8-day PHI), and <0.01 (<0.01) ppm in/on seed (51- to 103-day PHI). Residues of 2,4-DCP (and per tria l average residues) were <0.01- 25.195 (<0.01-22.451) ppm in/on ea rly-harvest forage, <0.01-36.966 (<0.01-32.080) ppm in/on typical-harvest forage, <0.01-190.589 (<0.01-19 0.347) ppm in/on early-harvest hay, <0.01- 132.961 (<0.01-131.509) ppm in/on typical-harv est hay, and <0.01-0.054 (<0.01-0.047) ppm in/on seed. The submitted residue decline da ta indicate that resi dues of 2,4-D generally decreased in/on forage and hay with increasing PHI. Residues of 2,4-D in/on seed were <LOQ2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.1/7.4.2/OPPTS 860.1500/OECD IIA 6.3.1, 6.3.2, 6.3.3 and IIIA 8.3.1, 8.3.2, 8.3.3 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

DP Barcode D400085/MRID No. 48449802.de1 Page 21 of 21 

### in both studies; therefore, residue decline could not be assessed. Residues of 2,4-DCP in forage, hay, and seed generally decreased or di d not increase with increasing PHI.

An acceptable method was used for quantitation of 2,4-D and 2,4-DCP. Confirmatory storage stability data are required for 2,4-DCP reflectin g frozen storage for up to 13.3-15.2 months in/on soybean forage, hay, and seed in HDPE containers.

E. REFERENCES

DP#: 309452 Subject: 2,4-D: Health Effects Division (HED) Metabolism Assessment Review Committee (MARC) Decision Document - Revised. Chemical I.D. No. 030001. Case No. 0073. Meeting date: 9/3/03 From: T. Jimerson To: Y. Donovan Dated: 10/14/04 MRID(s): None

F. DOCUMENT TRACKING

DP Barcode(s): D400085 PC Code: 030001

Template Version June 2005.**~·I** 2,4-D / PC

... code 030001/Dow AgroSciences LLC


# ==- I DACO 6 .
., / 0PPTS 860.1300 / 0ECD II 6

. 2

. 2, 6.2.3 & lilA 8.2, 8.4.1

, 8.4.2 Nat ure of the Residues in Plants- 2,4-D

- Tolera nt Soybean (AAD

- 12

, Event pDAB4468

- 416)



### Reviewed and Approved by**'·-~ ~** 

-=- <---.!. -= ·

# ::**=:~ :::b** **~~:::::::::==::::~~::::r-::::;:::::;::::.__** ___ Date: 3/4/12 Danette Drew, Chemist, HED RABV

Note: This Data Evaluation Recor d ( DER) was originally p rep ared under contract b y Ve r sar , Inc. (6850 Versa r Center, Springfield, VA 22151 ; submitted (1/ 24 / 20 12 ). The DER has been reviewed b y HED and revised to reflect current Office of Pesticide Programs (OPP) po l icies.



### STUDY REPORT:



### 48449801. Graper, L.; 

### Balcer, J.; Smith, K. ; Hogan, P

. (2011) A Nature of the Residue Study with [14C]

- 2,4

- D DMA Applied to AAD-12 Soybeans Amended Report

. Lab Study Number: 090051. Unpublished study prepared by Dow AgroSciences LLC, Research for Hire and ABC Laboratories, Inc

. 193 p.

EXECUTIVE SUMMARY:



### Dow AgroSciences LLC has submitted a study investigating the metabolism of [phenyl

- U

14C]2,4

- dichlorophenoxy acetic acid dimethylammonium salt [2,4-D DMA; specific activity 10.92 J.LCi/mg for planting box and 101.3 7 J.LCi/mg for pots] in 2,4-D-to l erant soybean. The

so ybean seed used in the study wa s genetic ally modified aryloxyalkanoate dioxygenase-12 (AAD

- 12) soybe an (event -construct pDAB4468

- 416). The mod i fication makes the soybean resistant to 2,4

- D and certain other pheno xya cetate and pyridyloxyacetate auxin herbicides.

The radiolabeled test substance was formulated as a soluble conce n trate liquid (SLIL) and
app lied to 2,4-D-tole rant soybeans grown outdoors in a planting bo x or pots as a preemergence soil application at 1.04

- 1. 1 1 lb ae/ A (1.166-1.248 kg ae/ha) followed by two foliar appl ica tions at 0.98 -1. 11 lb ae/ A/application (1.1 03

- 1.243 kg ae/ha/application), for a total application rate of 3

. 13

- 3.19lb ae/A (3 .51 2

- 3.5 78 kg ae/ha)

. The preemergence application was made on the da y of planting, and foliar applications were made at the V 4 and R2 growth stages at retreatment intervals (RTis) of 34 and 21 days to soybean grown in the planting box and 22 and 20 days to soybean grown in pots. Samples of forage and hay were collected from the planting box 7 days after the second and third application s , respectively (days after treatment (DA T) 2 and 3; 7

- DAT2 for forage and 7-DAT3 for hay). Because the 7-DAT3 hay sample was not dried

, another sample of hay was collec ted 22 days after the third application (22-DAT3) and drie d

. Samples of seed were collected from the planting box and pots at 62 a nd 41 days after the thi r d applicati on (62

- DAT3 and 41-DAT3)

, respectively. The in

- l i fe phase ofthe study wa s conducted by Research For Hire (Porterville, CA), and the analytical phase of the study was conducted by ABC Laboratories (Columbia, MO)

.

Total radioactive residues (TRR) determined by combustio n/LSC were 50

. 227 ppm in 7

- DAT2 forage, 35.565 ppm in 7-DAT3 hay, 37

. 008 ppm in 22-DAT3 hay

, 0.415 ppm in 62-DAT3 seed, and 0

. 372 ppm in 41-DAT3 seed.

A subsample of forage was subjected to conventional extraction with methanol/water and methanol/1 N NaOH

, which release d the majority of the radioactivity from forage (8 7. 6% and

DP# 400085/MRID No. 4 84 49 801 Page 1 of262,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

DP# 400085/MRID No. 48449801 Page 2 of 26 

### 6.1% TRR, respectively). Nonextractable resi dues following these extraction procedures were 2.6% TRR (1.304 ppm).

An additional subsample of forage and samp les of 7- and 22-DAT3 hay and 62- and 41-DAT3 seed were subjected to accelerated solvent extrac tion (ASE) with a series of neutral solvents at temperature ranges of 40-110 °C (forage, hay, and s eed) and 40-70 °C (hay and seed). In forage and hay the majority of characterization work was done on samples subjected to ASE at 40-10 °C, via extraction with methanol at 40 °C , methanol/water at 70 °C and 110 °C, and methanol/acetonitrile (ACN)/water at 110 °C. Ex traction with methanol at 40 °C released the majority of radioactivity from forage (105.2% TRR) and hay (77.3-86.0% TRR). Subsequent ASE with methanol/water at 70 and 110 °C and me thanol/ACN/water at 110 °C each released an additional 0.4-3.7% TRR from forage, 0.7- 13.7% TRR from 7-DAT3 hay, and 1.3-17.6% TRR from 22-DAT3 hay. Nonextractable residues fo llowing these extraction procedures were 2.8% TRR (1.393 ppm) in forage and 4.2-8.2% TRR (1.478-3.027 ppm) in 7- and 22-DAT3 hay.

In seed, the majority of char acterization work was done on 41-DAT3 samples subjected to ASE at 40-70 °C, via extraction with hexane and meth anol at 40 °C and methanol/water at 70 °C. Extraction with hexane, methanol, and meth anol/water released ~10.0-10.2%, 11.0-17.0, and 13.9-19.4% TRR, respectively. Subsequent hydrolys is of the nonextracable residues with 2 N NaOH released an additional 50.1% TRR. Rema ining nonextractable residues were 2.6% TRR (0.010 ppm).

The ASE procedures adequately extracted the majo rity of the residues from soybean matrices. Accountabilities were 97.9-117% for forage and hay and 93.5% for seed.

Residues were identified and quantitated in soybean forage, hay, and seed extracts and hydrolysates by high performance liquid chromatogr aphy (HPLC); seed extracts were purified by solid phase extraction (SPE) prior to analysis. Hexane extracts from 41 -DAT3 seed were also subjected to thin layer chromatography (TLC) fo r residue characterizatio n/identification. In addition, identification of 2,4-D and 2,4-DCP was confirmed, and the glucose conjugates of metabolite 2,4-DCP, including Metabolites A (acetyl-glucose conjugate), B (X11963417), and C disaccharide conjugate), were identified in fo rage and hay extracts/ hydrolysates following isolation by SPE and analysis by liquid chro matography with tandem mass spectrometry (LC/MS/MS). Metabolite A was thought to be th e initial or primary conjugate of 2,4-DCP. Metabolites B and C were found to be minor resi due components as well as artifacts of the extraction/analysis procedures.

Samples of soybean forage, hay, and seed were extracted and initially analyzed 44-146 days (1.4-4.8 months) after harvest. 22-DAT3 hay a nd 41-DAT3 seed were s ubjected to additional extraction and HPLC analysis up to 357-398 days (11.7-13.1 months) after harvest. To confirm storage stability, the or iginal ASE extracts of forage and 22-DAT3 hay were re-analyzed 453 and 417 days (14.9 and 14.1 months) after harvest, and the in itial and additional ASE results for 22- DAT3 hay and 41-DAT3 seed were compared. The petitioner stated that re-extraction and/or additional HPLC analysis showed the same extrac tion results and/or chro matographic profiles as in samples analyzed earlier.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

DP# 400085/MRID No. 48449801 Page 3 of 26 

### The parent, 2,4-D, was the major residue iden tified in forage (85.8% TRR; 43.117 ppm) and hay (59.4-64.3% TRR; 21.978-22.885 ppm), while 2,4-DCP was present at 7.1% TRR (3.558 ppm) in forage and 1.4-3.6% TRR (0.518-1.295 ppm) in hay. Metabolite A accounted for 10.4% TRR (5.223 ppm) in forage and 12.6-13.4% TRR (4.474- 4.969 ppm) in hay. Metabolites B and C were present at 0.8% and 0.1% TRR (0.381 a nd 0.055 ppm), respectively, in forage and 5.4- 7.0% and 1.1-2.7% TRR (1.919-2.573 and 0.395-1.000 pp m), respectively, in hay. Remaining radioactivity, designate d “other” (radioactive peaks that did not co-chromatograph with standards and any radioactivity that was detected but not quan titated as a specific peak), accounted for 5.1% TRR in forage and 2.5% an d 22.3% TRR, respectively in 7- and 22-DAT3 hay.

In 41-DAT3 seed, following ASE with methanol a nd methanol/water, and base hydrolysis of the nonextractable residues, 2,4-D accounted for 1.4% TRR, free 2,4-DCP comprised only 0.1% of the TRR, and the 2,4-DCP conjugates Metabolite s B and C accounted for 3.0% and 4.6% TRR, respectively. TLC of the hexane extracts (10% TRR) indicated that >90% of the radioactivity was distributed in natural const ituents. These components were a ttributed to triglycerides and unsaturated fatty acids. “Other” radioactiv ity accounted for 56.9% TRR (0.211 ppm) and was found to be multicomponent and polar. Pr ecipitates following base hydrolysis of the nonextractable residues accounted for 13.4% TRR.

Based on the results of the metabolism study wi th AAD-12 soybean (Event 416), the petitioner proposed that the metabolism of 2,4-D DMA pro ceeds rapidly via dissociation to the acid (2,4- D). After cleavage of the acid side chain, th e resulting dichlorophenol (2,4-DCP) was rapidly conjugated with acetylated glucos e and other glucose conjugates. Low levels of free 2,4-DCP compared to conjugated 2,4-DCP indicated that the conjugation was rapid and a preferential route of metabolism. In seed, bound residues we re produced that liberated large numbers of polar metabolites upon acid and base hydrolysis. The petitioner noted that since 2,4-D has been demonstrated to degrade to CO 2 

### in soil, it was likely that any 2,4-D applied to the soil was converted to CO 2 

### and became incorporated into natura l plant constituents. Hydrolysis experiments conducted with extracted soybean lipi ds and bound residues indi cated that this was a likely route of metabolism. Previous plan t metabolism studies conducted on AAD-1 corn also concluded that metabolism of 2,4-D DMA pro ceeded to 2,4-DCP, glucose conjugates of 2,4- DCP, and to incorporation into natural plant constituents. Overall, the 14 

### C-residues in the AAD12 soybeans were similar to those observed on transgenic corn and conventional crops.

STUDY/WAIVER ACCEPTABILITY/ DEFICIENCIES/CLARIFICATIONS :
Under the conditions and parameters used in th e study, the plant metabolism data are classified as scientifically acceptable. The acceptability of this study fo r regulatory purposes is addressed in the forthcoming U.S. EPA Residue Chemistry Summary Document [DP# 400085].2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

DP# 400085/MRID No. 48449801 Page 4 of 26 

### COMPLIANCE :
Signed and dated Good Laboratory Practice (GLP), Quality Assurance and Data Confidentiality statements were provided. No deviations fr om regulatory requirement s were reported which would have an impact on the validity of the study.

A. BACKGROUND INFORMATION

2,4-Dichlorophenoxyacetic acid (2,4-D) is an alkyl chlorophenoxy herbicide used to control a variety of broadleaf weeds. These data we re submitted to support the proposed use for 2,4-D choline application to soybean containing the inserted aryl oxyalkanoate dioxygenase-12 (AAD12) gene. This soybean is identified as c ontaining DAS-68416-4. Expression of the AAD-12 protein encoded by the AAD-12 gene results in a trait that increas es the herbicide tolerance of soybean to 2,4-D via increased metabolism through a pathway involving the metabolite 2,4- dichlorophenol (2,4-DCP).

The chemical structure and nomenclature of 2,4-D, 2,4-D DMA, and 2,4-D choline are summarized in Table A.1, and the physicochemical properties are summarized in Table A.2.

TABLE A.1. Test Compound Nomenclature. 2,4-D

Common name 2,4-D acid IUPAC name (2,4-dichlorophenoxy)acetic acid CAS name 2-(2,4-dichlorophenoxy)acetic acid
CAS # 94-75-7
PC Code 030001 End-use product/(EP) Not applicable 2,4-D dimethylamine salt (DMA)

IUPAC name (2,4-dichlorophenoxy)ace tic acid - dimethylamine (1:1) CAS name 2-(2,4-dichlorophenoxy)acetic acid compound with N-methylmanethanamine (1:1)
CAS # 2008-39-1
PC Code 030019 End-use product/(EP) 3.8 lb ae/gal SL/L formul ation (GF-2654 TS Herbicide; EPA Reg. No. 62719-XXX)2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

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TABLE A.1. Test Compound Nomenclature. 2,4-D choline

IUPAC name 2-hydroxy-N,N,N-trimethyleth anaminium (2,4-dichlorophenoxy)acetate CAS name Ethanaminium, 2-hydroxy-N,N,N-trim ethyl-, 2-(2,4-dichlorophenoxy)acetic acid hydroxide (1:1:1)
CAS # 1048373-72-3
PC Code 051505

TABLE A.2. Physicochemical Properties of the T echnical Grade Test Co mpound: 2,4-D acid.
Parameter Valu e Reference Melting point/range 138-141 °C DP#309452, T. Jimerson, 10/13/04 pH Not available Density Specific gravity = 1.416 at 25 °C Water solubility ( __°C) 569 mg/L at 20 °C Solvent solubility (mg/L at __°C) g/100 g at 25 °C acetone 85.0 benzene 1.07 diethyl ether 220 ethanol 130.0 isopropanol 31.6 toluene 0.067 xylene 0.58 Vapor pressure at __°C 1.4 x 10 -7 mm Hg at 25 °C Dissociation constant (pK a ) Not available Octanol/water partition coefficient Log(K OW ) In 0.001 M solution: 2.14 at pH 5 0.177 at pH 7 0.102 at pH 9 UV/visible absorption spectrum Not available2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

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### B. EXPERIMENTAL DESIGN

B.1. Test Site and Crop Information

Soybean seed containing the AAD-12 gene was planted in a planting box and two pots filled with sandy clay loam soil and maintained outdoors at Research for Hire (RFH; Porterville, CA). Samples of forage, hay, and seed were collected from the planting box; samples of seed only were collected from the pots.

The original two pots had poor growth and were discarded. Two additional pots were replanted and buried into soil to approximately one inch fro m the top of the pot to reduce the temperature of the soil in the pots. The study did not mention a control plot or pot. The soybean plants were watered as needed and fertiliz ed to ensure normal crop growth. Monthly climatic conditions (temperature and humidity maximums and minimu ms; and rainfall) were reported. There were no meteorological abnormalities that may have impact ed the study. A summary of the test site information is provided in Table B.1.1, and a summ ary of the crop information is provided in Table B.1.2.

TABLE B.1.1. Test Site Information.
Type Method Soil characteristics
Type %OM pH CEC (meq/100g)
One soil application and two foliar applications Soybean was grown from seed outdoors in one planting box (5’ x 3’ x 1.5’ deep) and two pots (each 12” in diameter). Sandy clay loam Not reported

TABLE B.1.2. Crop Information. 1 Crop/crop group Variety Growth stage at application Growth stage at harvest Harvested Matrix Soybean; Vegetable, legume, group 6; and Vegetable, foliage of legume, group 7 AAD-12;

event-construct pDAB4468-416 1. Preemergence (soil) 2. V4 (foliar) 3. R2 (foliar) Box: 7-DAT2 forage: BBCH 60; first flowers open 7-DAT3 hay: R3; beginning pod 22-DAT3 hay: R4; full bloom, open flower at one of the two uppermost nodes on the main stem with a fully developed leaf 62-DAT3 seed: R8; majority of pods ripe and beans final color, dry and hard 7-DAT2 forage 7-DAT3 hay 22-DAT3 hay 62-DAT3 seed

Pots: 41-DAT3 seed: R8; majority of pods ripe and beans final color, dry and hard 41-DAT3 seed

1 DAT = Days after treatment. Forage was harvested 7 days after the second application (DAT2) ; hay and seed were harvested following the third application (DAT3).2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

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### B.2. Test Materials

The radiolabeled test substance was diluted in me thanol and ACN, then isotopically diluted with nonradiolabeled 2,4-D DMA and form ulated as an SL/L using formulation blank. The dose for the pots was prepared with a highe r specific activity than that pr epared for the planting box. The characteristics of the test substa nce are presented in Table B.2.1.

TABLE B.2.1. Test Mat erial Characteristics.
Chemical structure

Radiolabel position [phenyl-U- 14 C]2,4-D DMA
Lot No. INV027475-0002 Purity 99.1% Specific activity As received: 39.4 mCi/mmol (148 μCi/mg) Formulated test substance: 10.92 μ Ci/mg (for planting box) and 101.37 μ Ci/mg (for pots)



### B.3. Study Use Pattern

The formulated test substance was diluted with water and ACN and applied as a preemergence soil application at 1.04 -1.11 lb ae/A (1.166-1.248 kg ae/ha) followe d by two foliar applications at 0.98-1.11 lb ae/A/application (1.103- 1.243 kg ae/ha/application), for a total application rate of 3.13-3.19 lb ae/A (3.512-3.578 kg ae/ha) to a plan ting box or pots. The preemergence application was made on the day of planting, and fo liar applications were made at the V4 and R2 growth stages at RTIs of 34 and 21 days to s oybean grown in the plantin g box, and at RTIs of 22 and 20 days to soybeans grown in pots. The speci fic activity of the test substance was higher for the pots to potentially aid in metabolite identifica tion in seed. Applications were made using an R&D wand sprayer pressurized with CO 2


### . Samples of forage and hay were harvested by cutt ing plants just above the soil surface (forage) or 1-1.5” above the soil surface (hay). Sample s of soybean seed were collected by cutting mature stalks ~1” above the soil surface; the pod s were removed from the stalks, and the seed was separated from the pods by hand. Samples of fo rage, hay, and seed were harvested from the planting box, and samples of seed only we re harvested from the pots as follow.

Planting box : Samples of forage (BBCH 60 growth st age; first flowers opened) and hay (R3 growth stage; beginning pod) were collected 7 da ys after the second and th ird foliar applications, respectively (7-DAT2 forage and 7- DAT3 hay). The first hay sample was not dried; therefore, a second sample of hay was cut at the R4 growth st age (full bloom, open flow er at one of the two uppermost nodes on the main stem with fully devel oped leaf) 22 days after the third application and allowed to dry in a greenhouse (22-DAT3 hay) . Samples of seed were collected from mature soybean plants (R8 growth stage; major ity of pods are ripe; beans final color, dry and hard) 62 days after the second fo liar application (62-DAT3 seed).2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

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### Pots : Samples of seed were collected from mature soybean plants (R8 growth stage) 41 days after the second foliar app lication (41-DAT3 seed).

Details of the study use pattern are presented in Table B.3.1.

TABLE B.3.1. Use Pa ttern Information. Chemical name [phenyl-U- 14 C]2,4-D DMA Application method The test substance was mixed with soluble liqui d (SL/L) formulation blank, diluted with water and ACN, and applied as a single preemergence soil application, followed by two foliar applications to so ybean plants using an R&D wand sprayer pressurized with CO 2 . Application rate Box: Preemergence: 1.11 lb ae/A (1.248 kg ae/ha) Foliar: 1.03-1.05 lb ae /A/application (1.153-1.177 kg ae/ha/application) Total: Forage (2 applications): 2.14 lb ae/A (2.401 kg ae/ha) Hay and seed (3 applications): 3.19 lb ae/A (3.578 kg ae/ha)
Pots: Preemergence: 1.04 lb ae/A (1.166 kg ae/ha) Foliar: 0.98-1.11 lb ae /A/application (1.103-1.243 kg ae/ha/application) Total: Seed (3 applications): 3. 13 lb ae/A (3.512 kg ae/ha) Number of applications 3: 1 soil application and 2 fo liar applications Timing of applications Box: Preemergence: 0 day post-planting Foliar: 34- and 21-days retreatment intervals
Pots: Preemergence: 0 day post-planting Foliar: 22- and 20-days retreatment intervals
PHI Box: 7-DAT2 Forage: 7 days after second application 7-DAT3 Hay: 7 days after third application 22-DAT3 Hay: 22 days after third application 62-DAT3 Seed: 62 days after third application.
Pots: 41-DAT3 Seed: 41 days after third application.



### B.4. Identification/ Characterization of Residues

B.4.1. Sample Handling and Preparation

Following harvest, all samples were stored frozen at the field site and were shipped on dry ice to the analytical laboratory, ABC Laboratories (Colum bia, MO), within 0-9 days of harvest. At ABC Laboratories, samples were prepared for ex traction by grinding in th e presence of dry ice using a grinding mill, then returned to frozen storage (~-20 °C).

One subsample of 7-DAT2 forage was extracted w ith methanol:water (90:10, v:v), then vacuum filtered; an aliquot of the extract was reserved for HPLC analysis. Remaining residues were further extracted with methanol:1.0 N NaOH (90: 10, v:v), at 50 °C for 1 hour, then at room temperature for 1 hour. The resulting extract wa s, centrifuged, vacuum f iltered and reserved for HPLC analysis.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

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### Subsamples of 7-DAT2 forage, 7- and 22-DAT3 hay and 62- and 41-DAT seed were subjected to ASE with a series of neutral solvents at temperature ranges of 40-110 °C (forage, hay, and seed) and 40-70 °C (hay and seed). ASE at 40- 110 °C involved extraction with hexane at 40 °C (seed only), acetone at 40 °C (seed only), metha nol at 40 °C, methanol:w ater (80:20, v:v) at 70 °C and 110 °C, and finally methanol:ACN:water (containing ammonium hydroxide at 2% of entire volume; 40:40:20, v:v) at 110 °C. ASE at 40-70 °C involved extraction with hexane (2x) at 40 °C (seed only), methanol ( 2x) at 40 °C, and methanol:water (80:20, v:v) (2 x) at 70 °C. Forage and hay extracts were reserved for HPLC; seed extracts were subjected to solid-phase extraction (SPE) procedures prior to HPLC analysis.

All seed extracts and hydrolysate s were subjected to SPE prior to further analysis. SPE was conducted on Strata-X SPE cartridges. Extracts were adjusted to pH 2-3 then applied to the cartridge; residues were eluted sequentially with methanol:wat er:1 N HCl (50:49:1, v:v:v), cyclohexane, and methanol:1 N NaOH (99:1, v:v).

Because of the concern that nonextractable radi oactivity might exist as conjugated 2,4-D or 2,4- DCP, the following extractable and nonextractab le residues were subj ected to acid and base hydrolysis with 2 N HCl and 2 N NaOH: 40 °C methanol extracts of 7-DAT2 forage and 22- DAT3 hay from the 40-100 °C ASE; combined 40 °C hexane extracts of 41-DAT3 seed from the 40-70 °C ASE; and nonextractable residues of 41-DAT3 seed following 40-70 °C ASE. Because results were similar for hydrolysis of fo rage and hay extracts, only hay results were reported in the Study Report.

For acid/base hydrolysis of forage and hay extrac ts, two separate aliquots of the 40 °C methanol extracts of 7-DAT2 forage and 22-DAT3 hay were adjusted to pH 11 with 2 N NaOH, then heated with 2 N HCl or 2 N NaOH (at 90 °C for 2 hours). The hydrolysates were adjusted to pH 4-5 with glacial acetic acid or 10 N NaOH, dilu ted with methanol and ACN, and centrifuged. The supernatant was filtered and reserved for HPLC analysis.

For hydrolysis of seed extracts, two separate aliquots of the combined 40 °C hexane extracts of 41-DAT3 seed were heated with 2 N HCl or 2 N NaOH (at 90 °C for 2 hours). The vials were rinsed with methanol, dichloromethane, and ACN to clarify the samples, and the acid hydrolysate was adjusted to pH 9-12. The hydrolys ates were concentrated to dryness or near dryness, then combined with wate r and adjusted to pH 2-3 using concentrated HCl in preparation for SPE as described above. The cyclohexa ne and methanol/1N NaOH eluates following SPE were concentrated. The cyclohe xane fraction was redissolved in hexane, dichloromethane, and methanol, and reserved for TLC analysis. The methanol/1 N NaOH eluate was dissolved in water: methanol (90:10, v:v) and adjusted to pH 3-4 with glacial acetic acid, then centrifuged, yielding an oily upper layer. This oily layer was combined with the methanol and dichloromethane rinses of the vials and reserved for TLC analysis.

For hydrolysis of the nonextractable residues of seed, two separate aliquots of the nonextractable residues of 41-DAT3 seed following 40-70 °C ASE were heated in a water bath for ~5 hours with 2 N HCl or 2 N NaOH (at 52-78 °C for fi rst 2.73 hours and at 80 °C for latter 2.17 hours),2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

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### then centrifuged. The solids were subsequently resuspended and centrifuged with water (2x) and methanol (3x). The methanol supernatants we re combined, adjusted to pH 9-10 with NaOH, then concentrated by rotary evaporation and co mbined with the aqueous supernatants following acid hydrolysis. The combined aqueous + metha nol supernatants following acid hydrolysis and the combined aqueous supernatants following base hydrolysis were prepared for SPE as described above by adjusting to pH 2-3 followed by several centrifugation and precipitate-rinsing steps. The supernatants and precipitates from th e acid and base treatment from this process were reserved for LSC. The supernatants were s ubjected to SPE cleanup and analyzed by HPLC or TLC.

Representative flowcharts (copied without alteration from MRID 48449801) of the extraction and characterization and hydrolysis procedures for soybean forage, hay, and seed are presented below in Figures B.4.1.1. and B.4.1.2.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

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### FIGURE B.4.1.1. Extraction Procedures for Soybean Forage, Hay, and Seed. 1

1 

### We note that the diagram appears to be mislab eled. The procedures for hay and seed on the far right should be desi gnated 40-70 °C ASE.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

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### FIGURE B.4.1.2. Acid and Base Hydrolysis Procedures.

B.4.2. Analytical Methodology

TRR in soybean forage, hay, and seed were dete rmined by combustion/LSC. TRR in extracts and hydrolysates were determined in quintuplicat e by direct liquid scinti llation counting (LSC), and TRR in nonextractable residues were determ ined by combustion/LSC. The reported limit of quantitation for TRR determina tions in tissue was 0.0107 ppm.

Extracts and SPE eluates were prepared for HPLC or TLC analysis by: (1 ) dilution with water: glacial acetic acid (100:0.1, v:v) to an organic solvent concentr ation of 30% (pH ~4-5); (2) concentration by rotary evaporation followed by r econstitution in methanol and/or ACN (pH ~3- 5); or (3) adjustment to pH ~9-12 with NaOH prior to concentration by vacuum/rotary evaporation, followed by adjustment to pH 3-5 with glacial acetic acid or HCl for analysis. The prepared extracts were centrifuged prior to HPLC or TLC analysis.

Extracts and hydrolysates of soybean forage, hay, and seed were analyzed by primary and secondary reverse-phase HPLC methods for identi fication and quantitation of metabolites. The primary HPLC system (Program A) was equippe d with a Synergi 4 μm Hydro-RP column, a UV detector (280 nm), and a radiodetector, and utili zed a gradient mobile phase of 0.1% acetic acid in water and 0.1% acetic acid in ACN:methanol (80:20, v:v). The s econdary HPLC system (Program B) was equipped with a Synergi 4 μm Polar-RP column, a UV detector (280 nm), and a radiodetector, and utilized a gradient mobile phase of 0.1% formic acid in water and 0.1% formic acid in ACN. Metabolites were identifie d by retention time compar isons with reference standards. Chemical names and structures for the reference standards ar e presented in Appendix I.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

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### The hexane extract and acid a nd base hydrolysates from the hexane extracts of 41-DAT3 seed were subjected to TLC analysis for identificatio n and quantitation of metabolites. TLC analyses were conducted using silica gel 60F 254 

### plates and solvent sy stem of hexane:methyl tert -butyl ether (9:1, v:v). Metabolites were identified by co-chromat ography and/or comparison of retention times with thos e of nonlabeled and radiolabeled re ference standards. Residues were visualized under UV light (254 nm) following ex posure to iodine vapor; radioactivity was detected by scraping the plates and subjecting the scrapings to radioassay by LSC. Chemical names and structures for the reference st andards are presented in Appendix I.

For further identification of metabolites, the 40 °C methanol extract of forage from ASE at 40- 110 °C that had been concentrated at acidic and ba sic pH for HPLC analysis, as well as the base hydrolysate of the 40 °C methanol extract of 22-DAT3 hay, were analyzed LC/MS and LC/MS/MS with electrospray ionization (ESI). A Berthold radioactivity monitor (RAM) was used to assist in location of the metabolite peaks.

C. RESULTS AND DISCUSSION

The storage conditions and durati ons for soybean matrices are presented in Table C.1. Samples of soybean forage, hay, and seed were extracted and initially analyzed 44-146 days (1.4-4.8 months) after harvest. 22-DAT3 hay and 41-DAT3 were subjected to additional extraction and HPLC analysis up to 357-398 days (11.7-13.1 mont hs) after harvest. To confirm storage stability, the original ASE extracts of fora ge and 22-DAT3 hay were re-analyzed 453 and 417 days (14.9 and 14.1 months) after harvest, and the initial and additional ASE results for 22- DAT3 hay and 41-DAT3 seed were compared. The petitioner stated that re-extraction and/or additional HPLC analysis showed the same extrac tion results and/or chro matographic profiles as in samples analyzed earlier.

TRR in AAD-12 (event 416) soybean matrices are reported in Table C.2.1. Following a single preemergence soil application, followed by two folia r applications to soyb ean plants grown in a planting box, for a total app lication rate of 3.19 lb ae/A, TRR were 50.227 ppm in 7-DAT2 forage (two applications; 2.14 lb ae/A), 35.565 ppm in 7-DAT3 hay, 37.008 ppm in 22-DAT3 hay, and 0.415 ppm in 62-DAT3 seed. Following a single preemergenc e soil application, followed by two foliar applications to soybean plants grown in pots, for a total application rate of 3.13 lb ae/A, TRR were 0.372 ppm in 41-DAT3 seed.

The distribution of radioactivity in the extracts of soybean matrices is presented in Tables C.2.2.1 (forage and hay) and C.2.2.2 (seed). A subsample of forage was subjected to conventional extraction with methanol/water and methanol/1 N NaOH, which released the majority of the radioactivity from forage (87.6 % and 6.1% TRR, respectively). Nonextractable residues following these extraction pr ocedures were 2.6% TRR (1.304 ppm).

An additional subsample of forage and samp les of 7- and 22-DAT3 hay and 62- and 41-DAT3 seed were subjected to accelerated solvent extrac tion (ASE) with a series of neutral solvents at temperature ranges of 40-110 °C (forage, hay, and s eed) and 40-70 °C (hay and seed). In forage2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

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### and hay the majority of characterization work was done on samples subjected to ASE at 40-10 °C, via extraction with methanol at 40 °C , methanol/water at 70 °C and 110 °C, and methanol/acetonitrile (ACN)/water at 110 °C. Ex traction with methanol at 40 °C released the majority of radioactivity from forage (105.2% TRR) and hay (77.3-86.0% TRR). Subsequent ASE with methanol/water at 70 a nd 110 °C and methanol/ACN/water at 110 °C each released an additional 0.4-3.7% TRR from forage, 0.7- 13.7% TRR from 7-DAT3 hay, and 1.3-17.6% TRR from 22-DAT3 hay. Nonextractable residues fo llowing these extraction procedures were 2.8% TRR (1.393 ppm) in forage and 4.2-8.2% TRR (1.478-3.027 ppm) in 7- and 22-DAT3 hay.

In seed, the majority of char acterization work was done on 41-DAT3 samples subjected to ASE at 40-70 °C, via extraction with hexane and meth anol at 40 °C and methanol/water at 70 °C. Extraction with hexane, methanol, and meth anol/water released ~10.0-10.2%, 11.0-17.0, and 13.9-19.4% TRR, respectively. Subsequent hydrolys is with 2 N NaOH released an additional 50.1% TRR. Remaining nonextractable residues were 2.6% TRR (0.010 ppm).

The ASE procedures adequately extracted the majo rity of the residues from soybean matrices. Accountabilities were 97.9-117% for forage and hay and 93.5% for seed.

Residues were identified and quantitated in soybean forage, hay, and seed extracts and hydrolysates by HPLC; seed extract s were purified by SPE prior to analysis. Hexane extracts from 41-DAT3 seed were also subjected to TLC for residue characteriza tion/identification. In addition, identification of 2,4-D and 2,4-DCP was confirmed, and the glucose conjugates of metabolite 2,4-DCP, including Metabolites A (acetyl-glucose conjugate), B (X11963417), and C (disaccharide conjugate), were identified in forage and hay extracts/hydrolysates following isolation by SPE and analysis by liquid chro matography with tandem mass spectrometry (LC/MS/MS). Metabolite A was thought to be th e initial or primary conjugate of 2,4-DCP. Metabolites B and C were found to be minor resi due components as well as artifacts of the extraction/analysis procedures.

The characterization and identifica tion of residues in soybean matr ices are summarized in Tables C.2.3.1. (forage and hay) and C.2.3.2. (seed). The parent, 2,4-D, was the major residue identified in forage (85.8% TRR; 43.117 ppm) and hay (59.4-64.3% TRR; 21.978-22.885 ppm), while 2,4-DCP was present at 7.1% TRR (3.558 ppm) in forage and 1.4-3.6% TRR (0.518-1.295 ppm) in hay. Metabolite A accounted for 10.4% TRR (5.223 ppm) in forage and 12.6-13.4% TRR (4.474-4.969 ppm) in hay. Metabolites B and C were present at 0.8% and 0.1% TRR (0.381 and 0.055 ppm), respectively, in fora ge and 5.4-7.0% and 1.1-2.7% TRR (1.919-2.573 and 0.395-1.000 ppm), respectively, in hay. Re maining radioactivit y, designated “other” (radioactive peaks that did not co-chromatograph with standards and any radioactivity that was detected but not quantitated as a specific peak), accounted for 5.1% TRR in forage and 2.5% and 22.3% TRR, respectively in 7- and 22-DAT3 hay.

The presence of Metabolites B and C as both mi nor residue components as well as artifacts of the extraction/analysis procedures was confirme d by comparing results following preparation for HPLC analysis by concentration at acidic or basic PH. These results also indicated that Metabolite A and other minor components gene rally converted to Metabolites B and C on2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

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### concentration under basic conditions. Further acid and base hydrolysis of the methanol extract of hay indicated that 2,4-D wa s converted to 2,4-DCP under acidic or basic conditions and that most of the extractable radioactivity that was not present as 2,4-D or 2,4-DCP was present as conjugated 2,4-DCP.

In 41-DAT3 seed, following ASE with methanol a nd methanol/water, and base hydrolysis of the nonextractable residues, 2,4-D accounted for 1.4% TRR, free 2,4-DCP comprised only 0.1% of the TRR, and the 2,4-DCP conjugates Metabolite s B and C accounted for 3.0% and 4.6% TRR, respectively. TLC of the hexane extracts (10% TRR) indicated that >90% of the radioactivity was distributed in natural const ituents. These components were a ttributed to triglycerides and unsaturated fatty acids. “Other” radioactivity accounted for 56.9% TRR (0.211 ppm). Most of the radioactivity in the 40 °C methanol a nd 70% methanol/water extracts (~15.6% TRR) was comprised of 35 different peaks, with the larg est accounting for 5.1% T RR; remaining “other” radioactivity in seed was found to be multicompo nent and polar. Precipitates following base hydrolysis of the nonextractable residues accounted for 13.4% TRR.

C.1. Storage Stability

TABLE C.1. Summary of Storage Conditions.
Matrix 1 Storage Temperature (°C) Actual Storage Duration Interval of Demonstrated Storage Stability

7-DAT2 Forage -20 HPLC (conventional): 44 days (1.4 months) HPLC (ASE): 146 days (4.8 months) HPLC (re-analysis ASE extracts): 453 days (14.9 months) MS: 468 days (15.4 months) 7-DAT3 Hay HPLC (ASE): 111 days (3.7 months) HPLC (re-analy sis ASE extracts): 417 days (13.7 months) MS: 432 days (14.2 months) 22-DAT3 Hay HPLC (ASE): 97 days (3.2 months) HPLC (ASE additional): 398 days (13.1 months) 62-DAT3 Seed Initiate extraction: 56 days (1.8 months) Additional extraction: 86 days (2.8 months) HPLC (re-extraction and ASE): 357 days (11.7 months) 41-DAT3 Seed HPLC (ASE): 79 days (2.6 months) HPLC (ASE additional): 357 (11.7 months)

1 DAT = Days after treatment. Forage was harvested 7 days after the second application (DAT2) ; hay and seed were harvested following the third application (DAT3). Fo rage and hay were harvested from the plan ting box only; seed was harvested 62 DAT from the planting box and 41 DAT from pots.



### C.2. Identification, Characterizati on, and Distribution of Residues

TABLE C.2.1. Total Radioactive Residues (T RR) in AAD-12 Soybean (event 416) Matrices

. 1 Matrix Timing, Applic. No.: Rate PHI (days) TRR (ppm ae)

7-DAT2 Forage Preemergence + 1 Foliar: 2.14 lb ae/A (2.401 kg ae/ha) 7 50.227

7-DAT3 Hay Preemergence + 2 Foliar: 3.19 lb ae/A (3.578 kg ae/ha) 7 35.565 22-DAT3 Hay 22 37.008
62-DAT Seed 62 0.4152,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

DP# 400085/MRID No. 48449801 Page 16 of 26

41-DAT Seed Preemergence + 2 Foliar: 3.13 lb ae/A (3.512 kg ae/ha) 41 0.372

1 DAT = Days after treatment. Forage was harvested 7 days after the second application (DAT2) ; hay and seed were harvested following the third application (DAT3). Fo rage and hay were harvested from the plan ting box only; seed was harvested 62 DAT from the planting box and 41 DAT from pots.

TABLE C.2.2.1. Distribution of the Parent and the Meta bolites in AAD-12 Soybean (event 416) Forage and Hay Following Application of [ 14 C]2,4-D DMA at 2.14 lb ae/A (2.401 kg ae/ha) for forage and 3.19 lb ae/A (3.578 kg ae/ha) for hay. 1

Metabolite Fraction 7-DAT2 Fo rage 7-DAT3 Hay 22-DAT3 Hay TRR = 50.227 ppm TRR = 35.565 ppm TRR = 37.008 ppm %TRR ppm %TRR ppm %TRR ppm %TRR ppm Neutral Organic & Methanolic Base Extraction Subsample #1

Methanol/water 87.6 44.011
Methanol/1 N NaOH 6.1 3.044
Combined extracts 93.7 47.055 2,4-D 73.5 36.932 2,4-DCP 4.5 2.282
Metabolite A (DCP-acetyl-glucose conjugate) 12.4 6.239

Metabolite B (X11963417) 2.1 1.079 Other (one peak) 1.0 0.523 Largest peak 0.6 0.322
Nonextractable 2.6 1.304 ASE 40-110 °C Subsample #2 Rep. A Rep. A Methanol, 40 °C 105.2 52.835 77.3 27.508 86.0 31.828

Concentrated for HPLC (pH 5) Subsample Subsample 2,4-D 85.1 42.762 51.9 19.222 2,4-DCP 2.4 1.216 0.8 0.314
Metabolite A (DCP-acetyl-glucose conjugate) 11.7 5.871 9.8 3.621

Metabolite B (X11963417) 1.8 0.923 2.9 1.055
Metabolite C -- -- 1.3 0.483
Other 2 3.7 1.834 21.8 8.053 Largest peak 0.9 0.429 2.1 0.772 Basified/conc. for HPLC (pH 11) Subsample Subsample 2,4-D 85.2 42.782 49.4 18.278 2,4-DCP 7.8 3.897 0.8 0.314 Metabolite B (X11963417) 13.9 6.974 19.8 7.332
Metabolite C 0.7 0.344 13.7 5.081
Other 3 2.1 1.052 5.3 1.952 Largest peak 0.5 0.234 1.3 0.470 Acid Hydrolysate (2 N HCl) Subsample Hydrolysate 72.8 26.947 2,4-D 44.2 16.367 2,4-DCP 26.3 9.740
Other 4 2.3 0.8402,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

DP# 400085/MRID No. 48449801 Page 17 of 26

TABLE C.2.2.1. Distribution of the Parent and the Meta bolites in AAD-12 Soybean (event 416) Forage and Hay Following Application of [ 14 C]2,4-D DMA at 2.14 lb ae/A (2.401 kg ae/ha) for forage and 3.19 lb ae/A (3.578 kg ae/ha) for hay. 1

Metabolite Fraction 7-DAT2 Fo rage 7-DAT3 Hay 22-DAT3 Hay TRR = 50.227 ppm TRR = 35.565 ppm TRR = 37.008 ppm %TRR ppm %TRR ppm %TRR ppm %TRR ppm Largest peak 0.6 0.213 Base Hydrolysis (2 N NaOH) Subsample Hydrolysate 81.6 30.181 2,4-D 44.1 16.316 2,4-DCP 19.4 7.172
Metabolite C 15.0 5.558
Other 5 3.1 1.136 Largest peak 0.6 0.240 Methanol/water, 70 °C 3.7 1.856 13.7 4.865 17.6 6.529 Methanol/water, 110 °C 1.0 0.509 2.0 0.717 3.8 1.401 Methanol/ACN/water, 110 °C 0.4 0.194 0.7 0.265 1.3 0.482
Summed ASE Extracts 6 Not reported Not reported Not reported 2,4-D 85.8 43.117 64.3 22.885 59.4 21.978 2,4-DCP 7.1 3.558 3.6 1.295 1.4 0.518
Metabolite A (DCP-acetyl-glucose conjugate) 10.4 5.223 12.6 4.474 13.4 4.969

Metabolite B (X11963417) 0.8 0. 381 5.4 1.919 7.0 2.573
Metabolite C 0.1 0.055 1.1 0.395 2.7 1.000
Other 7 5.1 2.557 2.5 0.901 22.3 8.256 Largest peak 2.2 1.088 5.6 1.975 1.5 0.537
Nonextractable 2.8 1.393 4.2 1.478 8.2 3.027
ASE 40-70 °C Rep. B Rep. C Methanol, 1, 40 °C 83.7 30.964 85.6 31.667 Methanol, 2, 40 °C 7.0 2.596 6.6 2.434 Methanol/water 1, 70 °C 11.1 4.094 11.5 4.255 Methanol/water 2, 70 °C 2.3 0.865 2.2 0.831
Nonextractable 10.9 4.048 11.0 4.088

1 Shading indicates that the extraction step and/or characteri zation analysis was not conducted fo r the matrix in question. DA T = Days after treatment. Forage was harvested 7 days after th e second application (DAT2); ha y was harvested 7 and 22 days following the third application (DAT3). ASE = Accelerated Solven t Extraction. “Other” includes radioactive peaks that did not co-chromatograph with any of the four metabolites or parent 2,4-D, plus any radioactivity that was detected but was not quantified as a specific peak.
2 Six peaks in forage; 29 peaks in 22-DAT3 hay.
3 Two peaks in forage; 5 peaks in 22-DAT3 hay.
4 Two peaks in 22-DAT3 hay.
5 Four peaks in 22-DAT3 hay.
6 Calculated by the pe titioner based on results from: 40 °C methanol a nd 70 and 110 °C methanol/water for forage and 22- DAT3 hay; and 40 °C methanol only for 7-DAT3 hay.
7 Twenty-three peaks in forage; 16 peaks in 7-DAT3 hay; and 60 peaks in 22-DAT3 hay.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

DP# 400085/MRID No. 48449801 Page 18 of 26

TABLE C.2.2.2. Distribution of the Parent and the Me tabolites in AAD-12 Soyb ean (event 416) Seed Following Application of [ 14 C]2,4-D DMA at 3.13-3.19 lb ae/A (3.512-3.578 kg ae/ha). 1

Metabolite Fraction 62-DAT3 Seed 41-DAT3 Seed TRR = 0.415 ppm TRR = 0.372 ppm %TRR ppm %TRR ppm %TRR ppm %TRR ppm ASE 40-110 °C Rep. A Rep B. Rep. A Hexane, 40 °C 12.7 0.053 12.7 0.053 9.1 0.034 Acetone, 40 °C 1.9 0.008 2.1 0.009 2.1 0.008 Methanol, 40 °C 7.4 0.031 7.7 0.032 8.5 0.031 Methanol/water, 70 °C 13.4 0.056 12.7 0.053 19.4 0.072 Methanol/water, 110 °C 6.0 0.025 6.6 0.028 4.7 0.018 Methanol/ACN/water, 110 °C 1.0 0.004 0.8 2 0.003 1.0 0.004
Nonextractable 70.5 0.293 67.4 0.280 65.5 0.244
ASE 40-70 °C Rep. B Rep. C Hexane, 1, 40 °C 8.9 0.033 9.0 0.033 Hexane, 2, 40 °C 1.1 0.004 1.2 0.005 Acid hydrolysis (2 N HCl) Subsample Hydrolysate 9.9 0.037 Base hydrolysis (2 N NaOH) Subsample Hydrolysate 9.8 0.037 Methanol, 1, 40 °C 11.6 0.043 11.0 0.041 Methanol, 2, 40 °C 5.4 0.020 -- 3 Combined methanol extracts, 40 °C Not reported 2,4-D 1.1 0.004 2,4-DCP 0.1 <0.001 Metabolite B (X11963417) 1.3 0.005
Metabolite C 1.9 0.007
Other 4 7.2 0.027 Largest other peak 3.5 0.013 Methanol/water, 1, (80:20, v:v), 70 °C 9.2 0.034 14.1 0.053 Methanol/water, 2, (80:20, v:v), 70 °C 4.7 0.018 5.3 0.020 Combined methanol/water extracts, 70 °C Not reported 2,4-D 0.3 0.001 Metabolite B (X11963417) 0.8 0.003
Metabolite C 1.5 0.006
Other 5 8.4 0.031 Largest other peak 5.1 0.109
Nonextractable 68.9 0.256 70.2 0.261 Acid hydrolysis (2 N HCl) Subsample Hydrolysate 33.6 0.125 Metabolite B (X11963417) 0.2 0.001
Other 6 27.6 0.103 Largest other peak 8.5 0.031 Precipitates 6.8 0.025
Nonextractable 23.2 0.086 Base hydrolysis (2 N NaOH) Subsample2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

DP# 400085/MRID No. 48449801 Page 19 of 26

TABLE C.2.2.2. Distribution of the Parent and the Me tabolites in AAD-12 Soyb ean (event 416) Seed Following Application of [ 14 C]2,4-D DMA at 3.13-3.19 lb ae/A (3.512-3.578 kg ae/ha). 1

Metabolite Fraction 62-DAT3 Seed 41-DAT3 Seed TRR = 0.415 ppm TRR = 0.372 ppm %TRR ppm %TRR ppm %TRR ppm %TRR ppm Hydrolysate 50.1 0.186
Metabolite A (DCP-acetyl-glucose conjugate) 0.2 0.001

Metabolite B (X11963417) 0.9 0.003
Metabolite C 1.2 0.005
Other 7 41.3 0.153 Largest other peak 6.6 0.024 Precipitates 13.4 0.050
Nonextractable 2.6 0.010

1 Shading indicates that the extraction step and/or characteri zation analysis was not conducted fo r the matrix in question. DA T = Days after treatment. Seed was harvested following the third ap plication (DAT3) at a 62-day PHI in a planting box and a 41-day PHI in pots. ASE = Accelerated solvent extraction. “Other” in cludes radioactive peaks that di d not co-chromatograph with any of the four metabolites or parent 2,4-D, plus any radioactivit y that was detected but was not quantified as a specific peak.
2 The solvent methanol/water (0.02 M EDTA, 1:1; v:v) was accidentally used instead of methanol/ACN/water (2% NH 4 OH of entire volume; 40:40:20, v: v:v) for this sample.
3 Second extract not done due to instrument error.
4 Twenty peaks.
5 Fifteen peaks.
6 Twenty-four peaks.
7 Forty-two peaks.

TABLE C.2.3.1. Summary of Characterization and Id entification of Radioactive Residues in AAD-12 Soybean (event 416) Forage and Hay Following Application of [ 14 C]2,4-D DMA at 2.14 lb ae/A (2.401 kg ae/ha) for forage and 3.19 lb ae/A (3.578 kg ae/ha) for hay. 1

Metabolite Fraction 7-DAT2 Fo rage 7-DAT3 Hay 22-DAT3 Hay TRR = 50.227 ppm TRR = 35.565 ppm TRR = 37.008 ppm %TRR ppm %TRR ppm %TRR ppm 2,4-D 85.8 43.117 64.3 22.885 59.4 21.978 2,4-DCP 7.1 3.558 3.6 1.295 1.4 0.518
Metabolite A 10.4 5.223 12.6 4.474 13.4 4.969 Metabolite B (X11963417) 0.8 0. 381 5.4 1.919 7.0 2.573
Metabolite C 0.1 0.055 1.1 0.395 2.7 1.000
Other 5.1 2.557 2.5 0.901 22.3 8.256
Total identified 104.2 52.334 87.1 30.968 83.9 31.038
Total characterized 5.1 2. 557 2.5 0.901 22.3 8.256
Total extractable 2 110.3 55.394 93.8 33.355 108.7 40.24 Unextractable (PES) 3 2.8 1.393 4.2 1.478 8.2 3.027 Accountability 4 113 97.9 117

1 Metabolite fractions were obtained from the summed AS E extracts (40-110 °C) as calculated by the petitioner.
2 Calculated by the study reviewer by summing ppm in ASE extracts (40-110 °C).
3 Residues remaining after exhaustive extractions.
4 Accountability = (Total ex tractable + Total unextractable)/(TRR from combus tion analysis; see TAB LE C.2.1) * 100. TRR were calculated by summing extractable and nonextractable radioactivity.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

DP# 400085/MRID No. 48449801 Page 20 of 26

TABLE C.2.3.2. Summary of Characterization and Id entification of Radioactive Residues in AAD-12 Soybean (event 416) Seed Following Application of [ 14 C]2,4-D DMA at 3.133 lb ae/A (3.512 kg ae/ha). 1

Metabolite Fraction 41-DAT3 Seed TRR = 0.372 ppm %TRR ppm 2,4-D 1.4 0.005 2,4-DCP 0.1 <0.001 Metabolite B (X11963417) 3.0 0.011
Metabolite C 4.6 0.018
Other 56.9 0.211 Hexane (triglycerides/f atty acids 10.0 0.037 Precipitates from base hydrolysis 13.4 0.050
Total identified 9.1 0.035
Total characterized 80.3 0.298
Total extractable 2 90.9 0.338 Unextractable (PES) 3 2.6 0.010 Accountability 4 93.5

1 Metabolite fractions were calculated by the study reviewer by summing results from ASE extraction (40-70 °C) for combined methanol 40 °C, combined methanol/water 70 °C, and base hydrolysis of nonextractable residues.
2 Calculated by the study reviewer by summing ppm in ASE extracts (40-70 °C) and ba se hydrolysis of nonextractable residues.
3 Residues remaining after exhaustive extractions.
4 Accountability = (Total ex tractable + Total unextractable)/(TRR from combus tion analysis; see TAB LE C.2.1) * 100. TRR were calculated by summing extractable and nonextractable radioactivity.



### C.3. Proposed Metabolic Profile

Based on the results of the metabolism study wi th AAD-12 soybean (Event 416), the petitioner proposed that the metabolism of 2,4-D DMA proceed s rapidly via dissociation to the acid. After cleavage of the acid side chai n, the resulting dichlorophenol ( 2,4-DCP) was rapidly conjugated with acetylated glucose and other glucose conjug ates. Low levels of free 2,4-DCP compared to conjugated 2,4-DCP indicated that the conjuga tion was rapid and a preferential route of metabolism. In seed, bound residues were produ ced that liberated la rge numbers of polar metabolites upon acid and base hydrolysis. The petitioner noted that since 2,4-D has been demonstrated to degrade to CO 2 

### in soil, it was likely that any 2,4-D applied to the soil was converted to CO 2 

### and became incorporated into natura l plant constituents. Hydrolysis experiments conducted with extracted soybean lipi ds and bound residues indi cated that this was a likely route of metabolism. Previous plan t metabolism studies conducted on AAD-1 corn also concluded that metabolism of 2,4-D DMA pro ceeded to 2,4-DCP, glucose conjugates of 2,4- DCP, and to incorporation into natural plant constituents. Overall, the 14 

### C-residues in the AAD12 soybeans were similar to those observed on transgenic corn and conventional crops.

The proposed metabolic pathway (copied withou t alteration from MRID 48449801) is presented in Figure C.3.1. We note that Metabolites B and C are incorrectly identified in the submitted pathway as noted below.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

DP# 400085/MRID No. 48449801 Page 21 of 26 

### FIGURE C.3.1. Proposed Metabolic Prof ile of 2,4-D DMA salt in AAD-12 Soybean.



# ↑ ↑ This compound is Metabolite B This compound is Metabolite C2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

DP# 400085/MRID No. 48449801 Page 22 of 26

TABLE C.3.1. Identification of Compounds from Metabolism Study
Common name/code Figure C.3.1 ID No. Chemical name Chemical structure

2,4-D 2,4-dichlorophenoxy acetic acid

2,4-DCP 2,4-dichlorophenol

Metabolite A; Acetyl-glucose-DCP conjugate; Peak D

Metabolite B (X11963417); Glucose conjugate of 2,4- DCP; Peak A 2,4-dichlorophenyl β -Dglucopyranoside

Metabolite C; 2,4-DCP disaccharide Peak E2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

DP# 400085/MRID No. 48449801 Page 23 of 26 

### D. CONCLUSION

The submitted AAD-12 soybean metabolism st udy is acceptable. Following a single preemergence soil application, followed by two folia r applications to soyb ean plants grown in a planting box, for a total app lication rate of 3.19 lb ae/A, TRR were 50.227 ppm in 7-DAT2 forage (two applications; 2.14 lb ae/A), 35.565 ppm in 7-DAT3 hay, 37.008 ppm in 22-DAT3 hay, and 0.415 ppm in 62-DAT3 seed. Following a single preemergenc e soil application, followed by two foliar applications to soybean plants grown in pots, for a total application rate of 3.13 lb ae/A, TRR was 0.372 ppm in 41-DAT3 seed.

The parent, 2,4-D, was the major residue identif ied in forage (85.8% TRR) and hay (59.4-64.3% TRR), while 2,4-DCP was present at 7.1% TRR inforage and 1.4-3.6% TRR in hay. Metabolite A accounted for 10.4% TRR in forage and 12.6-13.4% TRR in hay. Metabolites B and C were present at 0.8% and 0.1% TRR (0.381 and 0.055 ppm) , respectively, in forage and 5.4-7.0% and 1.1-2.7% TRR (1.919-2.573 and 0.395-1.000 ppm), respectively, in hay. Remaining radioactivity, designated “other” accounted for 5.1% TRR in forage and 2.5% and 22.3% TRR, respectively in 7- and 22-DAT3 hay.

In 41-DAT3 seed, following ASE with methanol a nd methanol/water, and base hydrolysis of the nonextractable residues, 2,4-D accounted for 1.4% TRR, free 2,4-DCP comprised only 0.1% of the TRR, and the 2,4-DCP conjugates Metabolite s B and C accounted for 3.0% and 4.6% TRR, respectively. TLC of the hexane extracts (10% TRR) indicated that >90% of the radioactivity was distributed in natural const ituents. These components were a ttributed to triglycerides and unsaturated fatty acids. “Other” radioactiv ity accounted for 56.9% TRR (0.211 ppm) and was found to be multicomponent and polar. Pr ecipitates following base hydrolysis of the nonextractable residues accounted for 13.4% TRR.

Based on the results of the metabolism study wi th AAD-12 soybean (Event 416), the petitioner proposed that the metabolism of 2,4-D DMA proceed s rapidly via dissociation to the acid. After cleavage of the acid side chai n, the resulting dichlorophenol ( 2,4-DCP) was rapidly conjugated with acetylated glucose and other glucose conjug ates. Low levels of free 2,4-DCP compared to conjugated 2,4-DCP indicated that the conjuga tion was rapid and a pr eferential route of metabolism. In seed, bound residues were produ ced that liberated la rge numbers of polar metabolites upon acid and base hydrolysis. The petitioner noted that since 2,4-D has been demonstrated to degrade to CO 2 

### in soil, it was likely that any 2,4-D applied to the soil was converted to CO 2 

### and became incorporated into natura l plant constituents. Hydrolysis experiments conducted with extracted soybean lipi ds and bound residues indi cated that this was a likely route of metabolism. Previous plan t metabolism studies conducted on AAD-1 corn also concluded that metabolism of 2,4-D DMA pro ceeded to 2,4-DCP, glucose conjugates of 2,4- DCP, and to incorporation into natural plant constituents. Overall, the 14 

### C-residues in the AAD12 soybeans were similar to those observed on transgenic corn and conventional crops.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

DP# 400085/MRID No. 48449801 Page 24 of 26 

### E. REFERENCES

DP#: 309452 Subject: 2,4-D: Health Effects Division (HED) Metabolism Assessment Review Committee (MARC) Decision Document - Revised. Chemical I.D. No. 030001. Case No. 0073. Meeting date: 9/3/03 From: T. Jimerson To: Y. Donovan Dated: 10/14/04 MRID(s): None

DP#: D384441 Subject: 2,4-D. Summary of Analytical Ch emistry and Residue Data for Use of 2,4-D Choline in/on Herbic ide Tolerant Field Corn C ontaining the Aryloxyalkanoate Dioxygenase-1 (ADD-1) Gene. From: A. LaMay To: M. Walsh Dated: 10/27/11 MRID(s): 48260701, 48260702, 48260703, 48260704

F. DOCUMENT TRACKING

DP Barcode(s): D400085 PC Code: 030001

Template Version June 2005.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

DP# 400085/MRID No. 48449801 Page 25 of 26 

### APPENDIX I. Chemical Names and Structu res of Reference Standards Used in the Soybean (AAD-12; Event 416) Metabolism Study.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 6.3/OPPTS 860.1300/OECD II 6.2.2, 6.2.3 & IIIA 8.2, 8.4.1, 8.4.2 Nature of the Residues in Plants - 2,4-D-Tolerant Soybean (AAD-12, Event pDAB4468-416)

DP# 400085/MRID No. 48449801 Page 26 of 26 

### APPENDIX I. Chemical Names and Structu res of Reference Standards Used in the Soybean (AAD-12; Event 416) Metabolism Study (continued).2,4

- D/PC Code 030001/Dow AgroScie n ces LLC DACO 7 .4 .5 /0PPTS 860

. 1520/0ECD IIA 6

. 5.4 and IliA 8.5 Pro cess ed Food and Feed

- 2,4

- D

- To 1 erant Soybean (AA D-1 2, Event 68416

- 4)



### Reviewed and Approved by

# ~**-- -=** **==~~ ~~2,:::._:::::=~.,.~~-~,..** **-----** Date: 3/4/12 Danette Drew, Chemist, HED RABV

Note : This DER was origina ll y p r epare d u nder contract by Ve r sar, Inc. (6850 Ve r sa r Center, Spr in gfie l d, VA 22151; sub mi tted l/24/12). T h e DER has been reviewed by t he Health E f fects D i vision (HED) an d re vised to reflect cur r ent Office of Pest i cide Programs (OPP) policies

.



### STUDY REPORTS:



### 48449802 Culligan, J. 

### (2011) Magnitude ofthe Re s idue of2 ,4

- D in/on Herbicide Tolerant Soybeans Containing the Aryloxyalkanoate Dioxygenase

- 12 (AAD

- 12) Gene

. Project Study Number: ARA-09-15

- 11; Dow AgroSciences Study No

. 090053. Unpublished study prepared by Ag Research Associates, LLC. 33 7 p.

EXECUTIVE SUMMARY:



### Dow AgroSciences L LC has submitted a processing study for 2,4

- D on soybean containing the inserted aryloxyalk anoa te dioxygenase

- 12 (AAD

- 12) gene (seed line Event DAS

- 68416

- 4). Expression of the AAD

- 12 protein encoded by the AAD

- 12 gene results in a trait that increases the herbicide tol e rance of soybeans to 2,4

- D via increa s ed metaboli s m through a pathway involving the metabolite 2,4

- dichlorophenol (2,4

- DCP). Two trial s were conducted in GA and NE during the 2009 growing season

.

In both trials, soybean seed was harvested 66 days (GA

- Ol) or 90 days (NE

- 16) following the last ofthree broadcast applications of a 3.79lb ae/gal solub l e concentrate liquid (SLIL) fo r mulation of2,4

- D dimethylamine s alt (2,4

- D DMA; Weedar® 64 Herbicide) at 1.93

- 2.02 lb ae/A/application, for a total seasonal application rate of 5.9 7- 6

. 00 lb ae/A. 2

, 4

- D was applied as

one preemergence application and two foliar applications

. Retreatment inter va ls (RTis) were 4

- 3 3 days between the first and second applications and 13 days between the second and third applications

. Applications were made using ground equipment in spra y v olumes of21.9-32.36 gall A and included a non-ionic surfactant (NIS) at all trial s i tes

. The petitioner noted that the application rate used in the study was only 2x that used in the corresponding crop field trials (refer to the 860

. 1500 DER for MRID 48449802) due to concerns over the potential for crop InJury

.

S am ples of soybean seed were processed into aspirated grain fractions ( AGF )

, meal

, hulls

, and alkali

- refined oil by GLP T echnologies (Navasota

, TX) using procedures simulating commercial
processmg.
Samples of soybean seed and processed commodities were analyzed for residues o f 2,4

- D and its metabolite 2,4-DCP using a high performance liquid chromatography method with tandem m a ss sp e ctrometry detection (LC/MS/MS). The limit o f quantitation ( L OQ, e qui va l ent to the lowest leve l of method validation; LLMV ) was 0

. 01 ppm each for 2,4

- D and 2,4-DCP in all matrices

. The method was adequate for data validation b a sed on acceptable concurrent recovery data. The

DP Barcode 400085/MRID No

. 4 844980 2 .de 2 Pa ge 1 of 132,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.5/OPPTS 860.1520/OECD IIA 6.5.4 and IIIA 8.5 Processed Food and Feed - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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### fortification levels used in concurrent method recovery were adequate to bracket expected residue levels. Concurrent recove ries were corrected for apparent residues in controls; residues in treated samples were not corr ected for residues in controls.

Samples were stored frozen ( ≤ -20 °C) from harvest/processing to extraction for 2,4-D and 2,4- DCP for 357-376 days (11.7-12.4 months) for soyb ean seed (RAC) and 276-312 days (9.1-10.3 months) for the processed commodities. Adequate storage stability data are available for 2,4-D indicating that residues are stable under frozen storage conditions for at least 12 months in/on soybean seed and in the processed field crop commodities of corn starch, flour, and oil; wheat flour; sugarcane, sugar, molasses, and bagasse; a nd rice bran and hulls. In terim storage stability data are available (DP #394948) indi cating residues of 2,4-DCP are st able for up to 8 months in soybean commodities. The available storage st ability data for 2,4-D are adequate to support sample storage conditions and durations; however , confirmatory storage stability data are required for 2,4-DCP demonstrating the stab ility of 2,4-DCP in/on soybean seed and representative processed commodities stored frozen in High-Density Polyethylene (HDPE) containers for up to ~12 months for seed and 10 months for processed commodities.

Residues of 2,4-D were below the LOQ (<0.01 ppm) in/on all samples of soybean seed RAC, meal, hulls, and alkali-refined oil; therefore, no processing factors were calculated. Residues of 2,4-D were 0.012-0.016 ppm in AGF. The processi ng data indicate that residues of 2,4-D concentrate in AGF (average processing factor of >1.4x). Average residues of 2,4-DCP were 0.012-0.109 ppm in/on seed prior to AGF ge neration (RAC), 0.074-0.831 ppm in AGF, 0.015- 0.155 ppm in meal, <0.01-0.050 in hulls, and below the LOQ (<0.01 ppm) in alkali-refined oil. The processing data indicate th at residues of 2,4-DCP concentrat e in AGF (average processing factor of 6.9x) and meal (average processing fa ctor of 1.3x) but do not concentrate in alkalirefined oil or hulls (average processing factors of <0.5x and <0.7x, respectively).

The processing factors calculated in this st udy were less than the maximum theoretical concentration factors of 11.3x for soybean hulls, 2.2x for meal, and 12.0x for oil (based separation of components; OPPTS 860.1520, Table 3).

STUDY/WAIVER ACCEPTABILITY/ DEFICIENCIES/CLARIFICATIONS :
Under the conditions and parameters used in the study, the field tria l residue data are tentatively classified as scientifically acceptable pending subm ission of confirmatory storage stability data demonstrating the stability of 2,4-DCP in/on s oybean seed and processed commodities stored frozen for up to ~12 months for seed and 10 months for processed commodities. The acceptability of this study for regulatory purposes is addressed in the forthcoming U.S. EPA Residue Chemistry Summ ary Document, DP# 400085.

COMPLIANCE :2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.5/OPPTS 860.1520/OECD IIA 6.5.4 and IIIA 8.5 Processed Food and Feed - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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### Signed and dated Good Laboratory Practice (GLP), Quality Assurance, a nd Data Confidentiality statements were provided. No deviations fr om regulatory requirement s were reported which would have an impact on the validity of the study.

A. BACKGROUND INFORMATION

2,4-Dichlorophenoxyacetic acid (2,4-D) is an alkyl chlorophenoxy herbicide used to control a variety of broadleaf weeds. These data we re submitted to support the proposed use for 2,4-D choline application to soybean s containing the inserted aryl oxyalkanoate dioxygenase-12 (AAD12) gene. This soybean is identified as c ontaining DAS-68416-4. Expression of the AAD-12 protein encoded by the AAD-12 gene results in a trait that increas es the herbicide tolerance of soybean to 2,4-D via increased metabolism through a pathway involving the metabolite 2,4- dichlorophenol (2,4-DCP).

The chemical structure and nomenclature of 2,4-D, 2,4-D DMA, and 2,4-D choline are summarized in Table A.1, and the physicochemical properties are summarized in Table A.2.

TABLE A.1. Test Compound Nomenclature. 2,4-D

Common name 2,4-D acid IUPAC name (2,4-dichlorophenoxy)acetic acid CAS name 2-(2,4-dichlorophenoxy)acetic acid
CAS # 94-75-7
PC Code 030001 End-use product/(EP) Weedar® 64 2,4-D dimethylamine salt (DMA)

IUPAC name (2,4-dichlorophenoxy)ace tic acid - dimethylamine (1:1) CAS name 2-(2,4-dichlorophenoxy)acetic ac id compound with N-methylethanamine (1:1)
CAS # 2008-39-1
PC Code 030019 End-use product/(EP) 3.79 lb ae /gal SL/L formulation (Weedar ® 64 Herbicide)2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.5/OPPTS 860.1520/OECD IIA 6.5.4 and IIIA 8.5 Processed Food and Feed - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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TABLE A.1. Test Compound Nomenclature. 2,4-D choline

IUPAC name 2-hydroxy-N,N,N-trimethyleth anaminium (2,4-dichlorophenoxy)acetate CAS name Ethanaminium, 2-hydroxy-N,N,N-trim ethyl-, 2-(2,4-dichlorophenoxy)acetic acid hydroxide (1:1:1)
CAS # 1048373-72-3
PC Code 051505 2,4-DCP

2,4-dichlorophenol

TABLE A.2. Physicochemical Properties of the T echnical Grade Test Co mpound: 2,4-D acid.
Parameter Valu e Reference Melting point/range 138-141 °C DP#309452, T. Jimerson, 10/13/04 pH Not available Density Specific gravity = 1.416 at 25 °C Water solubility ( __°C) 569 mg/L at 20 °C Solvent solubility (mg/L at __°C) g/100 g at 25 °C acetone 85.0 benzene 1.07 diethyl ether 220 ethanol 130.0 isopropanol 31.6 toluene 0.067 xylene 0.58 Vapor pressure at __°C 1.4 x 10 -7 mm Hg at 25 °C Dissociation constant (pK a ) Not available Octanol/water partition coefficient Log(K OW ) In 0.001 M solution: 2.14 at pH 5 0.177 at pH 7 0.102 at pH 9 UV/visible absorption spectrum Not available2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.5/OPPTS 860.1520/OECD IIA 6.5.4 and IIIA 8.5 Processed Food and Feed - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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### B. EXPERIMENTAL DESIGN

B.1. Application and Crop Information

Two trials were conducted on soybean contai ning the AAD-12 gene (Event DAS-68416-4) in GA and NE during the 2009 growing season. Due to limited availability of seed with Event DAS-68416-4, the untreated contro l plots were planted with the same variety of soybeans (Maverick) but the seed did not contain the inserted AAD-12 gene.

In both trials, soybean seed was harvested 66 days (GA-01) or 90 days (NE-16) following the last of three broadcas t applications of a 3.79 lb ae/gal SL/L formulation of 2,4-D DMA (Weedar ® 

### 64 Herbicide) at 1.93-2.02 lb ae/A/application, for a total seasonal application rate of 5.97-6.00 lb ae/A. 2,4-D was applied as one preemergence application and two foliar applications. RTIs were 4-33 days between th e first and second applications and 13 days between the second and third applications. Appl ications were made using ground equipment in spray volumes of 21.9-32.36 gal/A and included an NI S at all trial sites. The petitioner noted that the application rate used in the study was only 2x that used in the corresponding crop field trials (refer to the 860.1500 DER1 for MRID 48449802) due to concer ns over the potential for crop injury.

TABLE B.1.2. Study Use Pattern.
Location City, State; Year (Trial ID No.) EP 1 Application Tank Mix/ Adjuvants 3 Method/Timing Volume (gal/A) Rate (lb ae./A) RTI 2 (days) Total Rate (lb a.i./A) Sycamore, GA; 2009 (GA-01) 3.79 lb ae/gal SL 1. Broadcast/Pre-emergence 23.5 2.00 -- 6.00 NIS
2. Foliar broadcast/ R1 22.1 2.00 4
3. Foliar broadcast/ R2 21.9 2.00 13York, NE; 2009 (NE16) 3.79 lb ae/gal SL 1. Broadcast/ BBCH 00 25.19 1.93 -- 5.97 NIS
2. Foliar broadcast/ V6 32.18 2.0196 33
3. Foliar broadcast/ R2 32.36 2.0243 13

1 EP = End-use Product; Weedar 64, a 3.79 lb ae/gal SL/L formulation of 2,4-D DMA.
2 Retreatment Interval
3 NIS = Non-ionic surfactant; added to all spray mixtures at 0.25% v/v.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.5/OPPTS 860.1520/OECD IIA 6.5.4 and IIIA 8.5 Processed Food and Feed - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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### B.2. Sample Handling and Processing Procedures

One untreated and one treated bulk soybean seed sample (~1400-1450 lb each) were harvested 66 days (GA-01) or 90 days (NE-16) after the last application. Samples were placed in frozen storage at the GA trial within 1.3 hou rs of collection; samples from the NE trial were first placed in cold storage and then moved to a freezer within 3 days of collection. The samples were stored frozen for 3-11 days after collection then shi pped frozen via freezer truck to the processing facility, GLP Technologies (Navasota, TX). S oybean seed samples were stored frozen (~<-12 °C) at GLP Technologies until processing and were processed within 65-102 days of harvest. Samples from both trials were processed into AGF, meal, hulls, and alkali-refined oil using procedures simulating commercial processing. Samp les were stored frozen after processing and were shipped frozen overnight with dry ice to Dow AgroScien ces, LLC (Indianapolis, IN) for residue analysis. Samples were maintained frozen ( ≤ -20 °C) at the analytic al laboratory. In preparation for analysis, samples (except oi l) were ground in the presence of dry ice.

The petitioner submitted adequate descriptions of the processing procedures including material balance summaries. Soybean processing procedur es are summarized below in Figures 1 (AGF) and 2 (meal, hulls, and alkali-refined oil), whic h were copied without alteration from MRID 48449802.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.5/OPPTS 860.1520/OECD IIA 6.5.4 and IIIA 8.5 Processed Food and Feed - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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### FIGURE 1. Processing Fl owchart for Soybean AGF.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.5/OPPTS 860.1520/OECD IIA 6.5.4 and IIIA 8.5 Processed Food and Feed - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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### FIGURE 2. Processing Flowchart for Soybean Alkali-Refined Oil, Meal and Hulls.

B.3. Analytical Methodology

Samples of soybean and processed commodities we re analyzed for residues of 2,4-D and its metabolite 2,4-DCP by LC/MS/MS. A complete de scription of the method was included in the submission.

Briefly, samples were extracted with metha nol:1.0 N NaOH (90:10, v:v) and centrifuged. An aliquot of the extract was comb ined with a mixed stable isot ope internal standard of 2,4-D( 13 

### C 6 

### ) or 2,4-DCP( 13 

### C 6 

### ) and evaporated to near dryness. Th e sample was mixed with 2 N HCl (at 90 °C for 1 hour). After cooling, the extract was diluted with me thanol, filtered, and analyzed by2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.5/OPPTS 860.1520/OECD IIA 6.5.4 and IIIA 8.5 Processed Food and Feed - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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### on-line solid-phase extraction (SPE ; residues eluted with water:me thanol containing 0.1% formic acid (70:30, v:v)) and LC/MS/MS in the negative ion mode.

The reported LOQ (equivalent to the LLMV) was 0.01 ppm, and the LOD was 0.003 ppm for 2,4-D and 2,4-DCP in all matrices. The method was validated concurrently with analysis of the processing study samples.

C. RESULTS AND DISCUSSION

The storage conditions and dur ations for samples from the soybean processing study are presented in Table C.2. Samp les were stored frozen ( ≤ -20 °C) from harvest/processing to extraction for 2,4-D and 2,4-DCP for 357-376 days (11.7-12.4 months) for soybean seed (RAC) and 276-312 days (9.1-10.3 months) for the proce ssed commodities. Stability of 2,4-D has been demonstrated in a variety of field crop commodities for up to 12 months, including soybean seed and the processed commodities of corn starch, flour, and oil; wheat fl our; sugarcane, sugar, molasses, and bagasse; and rice br an and hulls (Residue Chemistr y Chapter of the 2,4-D RED; 10/12/04). Interim storage stab ility data are available (DP #394948) indicating residues of 2,4- DCP are stable for up to 8 months in soybean co mmodities. The available storage stability data for 2,4-D are adequate to support sample storage conditions and durations; confirmatory storage stability data are required for 2,4-DCP reflecting the stability of 2,4-DCP in/on soybean seed and representative processed commodities stored frozen in HDPE containers for up to ~12 months for seed and 10 months for processed commodities, as the samples from this study were stored in HDPE containers.

Concurrent method recovery data from the soybean processing study are pr esented in Table C.1. The LC/MS/MS method used to analyze soybean processing samples for residues of 2,4-D and 2,4-DCP was adequate for data collection based on acceptable concurrent method recovery data. Concurrent method recoveries were within th e acceptable range of 70-120% for soybean and processed commodities fortifie d with 2,4-D and 2,4-DCP each at 0.01 and 1 ppm for seed, AGF, hulls, and meal and at 0.01 and 0.1 ppm for refined o il. The fortification le vels were adequate to bracket residues found in treated samples. Appa rent residues of 2,4-D were observed in one sample of untreated AGF at 0.013 ppm; appa rent residues of 2,4-D and 2,4-DCP in/on all remaining untreated samples of soybean seed, AG F, meal, hulls, and refined oil. Concurrent recoveries were corrected for a pparent residues in controls; resi dues in treated samples were not corrected for residue s in controls.

Residue data for the soybean processing study ar e presented in Table C.3. Residues of 2,4-D were below the LOQ (<0.01 ppm) in/on all samples of soybean seed RAC, meal, hulls, and alkali-refined oil; therefore, no processing factors were calculated. Residues of 2,4-D were 0.012-0.016 ppm in AGF. The processing data indicat e that residues of 2,4-D concentrate in AGF (average processing factor of >1.4x). Average residues of 2,4-DCP were 0.012-0.109 ppm in/on seed prior to AGF generation (RAC), 0.074-0.831 ppm in AGF, 0.015-0.155 ppm in meal, <0.01-0.050 in hulls, and below the LOQ (<0.01 ppm) in alkali-refined oil. The processing data indicate that residues of 2,4-DC P concentrate in AGF (average processing factor of 6.9x) and2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.5/OPPTS 860.1520/OECD IIA 6.5.4 and IIIA 8.5 Processed Food and Feed - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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### meal (average processing factor of 1.3x) but do not concentrate in alkali-refined oil or hulls (average processing factors of <0.5x and <0.7x, respectively).

The processing factors calculated in this st udy were less than the maximum theoretical concentration factors of 11.3x for hulls, 2.2x for meal, and 12.0x for oil (based separation of components; OPPTS 860.1520, Table 3).

TABLE C.1. Summary of Concurrent Recoveries of 2,4-D from Soybean Processed Commodities. Matrix Spike level (ppm) Sample size (n) Recoveries (%) 1 Mean ± std dev 2 (%) 2,4-D Soybean seed (RAC) 0.01 8 79, 81, 83, 83, 85, 87, 91, 102 86 ± 7 1 8 79, 81, 86, 88, 94, 96, 96, 97 90 ± 7 Aspirated grain fractions 0.01 2 96, 102 99 1 2 93, 95 94 Meal 0.01 2 89, 106 98 1 2 95, 97 96 Hulls 0.01 2 89, 101 95 1 2 96, 98 97 Refined oil 0.01 2 91, 95 93 0.1 2 96, 99 98 2,4-DCP Soybean seed (RAC) 0.01 8 79, 90, 91, 93, 94, 98, 105, 109 95 ± 9 1 8 81, 82, 86, 92, 94, 97, 97, 103 92 ± 8 Aspirated grain fractions 0.01 2 70, 73 72 1 2 100, 104 102 Meal 0.01 2 98, 105 102 1 2 99, 107 103 Hulls 0.01 2 85, 94 90 1 2 91, 100 96 Refined oil 0.01 2 89, 92 91 0.1 2 89, 95 92

1 Recoveries were corrected for detectable residues in the untreated samples, if present.
2 Standard deviation is not applicable for sample sizes n < 3 samples.2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.5/OPPTS 860.1520/OECD IIA 6.5.4 and IIIA 8.5 Processed Food and Feed - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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TABLE C.2. Summary of Storage Condition s for Soybean Processed Commodities

. Matrix Storage Temperature (°C) Actual Storage Duration 1 Interval of Demonstrated Storage Stability

Soybean seed (RAC) Frozen at field sites

< -12 at processing facility

Approximately -20

at analytical lab 357-376 days (11.7-12.4 months) Adequa te storage stability data are available for 2,4-D indicating that residues are stable under frozen storage conditions for at least 12 months in/on soybean seed; corn starch, flour, and oil; wheat flour; sugarcane, sugar, molasses, and bagasse; and rice bran and hulls. 2 Interim storage stability data are available demonstrating residues of 2,4-DCP are stable in soybean commodities for up to 8 months 3

. Aspirated grain fractions 276 days (9.1 months) Hulls 287-312 days (9.4-10.3 months) Meal 282-304 days (9.3-10.0 months) Refined oil 280-302 days (9.2-9.9 months)

1 Duration from harvest to extraction; interval between extract ion and analysis was not provide d. Samples were processed within 65-102 days of harvest.
2 Residue Chemistry Chapter of the 2,4-D RED; 10/12/04.
3 DP #394948 of interim storage stability data review.

TABLE C.3. Residue Data from Soybean Processing Study with 2,4-D. RAC Processed Commodity Total Rate (lb ae/A) PHI (days) Residues (ppm) 1 [Average] Processing Factor 2
2,4-D 2,4-DCP 2,4-D 2,4-DCP

Sycamore, GA; 2009 (GA-01) Soybean RAC 3 : Seed (prior to AGF generation) 6.00 66 ND, ND, ND [<0.01] 0.121, 0.107, 0.099 [0.109] -- --

Seed (after AGF generation) ND, ND, ND [<0.01] 0.088, 0.101, 0.082 [0.090] -- --

AGF 0.016 0.831 >1.6x 7.6x Meal ND, ND [<0.01] 0.158, 0.152 [0.155] NC 4 1.4x

Hulls ND, ND [<0.01] 0.053, 0.046 [0.050] NC 0.5x

Alkali-refined oil ND, ND [<0.01] ND, ND [<0.01] NC <0.09x

York, NE; 2009 (NE-16) Soybean RAC 3 : Seed (prior to AGF generation) 5.97 90 ND, ND, ND [<0.01] 0.011, 0.013, 0.011 [0.012] -- --

Seed (after AGF generation) ND, ND, ND [<0.01] 0.011, (0.009), 0.012 [<0.011] -- --

AGF 0.012 0.074 >1.2x 6.2x Meal ND, ND [<0.01] 0.015, 0.015 [0.015] NC 1.3x

Hulls ND, ND [<0.01] (0.004), ND [<0.01] NC <0.8x

Alkali-refined oil ND, ND [<0.01] ND, ND [<0.01] NC <0.8x2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.5/OPPTS 860.1520/OECD IIA 6.5.4 and IIIA 8.5 Processed Food and Feed - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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TABLE C.3. Residue Data from Soybean Processing Study with 2,4-D. RAC Processed Commodity Total Rate (lb ae/A) PHI (days) Residues (ppm) 1 [Average] Processing Factor 2
2,4-D 2,4-DCP 2,4-D 2,4-DCP

Average Soybean RAC 3 : Seed (prior to AGF generation) 5.97-6.00 66-90 -- --

AGF >1.4x 6.9x
Meal NC 1.3x
Hulls NC <0.7x
Alkali-refined oil NC <0.5x

1 ND = Nondetectable (<LOD). The LOD was 0.003 ppm for all analytes in all ma trices; the LOQ was 0.01 ppm for all analytes. Residues between the LOD and LOQ are reported in parentheses. When calculating per trial averages, the LOQ was used for values reported as ND or between the LOD and LOQ.
2 Processing Factor = [Measured residue for analyte in the pro cessed fraction] / [Measured residue for analyte in the RAC].
3 The residues for RAC: Seed (prior to AGF generati on) were used for calculating processing factors.
4 NC = Not calculated. The processing fa ctor could not be calculated because resi dues were below the LOQ in the RAC and the processed fraction.



### D. CONCLUSION

Pending submission of confirmatory storage stab ility data for 2,4-DCP, the submitted soybean processing study is acceptable. Processing factor s for 2,4-D could not be calculated for alkalirefined oil, meal, and hulls because residues we re below the LOQ in/on all samples of soybean seed and processed commodities. The data indi cated that residues of 2,4-D may concentrate in AGF (average processing factor of >1.4x). Re sidues of 2,4-DCP may co ncentrate in AGF and meal (average processing factors of 6.9x and 1.3x, respectively) but do not concentrate in alkalirefined oil and hulls (processing factors of <1x). The observed concentration factors were less than the theoretical concentration factors.

An acceptable method was used for the determination of residues of 2,4-D and 2,4-DCP in soybean seed and processed commodities. Confir matory storage stability data are required demonstrating the stability of 2,4-DCP in/on soybean seed and representative processed commodities stored frozen in HDPE containers fo r up to ~12 months for seed and 10 months for processed commodities.

E. REFERENCES

DP#: 309452 Subject: 2,4-D: Health Effects Division (HED) Metabolism Assessment Review Committee (MARC) Decision Document - Revised. Chemical I.D. No. 030001. Case No. 0073. Meeting date: 9/3/03 From: T. Jimerson To: Y. Donovan Dated: 10/14/04 MRID(s): None2,4-D/PC Code 030001/Dow AgroSciences LLC DACO 7.4.5/OPPTS 860.1520/OECD IIA 6.5.4 and IIIA 8.5 Processed Food and Feed - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4)

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### F. DOCUMENT TRACKING

DP Barcode(s): D400085 PC Code: 030001

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