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 

Petition No.: NA Regulatory Action: Section 3 

Risk Assessment Type: NA 

TXRNo.:NA 

Case No.: NA 

CAS No.: 1048373-72-3, 94-75-7 

MRID No.: (See Table Below) 40 CFR: 40 CFR §180.142 

FROM: 

THROUGH: 

TO: 

Danette Drew, Chemist~ Risk Assessment Branch V 

Health Effects Division (7509P) 

/fk; 

Ideliz Negron-Encarnacion, Chem'f.·sr j ~//) J ~ichael Metzger, Branch Chief h td'-M.. _ . ·' 

Rtsk Assessment Branch V Nil " 

Health Effects 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; 48449801 .der.doc 

Soybean 

48449802 860.1500 Field Trial! Residue Decline- Soybean New DER: 48449802.derl.doc 

860.1520 Processed Food and Feed - Soybean New DER; 48449802.der2.doc 

491 22501 860.13 80 Storage Stability- Soybean New DER; 491 22501 

Page 1 of23 
2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085 

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Executive Summary

2,4-Dichlorophenoxyacetic acid (2,4-D) is an alkylchlorophenoxy herbicide used to control a 

variety of broadleaf weeds. The proposed use 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 herbicide 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 existing 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 petition 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-emergence (when soybean is no later than the R2, 

or full flowering, growth stage) application to soybean at a maximum single application 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 between applications. The maximum 

seasonal 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 postemergence applications per use season. Aerial application and chemigation 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 elucidate 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 identified 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 rapidly via dissociation to the acid. After 

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 that conjugation 

is rapid and is a preferential route of metabolism. Metabolism proceeds through incorporation of 

the radiolabeled carbon into natural plant constituents. Previous 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 incorporation into natural plant constituents. 

Overall, the 14C-residues in the AAD-12 soybeans were similar 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 species. Based on the 

available data, on 9/3/03, the Agency determined that 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 tolerances 
2,4-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 harvesting (forage and hay) restrictions are 

maintained on the label. 

Adequate methods are available for data collection. In addition, the 10/97 edition of FDA PAM 

Volume I, Appendix I indicates that 2,4-D is partially recovered (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) from harvest to extraction for 2,4-D 

and 2,4-DCP for up 15.2 months for forage, 15.1 months 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 stability 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 months 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 support the storage durations 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 postemergence 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 dissociate into 2,4-D 

parent and the cation. Based on the study results, residues (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-harvest 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 label should be submitted which specifies a 

minimum PHI of 50 days for soybean. The proposed 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, and alkali-refined oil; therefore, no processing 

factors were calculated for these commodities. The 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 (average 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 hulls 
2,4-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 studies 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 submitted 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. tolerance for soybean seed (0.02 ppm) 

should be decreased to 0.01 ppm. This revised tolerance level will also support the proposed 2,4-

D choline use on soybean. The established tolerance for AGF will cover residues of 2,4-D in/on 

soybean AGF. 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 at 

this time. 

Recommended Tolerances 

Table 1. Tolerance 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 revises the proposed label for GF-2654-TS (56.3% 2,4-D choline; Reg. 

no. 62719-AUU) end use products as described below, and pending submission of adequate 

storage stability data described below, there are no residue chemistry deficiencies that would 

preclude the requested registration for the use of 2,4-D choline on AAD-12 soybean. HED has 

determined that the residue of concern for tolerance expression is 2,4-D. The existing tolerances 

for 2,4-D are adequate to support the proposed new use. However, HED recommends that the 

existing tolerance for soybean seed be decreased to 0.01 ppm to allow harmonization with 

Codex. The tolerance of 0.01 ppm on soybean seed will also support the proposed 2,4-D choline 

use on soybeans. Tolerances for 2,4-D residues are listed in 40 CFR §180.142. 

A human health risk assessment is forthcoming. 

Residue Chemistry Deficiencies 

The following label modifications are needed prior 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 specifies a minimum preharvest interval of 50 

days for soybean. The proposed label currently specifies a 30 day PHI which does not 

reflect the minimum PHI (51 days) used for soybean seed in the crop field trials. 
2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085 

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Background

2,4-Dichlorophenoxyacetic acid (2,4-D) is an alkylchlorophenoxy herbicide used to control a 

variety of broadleaf weeds. The proposed use 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 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, 2,4-D choline and 2,4-DCP are 

presented in Table 2. Physicochemical properties of 2,4-D are summarized in Table 2. The 

dissociation constant for 2,4-D choline is listed in Table 3. 

2,4-D choline is expected to readily dissociate into parent 2,4-D and choline cation when mixed 

with water. There is no toxicological concern for choline. 2,4-D is the dissociation product of 

concern for the formulation associated 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)acetic acid - dimethylamine (1:1) 

CAS name 2-(2,4-dichlorophenoxy)acetic acid 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-trimethylethanaminium (2,4-dichlorophenoxy)acetate 
2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085 

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Table 2. Test Compound Nomenclature.

CAS name Ethanaminium, 2-hydroxy-N,N,N-trimethyl-, 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 Technical Grade Test Compound: 2,4-D acid. 

Parameter Value 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 (pKa) Not available 

Octanol/water partition coefficient Log(KOW) 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 Value 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 petition 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) application to soybean at a maximum single application 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 between applications. The maximum seasonal 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 prohibited. The proposed preharvest 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 

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Table 5. Summary of Directions for Use 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 applications with a 

minimum of 12 days between applications. 

HED Conclusion: The use directions provided in the petition 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. The proposed label currently 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 available reflecting the application of [phenyl-U-14C]-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 concentrate liquid (SL/L) and 

applied to 2,4-D-tolerant soybeans 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) followed 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 applications, 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 days 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 chromatography (HPLC); seed extracts were purified 
2,4-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) for residue characterization/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 (disaccharide 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). Structures of the metabolites may be found in 

Attachment 1 to this memorandum. 

The parent, 2,4-D, was the major residue identified 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 forage and 2.5% and 22.3% TRR, respectively in 

7- and 22-DAT3 hay. 

In 41-DAT3 seed, following accelerated solvent 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, respectively. 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 found to be multicomponent and polar. 

Precipitates following base hydrolysis of the nonextractable residues accounted for 13.4% TRR. 

Based on the results of the metabolism study with AAD-12 soybean (Event 416), the petitioner 

proposed that the metabolism of 2,4-D DMA proceeds rapidly via dissociation to the acid: After 

cleavage of the acid side chain, the resulting dichlorophenol (2,4-DCP) was rapidly conjugated 

with acetylated glucose 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 were 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 CO2 in soil, it was likely that any 2,4-D applied to the soil was 

converted to CO2 and became incorporated into natural plant constituents. Hydrolysis 

experiments conducted with extracted soybean lipids and bound residues indicated 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 [14C]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 Forage 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 extractable2 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 

Accountability4 113 97.9 117 1

 Metabolite fractions were obtained from the summed ASE 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 extractable + Total unextractable)/(TRR from combustion analysis; see TABLE 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 [14C]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/fatty 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 extractable2 90.9 0.338 

Unextractable (PES)3 2.6 0.010 

Accountability4 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 base hydrolysis of nonextractable residues. 3

 Residues remaining after exhaustive extractions. 

4

 Accountability = (Total extractable + Total unextractable)/(TRR from combustion analysis; see TABLE 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 conjugate of 2,4-DCP). In soybean seed the 

primary identified residues were 2,4-D, Metabolite 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 pathway of metabolism of 2,4-D in AAD-12 soybean 

proceeds rapidly via dissociation to the acid. After 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 that conjugation is rapid and is a preferential route of 

metabolism. Metabolism proceeds through incorporation of the radiolabeled carbon into natural 

plant constituents. Previous 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 

incorporation into natural plant constituents. Overall, the 14C-residues in the AAD-12 soybeans 

were similar 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. 

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 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). 

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 method was included in the submission. 

Briefly, samples were extracted with methanol:1.0 N NaOH (90:10, v:v) and centrifuged. An 

aliquot of the extract was combined with a mixed stable isotope internal standard of 2,4-D(13C6) 

or 2,4-DCP(13C6) and evaporated to near dryness. The sample was mixed with 2 N HCl (at 90 

°C for 1 hour). After cooling, the extract was diluted with methanol, filtered, and analyzed by 

on-line solid-phase extraction (SPE; residues eluted with water:methanol 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 level 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 presented 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 recovery data. Concurrent recoveries were generally within the 

acceptable range of 70-120% in soybean 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 treated samples. Apparent residues of 2,4-D were below the LOQ 

(<0.01 ppm) in/on all samples of untreated soybean 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 untreated 
2,4-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; apparent 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 residues of 2,4-D and 2,4-DCP found in the untreated 

samples did not appear to be the result 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. 

Table 8. Summary of Concurrent Recoveries of 2,4-D and 2,4-DCP from Soybean.

Matrix Spike level 

 (ppm) 

Sample size 

 (n) 

Recoveries 

 (%)1

Mean ± std dev2

(%) 

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 apparent residues in the untreated samples. 2

 Standard deviation is not applicable for sample sizes n < 3 samples. 

Processing Study 

Samples of soybean and processed commodities were analyzed for residues of 2,4-D and its 

metabolite 2,4-DCP using the analytical method described 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 the acceptable range of 70-120% for soybean and 
2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085 

Page 12 of 23 

processed commodities fortified 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 oil (see Table 9). The fortification levels 

were adequate to bracket residues found in treated samples. Apparent residues of 2,4-D were 

observed in one sample of untreated AGF at 0.013 ppm; apparent residues of 2,4-D and 2,4-DCP 

were below the LOQ in/on all remaining untreated samples of soybean seed, AGF, meal, hulls, 

and refined oil. Concurrent recoveries were corrected for apparent residues in controls; residues 

in treated samples were not corrected 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 dev2

(%) 

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 plants (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 processing studies have been adequately validated 

for data collection purposes based on acceptable concurrent fortification recovery data. 

860.1360 Multiresidue Methods
2,4-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 Methods Section 402 E1 and 402 E2. Conjugated metabolites 

would not be determined by these multiresidue methods. 

HED Conclusion: Multi-residue method testing 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) from harvest to extraction for 2,4-D 

and 2,4-DCP for up 15.2 months for forage, 15.1 months 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 that residues are stable 

under frozen storage conditions for at least 12 months in/on a variety of field crop commodities 

including corn grain, forage, and fodder; sorghum grain; wheat grain, forage, and straw; rice 

grain; grass forage and hay. 

In addition, 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) 

stored frozen in High-Density Polyethylene (HDPE) containers (as used the field trial studies). 

The available storage stability data 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 months 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 residues 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; sugarcane, sugar, molasses, and bagasse; and rice bran 

and hulls) and for at least 27 months in the soybean 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 stability 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 durations (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; wheat grain, forage, and straw; 

rice grain; grass forage and hay; and soybean seed2

, 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 months 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 stable 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 extraction 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 Conditions for Soybean Processed Commodities.

Matrix Storage 

Temperature 

 (°C) 

Actual Storage Duration1 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) 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; corn starch, flour, and oil; 

wheat flour; sugarcane, sugar, molasses, 

and bagasse; and rice bran and hulls2 

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 extraction and analysis was not provided. 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 conditions 
2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085 

Page 15 of 23 

and durations in the soybean field trials. Storage stability data are available to support the 

storage durations (up to 10.3 months) for the processed 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 grain 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 and the harvesting of forage and hay, those are not considered 

feedstuffs associated with the proposed use. Soybean meal and AGF are included in the livestock 

dietary burden determination (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 the established tolerances for 2,4-D residues in 

livestock feedstuffs, the maximum reasonably 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 the tolerance level for grass forage and hay. The 

MRBD for poultry is 1.5 ppm and for swine is 0.9 ppm, based primarily 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 soybean 
2,4-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 cover the use on feedstuffs of AAD-12 soybeans, provided the grazing 

and harvesting (forage and hay) restrictions are maintained on the label. 

860.1500 Crop Field Trials 

48449802.de1 

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). 

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 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 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 applications of a 3.79 lb ae (acid equivalents)/gal soluble concentrate liquid (SL/L) 

formulation of 2,4-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 of 2.97-3.13 lb ae/A. 2,4-D was applied as 

one preemergence application and two foliar applications, made ~12 days before the soybeans 

reached the R2 growth stage (full flowering stage), and at the ~R2 growth stage. Retreatment 

intervals (RTIs) were 4-64 days between the first 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-ionic surfactant (NIS) at all trial sites. 

Soybean forage and hay were harvested at two preharvest intervals (PHIs), reflecting early 

harvest (targeted 7-day PHI following the second application) 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 was 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 and 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 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 of all RACs, residues 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), <0.01-18.424 (<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 typical-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-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-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, 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 decline 

studies; therefore, residue 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 from Soybean Field Trials with 2,4-D.

Commodity Total 

Applic. 

Rate 

 (lb ae/A) 

PHI 

(days) 

Residue Levels1

 (ppm) 

n Sample

Min. 

Sample

Max. 

LAFT2 HAFT2 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 calculation of median, mean, and 

standard deviation, the LOQ (0.01 ppm) was 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 label reflecting a PHI of 50 days for 

soybean, the submitted field trial data are adequate 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 application #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 application #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 residues) were up to 25.2 (22.4) ppm 

in/on early-harvest forage, up to 40.0 (32.1) ppm in/on typical-harvest forage, up to 190 (190) 

ppm in/on early-harvest hay, up to133 (132) ppm in/on typical-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 decline could not be assessed. Residues of 2,4-DCP in forage, hay, and seed 

generally decreased or did not increase with increasing PHI. 

The trial numbers and geographical locations are 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 trial residue data are classified 

as scientifically acceptable pending submission of a revised label 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 processing 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-DCP). 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 broadcast applications of a 3.79 lb ae/gal soluble concentrate liquid (SL/L) 

formulation of 2,4-D dimethylamine salt (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. Retreatment intervals (RTIs) were 4-

33 days between the first and second applications and 13 days between the second and third 

applications. Applications were made using ground equipment in spray volumes of 21.9-32.36 

gal/A and included a non-ionic surfactant (NIS) 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 48449802.de1) due to concerns over the potential for crop injury

Samples of soybean seed were processed into aspirated 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 processing 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 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-DCP concentrate in AGF (average processing 

factor of 6.9x) and meal (average processing factor 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 study 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 processing study is presented in Table 14. 

Table 14. 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 Factor2

2,4-D 2,4-DCP 2,4-D 2,4-DCP 

Sycamore, GA; 2009 (GA-01) 

Soybean RAC3: 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] 

NC4 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 RAC3: 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 <0.8x 
2,4-D Summary of Analytical Chemistry and Residue Data DP#: 400085 

Page 20 of 23 

Table 14. 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 Factor2

2,4-D 2,4-DCP 2,4-D 2,4-DCP 

Average 

Soybean RAC3: 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 matrices; 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 processed fraction] / [Measured residue for analyte in the RAC]. 3

 The residues for RAC: Seed (prior to AGF generation) were used for calculating processing factors. 4

 NC = Not calculated. The processing factor could not be calculated because residues 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 processed commodities. The data indicated that 

residues of 2,4-D may concentrate in AGF (average processing 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 concentrate in alkali-refined 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. 

860.1650 Submittal of Analytical Reference Standards 

Analytical standards for 2,4-D are currently available in the EPA National Pesticide Standards 

Repository (personal communication with Theresa Cole of the US EPA National Pesticide 

Standard Repository, 08/31/2011). 

860.1850/860.1900 Confined/Field Accumulation 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 14Cresidues in rotated crops are due primarily to the incorporation of radioactivity into natural plant 

constituents. Limited field studies and rotational crop tolerances are not required for residues of 

2,4-D. No plant-back restriction following application 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 rotational 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 current tolerance of 0.02 ppm for residues of 2,4-D 

on soybean seed. However, revisions to the current tolerance are recommended for international 

harmonization. In order to harmonize with the Codex maximum residue levels (MRLs) for 

soybean seed the existing U.S. tolerance for soybean seed (0.02 ppm) should be decreased to 

0.01 ppm. This revision will also support the proposed 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; therefore, the current tolerances for residues of 2,4-D on soybean forage and 

hay are not being reevaluated at this time. 

References 

2,4-D. Summary of Analytical Chemistry and Residue 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 Assessment 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 Chemistry 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 choline 

IUPAC Name: 2-hydroxy-N,N,Ntrimethylethanaminium (2,4-

dichlorophenoxy)acetate 

CAS Name: Ethanaminium, 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.: 2008-39-1 
2,4-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 E 
2,4-D/PC Code 030001/Dow AgroSciences LLC 

DACO 7.3/0CSPP 860.1380/0ECD 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=====---

This DER was originally prepared by Summitec Corporation (9724 Kingston Pike, Suite 602, Knoxville, TN 37922; submitted 

6/13/13) and subcontractor CSS-Dynamac Corporation (10301 Democracy Lane, Suite 300, Fairfax, VA 22030). The DER has 

been reviewed by the Health Effects Division (HED) and revised to reflect current Office of Pesticide 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 by 

Dow AgroSciences LLC. 52 pages. 

Note that MRID 49122501 (amended report) replaces MRID 49075901 (not reviewed). 

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 

-20 °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 

chromatography 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. 

Interim results of the storage stability study through 8 months were previously submitted and 

reviewed under DP# 394948 (P. Savoia, 10/27111). The final storage stability 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 27.8 months. 

STUDY fW AIVER ACCEPTABILITY /DEFICIENCIES/CLARIFICATIONS: 

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), Quality Assurance, and Data Confidentiality 

statements were provided. No deviations from regulatory requirements were reported which 

would have an impact on the validity ofthe study. 

DP# 411873/MRID No. 49 122501 Page 1 of 8 
 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 2 of 8

A. BACKGROUND INFORMATION

2,4-Dichlorophenoxyacetic acid (2,4-D) is an alkylchlorophenoxy herbicide used to control a 

variety of broadleaf weeds. It is a longstanding active ingredient registered for a variety of 

food/feed uses. It may be applied as broadcast, banded, or directed (spray or wiper) treatments 

during dormancy or preplant, preharvest, preemergence, emergence, postemergence, or 

postharvest using ground or aerial 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 grade 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 applicable 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 Value 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 °C 
 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 3 of 8

TABLE A.2. Physicochemical Properties of the Technical Grade Test Compound: 2,4-D. 

Parameter Value 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 (pKa) Not available 

Octanol/water partition coefficient Log(KOW) 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 study (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 indicated that samples from that study were 

ground and blended. For each commodity and sampling interval, three sub-samples per analyte 

were designated for storage samples; in addition, 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 unfortified 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 methanol. Sample sets were prepared and 

fortified on three separate dates to allow for the simultaneous extraction and 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 presented 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 27.8 
 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 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 interval in a storage stability study is to establish 

the residue levels present at the time samples 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 Sciences 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 13C6-2,4-D and 13C6-2,4-DCP 

was added. The mixture was concentrated, acidified with 2 N hydrochloric acid, and heated at 

90 °C for one hour. Upon cooling, the extract was diluted with methanol 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 concurrent recoveries for each analyte were 

conducted at the same level as the fortified samples (0.10 ppm). 

C. RESULTS AND DISCUSSION 

Concurrent recovery data for the storage stability study are reported in Table C.1. The data 

indicate that the LC/MS/MS method was adequate for the determination of residues of 2,4-D and 

2,4-DCP in soybean forage, hay, and seed. Concurrent 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 the 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 stability study are presented in Table C.2. Graphs of residue stability 

of 2,4-D and 2,4-DCP in soybean commodities are presented in Figures C.1 and C.2. The 

storage stability results indicate that residues of 2,4-D and 2,4-DCP 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 Interval1

 (months) 

Sample 

Size (n) 

Recoveries2 

 (%) 

Mean Recovery3

(%) 

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 concurrently and analyzed with samples for the 8.1-month storage 

interval; samples for the 2.0-month storage interval were analyzed with samples for the 25.3-month storage interval; and samples 

for the 4.5-month storage interval were analyzed with 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 30

Corrected Recovery (%)

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 30

Corrected Recovery (%)

Storage Interval (Months)

2,4‐DCP Stability in Soybean Commodities

Forage

Hay

Seed
 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 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 

Residues2

 (ppm) 

Mean Recovery2

(%) 

Corrected 

Recovery3 (%)

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, 0.1284 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 87 
 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 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 

Residues2

 (ppm) 

Mean Recovery2

(%) 

Corrected 

Recovery3 (%)

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 other two recoveries and was not used in determining the average 

recovered residues. 

D. CONCLUSION

The storage stability data are adequate and demonstrate that residues of 2,4-D and 2,4-DCP are 

relatively stable in/on soybean forage, hay, and seed 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 AgroSciences LLC 

DACO 7.4.117.4.2/0PPTS 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 the Health Effects Division (HED) and revised to reflect 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). 

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 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 applications 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 as 

one preemergence application and two foliar applications, made ~ 12 days before the soybeans 

reached the R2 growth stage (full flowering stage), and at the ~R2 growth stage. Retreatment 

intervals (RTis) were 4-64 days between the first 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-ionic surfactant (NIS) at all trial sites. 

Soybean forage and hay were harvested at two preharvest intervals (PHis), reflecting early 

harvest (targeted 7-day PHI following the second application) 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 was 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 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; additional seed samples for residue decline 

were collected at PHis of 37-56, 44-63, 51-70, 58-77, and 65-84 days. 

DP Barcode 0400085/MRID No. 48449802.del Page I of21 
 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 2 of 21 

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 lowest 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 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 months) for hay, and 405 days (13.3 months) 

for seed. The interval between extraction and analysis was not reported. 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 a variety 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 stability data (DP #394948) are available indicating 

residues of 2,4-DCP are stable for up to 8 months in soybean commodities. The available 

storage stability 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 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) containers (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 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 of all RACs, residues 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), <0.01-18.424 (<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 typical-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-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-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, 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 decline 

studies; therefore, residue 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) 

DP Barcode D400085/MRID No. 48449802.de1 Page 3 of 21 

Under the conditions and parameters used in the study, the field trial residue data are tentatively 

classified as scientifically acceptable pending submission 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, and Data Confidentiality 

statements were provided. No deviations from regulatory requirements 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 alkylchlorophenoxy herbicide used to control a 

variety of broadleaf weeds. These data were submitted to support the proposed use for 2,4-D 

choline application to soybeans containing the inserted aryloxyalkanoate dioxygenase-12 (AAD12) 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 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/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 4 of 21 

TABLE A.1. Test Compound Nomenclature.

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 # 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-trimethylethanaminium (2,4-dichlorophenoxy)acetate 

CAS name Ethanaminium, 2-hydroxy-N,N,N-trimethyl-, 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 Technical Grade Test Compound: 2,4-D acid. 

Parameter Value 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 (pKa) Not available 
 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 5 of 21 

TABLE A.2. Physicochemical Properties of the Technical Grade Test Compound: 2,4-D acid. 

Parameter Value Reference 

Octanol/water partition coefficient Log(KOW) 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 conducted 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 untreated 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/gal SL/L formulation of 2,4-D DMA (Weedar® 64 

Herbicide) at 0.95-1.10 lb ae/A/application, for a total seasonal application rate of 2.97-3.13 lb 

ae/A. 2,4-D was applied as one preemergence application and two foliar applications, made ~12 

days before the soybeans reached the R2 growth stage, and at the ~R2 growth stage. RTIs were 

4-64 days between the first 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 an NIS at all trial sites. 

Soybean forage and hay were harvested at two PHIs, reflecting early harvest (targeted 7-day PHI 

following application 2) 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 was 

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 and 

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. 

Crops were grown and maintained using standard agricultural practices to yield commercially 

representative soybeans. Any maintenance pesticides or fertilizers used were reported. For each 

field trial, the petitioner presented the average 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 generally 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 cooler than normal; conditions at trials IL-08, IL09, IN-10, IA-18, MO-19, and MO-22 (during October; seed harvested 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 caused 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 significant 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 weather 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. The study use pattern is presented in Table B.1.2, 

and the crop varieties grown are 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. Study Use Pattern.

Location 

City, State; Year 

(Trial ID No.) 

EP1 Application Tank Mix/ 

Adjuvants3

 Method/Timing Volume

(gal/A) 

 Rate 

(lb ae/A) 

RTI2

(days) 

Total Rate 

(lb ae/A) 

Sycamore, GA; 2009 

(GA-01) 

3.79 lb 

ae/gal 

SL/L 

1. Broadcast/Preemergence 21.7 0.99 -- 2.99 NIS 

2. Foliar broadcast/ R1 21.6 1.00 4 

3. Foliar broadcast/ R2 21.5 1.00 13 
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 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4) 

DP Barcode D400085/MRID No. 48449802.de1 Page 7 of 21 

TABLE B.1.2. Study Use Pattern.

Location 

City, State; Year 

(Trial ID No.) 

EP1 Application Tank Mix/ 

Adjuvants3

 Method/Timing Volume

(gal/A) 

 Rate 

(lb ae/A) 

RTI2

(days) 

Total Rate 

(lb ae/A) 

Chula, GA; 2009 

(GA-02) 

3.79 lb 

ae/gal 

SL/L 

1. Broadcast/Preemergence 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/Preemergence 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/Preemergence 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/Preemergence 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 12 
 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 8 of 21 

TABLE B.1.2. Study Use Pattern.

Location 

City, State; Year 

(Trial ID No.) 

EP1 Application Tank Mix/ 

Adjuvants3

 Method/Timing Volume

(gal/A) 

 Rate 

(lb ae/A) 

RTI2

(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 13 

York, 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) 

DP Barcode D400085/MRID No. 48449802.de1 Page 9 of 21 

TABLE B.1.3. Trial Numbers and Geographical Locations. 

NAFTA 

Growing Zones 

Soybean 

Submitted Requested1 

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 Regulatory Directive - Dir98-02, Section 9 - Crop Field Trials, Appendix 

III, Table 2 for soybean. 

B.2. Sample Handling and Preparation

Single untreated and duplicate treated samples were collected from each trial. Samples of 

forage, hay, and seed were collected and placed in frozen storage at the field sites within 4 hours 

of collection, or chilled within 4 hours of collection using a cooler with blue, dry, or wet ice, 

after which samples were placed in a freezer within 8 hours of collection. The hay samples were 

allowed to dry in the field to a moisture content of ~<20% for 0-12 days prior to collection. The 

samples were shipped frozen via freezer truck within 1-134 days of harvest to the analytical 

laboratory, Dow AgroSciences (Indianapolis, IN) for residue analysis. Samples were maintained 
 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 10 of 21 

frozen (approximately -20 °C) at the analytical laboratory. In preparation 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 method was included in the submission. 

Briefly, samples were extracted with methanol:1.0 N NaOH (90:10, v:v) and centrifuged. An 

aliquot of the extract was combined with a mixed stable isotope internal standard of 2,4-D(13C6) 

or 2,4-DCP(13C6) and evaporated to near dryness. The sample was mixed with 2 N HCl (at 90 

°C for 1 hour). After cooling, the extract was diluted with methanol, filtered, and analyzed by 

on-line solid-phase extraction (SPE; residues eluted with water:methanol 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 soybean 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 months) for hay, and 405 days (13.3 months) for 

seed. The interval between extraction and analysis 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 grain, forage, and straw; rice grain; grass forage 

and hay; and soybean seed (Residue Chemistry 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 available storage stability data for 2,4-D are adequate 

to support sample storage conditions and durations; however, confirmatory storage stability data 

are required for the metabolite 2,4-DCP reflecting 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 method recovery data. Concurrent recoveries were generally 

within the acceptable range of 70-120% in soybean 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 treated samples. Apparent residues of 2,4-D were 

below the LOQ (<0.01 ppm) in/on all samples of untreated soybean 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 untreated seed at 0.014-0.312 ppm. Apparent residues of 2,4-DCP were below the LOQ 
 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 11 of 21 

(<0.01 ppm) in/on all samples of untreated soybean seed; apparent 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 residues of 2,4-D and 2,4-DCP found in 

the untreated samples did not appear to be the result 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 reported in Table C.3. A summary of residue data for 

soybean is presented in Table C.4. 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 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), <0.01-18.424 (<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-day 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-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, 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 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 C.1. Summary of Concurrent Recoveries of 2,4-D and 2,4-DCP from Soybean.

Matrix Spike level 

 (ppm) 

Sample size 

 (n) 

Recoveries 

 (%)1

Mean ± std dev2

(%) 

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-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 12 of 21 

TABLE C.1. Summary of Concurrent Recoveries of 2,4-D and 2,4-DCP from Soybean.

Matrix Spike level 

 (ppm) 

Sample size 

 (n) 

Recoveries 

 (%)1

Mean ± std dev2

(%) 

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 apparent residues in the untreated samples. 2

 Standard deviation is not applicable 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; wheat grain, forage, and straw; 

rice grain; grass forage and hay; and soybean seed.2 Interim 

storage stability data are available demonstrating residues of 

2,4-DCP are stable in soybean commodities for up to 8 

months3

. 

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 extraction 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) 

PHI1

 (days) 

Residues2

, 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) 

DP Barcode D400085/MRID No. 48449802.de1 Page 13 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) 

PHI1

 (days) 

Residues2

, 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) 

DP Barcode D400085/MRID No. 48449802.de1 Page 14 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) 

PHI1

 (days) 

Residues2

, 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] 

83

(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) 

DP Barcode D400085/MRID No. 48449802.de1 Page 15 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) 

PHI1

 (days) 

Residues2

, 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) 0.0374, 0.041 

[0.039] 

13.0284, 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 

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 Crop Field Trial/ Residue Decline - 2,4-D-Tolerant Soybean (AAD-12, Event 68416-4) 

DP Barcode D400085/MRID No. 48449802.de1 Page 16 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) 

PHI1

 (days) 

Residues2

, 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] 

83

(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) 

DP Barcode D400085/MRID No. 48449802.de1 Page 17 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) 

PHI1

 (days) 

Residues2

, 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] 

0.4115 31.8105

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) 

PHI1

 (days) 

Residues2

, 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] 
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 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) 

PHI1

 (days) 

Residues2

, 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 application 2; for “Typical” harvest, samples were collected following 

final application. The number of days hay was allowed to dry prior to collection is presented in parentheses. 2

 ND = Nondetectable (<LOD). The LOD was 0.003 ppm for all analytes in all matrices; 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 control and treated values were switched. The appropriate value for 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. 
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 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 from Soybean Field Trials with 2,4-D.

Commodity Total 

Applic. 

Rate 

 (lb ae/A) 

PHI 

(days) 

Residue Levels1

 (ppm) 

n Sample

Min. 

Sample

Max. 

LAFT2 HAFT2 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 calculation of median, mean, and 

standard deviation, the LOQ (0.01 ppm) was 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 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 (Event DAS-68416-

4) 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-

18.424 (<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-day 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-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, 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 
 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 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 did 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 reflecting 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 

Nature of the Residues in Plants- 2,4-D-Tolerant 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 Record (DER) was originally prepared under contract by Versar, Inc. (6850 Versar Center, 

Springfield, VA 22151 ; submitted (1/24/20 12). The DER has been reviewed by HED and revised to reflect current Office of 

Pesticide Programs (OPP) policies. 

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-U14C]2,4-dichlorophenoxy acetic acid dimethylammonium salt [2,4-D DMA; specific activity 

10.92 J.LCi/mg for planting box and 101.37 J.LCi/mg for pots] in 2,4-D-tolerant soybean. The 

soybean seed used in the study was genetically modified aryloxyalkanoate dioxygenase-12 

(AAD-12) soybean (event-construct pDAB4468-416). The modification makes the soybean 

resistant to 2,4-D and certain other phenoxyacetate and pyridyloxyacetate auxin herbicides. 

The radiolabeled test substance was formulated as a soluble concentrate liquid (SLIL) and 

applied to 2,4-D-tolerant soybeans 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) followed by two foliar applications 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 .512-3.578 kg ae/ha). The preemergence application was made on the day 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 applications, 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 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 days after the third 

application (62-DAT3 and 41-DAT3), respectively. The in-life phase ofthe study was 

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 combustion/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 released the majority of the radioactivity from forage (87.6% and 

DP# 400085/MRID No. 48449801 Page 1 of26 
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DP# 400085/MRID No. 48449801 Page 2 of 26

6.1% TRR, respectively). Nonextractable residues following these extraction procedures were 

2.6% TRR (1.304 ppm). 

An additional subsample of forage and samples of 7- and 22-DAT3 hay and 62- and 41-DAT3 

seed were subjected to accelerated solvent extraction (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). 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. Extraction 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 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 following 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 characterization work was done on 41-DAT3 samples subjected to ASE 

at 40-70 °C, via extraction with hexane and methanol at 40 °C and methanol/water at 70 °C. 

Extraction with hexane, methanol, and methanol/water released ~10.0-10.2%, 11.0-17.0, and 

13.9-19.4% TRR, respectively. Subsequent hydrolysis of the nonextracable residues 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 majority 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 chromatography (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) for residue characterization/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 chromatography with tandem mass spectrometry 

(LC/MS/MS). Metabolite A was thought to be the initial or primary conjugate of 2,4-DCP. 

Metabolites B and C were found to be minor residue 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 and 41-DAT3 seed were subjected to additional 

extraction and HPLC analysis up to 357-398 days (11.7-13.1 months) after harvest. To confirm 

storage stability, the original 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 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 extraction results and/or chromatographic profiles as 

in samples analyzed earlier. 
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DP# 400085/MRID No. 48449801 Page 3 of 26

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 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” (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. 

In 41-DAT3 seed, following 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, 

respectively. 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 

found to be multicomponent and polar. Precipitates following base hydrolysis of the 

nonextractable residues accounted for 13.4% TRR. 

Based on the results of the metabolism study with AAD-12 soybean (Event 416), the petitioner 

proposed that the metabolism of 2,4-D DMA proceeds rapidly via dissociation to the acid (2,4-

D). After cleavage of the acid side chain, the resulting dichlorophenol (2,4-DCP) was rapidly 

conjugated with acetylated glucose 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 were 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 CO2 in soil, it was likely that any 2,4-D applied to the soil was 

converted to CO2 and became incorporated into natural plant constituents. Hydrolysis 

experiments conducted with extracted soybean lipids and bound residues indicated that this was 

a likely route of metabolism. Previous 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 incorporation into natural plant constituents. Overall, the 14C-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 the study, the plant metabolism 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]. 
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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 from regulatory requirements 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 alkylchlorophenoxy herbicide used to control a 

variety of broadleaf weeds. These data were submitted to support the proposed use for 2,4-D 

choline application to soybean containing the inserted aryloxyalkanoate dioxygenase-12 (AAD12) 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 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)acetic 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 formulation (GF-2654 TS Herbicide; EPA Reg. No. 62719-XXX) 
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TABLE A.1. Test Compound Nomenclature.

2,4-D choline 

IUPAC name 2-hydroxy-N,N,N-trimethylethanaminium (2,4-dichlorophenoxy)acetate 

CAS name Ethanaminium, 2-hydroxy-N,N,N-trimethyl-, 2-(2,4-dichlorophenoxy)acetic acid 

hydroxide (1:1:1) 

CAS # 1048373-72-3 

PC Code 051505 

TABLE A.2. Physicochemical Properties of the Technical Grade Test Compound: 2,4-D acid. 

Parameter Value 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 (pKa) Not available 

Octanol/water partition coefficient Log(KOW) 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 
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DP# 400085/MRID No. 48449801 Page 6 of 26

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 from 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 fertilized to ensure normal crop growth. Monthly climatic conditions 

(temperature and humidity maximums and minimums; and rainfall) were reported. There were 

no meteorological abnormalities that may have impacted the study. A summary of the test site 

information is provided in Table B.1.1, and a summary 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). 
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DP# 400085/MRID No. 48449801 Page 7 of 26

B.2. Test Materials

The radiolabeled test substance was diluted in methanol and ACN, then isotopically diluted with 

nonradiolabeled 2,4-D DMA and formulated as an SL/L using formulation blank. The dose for 

the pots was prepared with a higher specific activity than that prepared for the planting box. The 

characteristics of the test substance are presented in Table B.2.1. 

TABLE B.2.1. Test Material Characteristics.

Chemical structure 

Radiolabel position [phenyl-U-14C]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 μCi/mg (for planting box) and 101.37 μCi/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) followed 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 planting box or pots. The preemergence 

application was made on the day of planting, and foliar applications were made at the V4 and R2 

growth stages at RTIs of 34 and 21 days to soybean grown in the planting box, and at RTIs of 22 

and 20 days to soybeans grown in pots. The specific activity of the test substance was higher for 

the pots to potentially aid in metabolite identification in seed. Applications were made using an 

R&D wand sprayer pressurized with CO2. 

Samples of forage and hay were harvested by cutting plants just above the soil surface (forage) 

or 1-1.5” above the soil surface (hay). Samples of soybean seed were collected by cutting 

mature stalks ~1” above the soil surface; the pods were removed from the stalks, and the seed 

was separated from the pods by hand. Samples of forage, hay, and seed were harvested from the 

planting box, and samples of seed only were harvested from the pots as follow. 

Planting box: Samples of forage (BBCH 60 growth stage; first flowers opened) and hay (R3 

growth stage; beginning pod) were collected 7 days after the second and third 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 stage (full bloom, open flower at one of the two 

uppermost nodes on the main stem with fully developed 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; majority of pods are ripe; beans final color, dry and 

hard) 62 days after the second foliar application (62-DAT3 seed). 
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DP# 400085/MRID No. 48449801 Page 8 of 26

Pots: Samples of seed were collected from mature soybean plants (R8 growth stage) 41 days 

after the second foliar application (41-DAT3 seed). 

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

TABLE B.3.1. Use Pattern Information.

Chemical name [phenyl-U-14C]2,4-D DMA 

Application method The test substance was mixed with soluble liquid (SL/L) formulation blank, diluted 

with water and ACN, and applied as a single preemergence soil application, 

followed by two foliar applications to soybean plants using an R&D wand sprayer 

pressurized with CO2. 

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 foliar 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 (Columbia, MO), within 0-9 days of harvest. At 

ABC Laboratories, samples were prepared for extraction by grinding in the presence of dry ice 

using a grinding mill, then returned to frozen storage (~-20 °C). 

One subsample of 7-DAT2 forage was extracted with 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 was, centrifuged, vacuum filtered and reserved for 

HPLC analysis. 
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DP# 400085/MRID No. 48449801 Page 9 of 26

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), methanol at 40 °C, methanol:water (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) (2x) 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 hydrolysates 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:water: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 radioactivity might exist as conjugated 2,4-D or 2,4-

DCP, the following extractable and nonextractable residues were subjected 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 forage and hay extracts, only hay results were 

reported in the Study Report. 

For acid/base hydrolysis of forage and hay extracts, 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, diluted 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 hydrolysates were concentrated to dryness or near 

dryness, then combined with water and adjusted to pH 2-3 using concentrated HCl in preparation 

for SPE as described above. The cyclohexane and methanol/1N NaOH eluates following SPE 

were concentrated. The cyclohexane 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 first 2.73 hours and at 80 °C for latter 2.17 hours), 
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DP# 400085/MRID No. 48449801 Page 10 of 26

then centrifuged. The solids were subsequently resuspended and centrifuged with water (2x) and 

methanol (3x). The methanol supernatants were combined, adjusted to pH 9-10 with NaOH, 

then concentrated by rotary evaporation and combined with the aqueous supernatants following 

acid hydrolysis. The combined aqueous + methanol 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 the acid and base treatment from this process were 

reserved for LSC. The supernatants were subjected 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. 
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DP# 400085/MRID No. 48449801 Page 11 of 26

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

1

 We note that the diagram appears to be mislabeled. The procedures for hay and seed on the far 

right should be designated 40-70 °C ASE. 
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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 determined by combustion/LSC. TRR in extracts 

and hydrolysates were determined in quintuplicate by direct liquid scintillation counting (LSC), 

and TRR in nonextractable residues were determined by combustion/LSC. The reported limit of 

quantitation for TRR determinations 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 concentration of 30% (pH ~4-5); (2) 

concentration by rotary evaporation followed by reconstitution 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 identification and quantitation of metabolites. The 

primary HPLC system (Program A) was equipped with a Synergi 4 μm Hydro-RP column, a UV 

detector (280 nm), and a radiodetector, and utilized a gradient mobile phase of 0.1% acetic acid 

in water and 0.1% acetic acid in ACN:methanol (80:20, v:v). The secondary 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 identified by retention time comparisons with reference 

standards. Chemical names and structures for the reference standards are presented in Appendix 

I. 
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DP# 400085/MRID No. 48449801 Page 13 of 26

The hexane extract and acid and base hydrolysates from the hexane extracts of 41-DAT3 seed 

were subjected to TLC analysis for identification and quantitation of metabolites. TLC analyses 

were conducted using silica gel 60F254 plates and solvent system of hexane:methyl tert-butyl 

ether (9:1, v:v). Metabolites were identified by co-chromatography and/or comparison of 

retention times with those of nonlabeled and radiolabeled reference standards. Residues were 

visualized under UV light (254 nm) following exposure 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 standards 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 basic 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 durations 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 months) after harvest. To confirm storage 

stability, the original 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 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 extraction results and/or chromatographic 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 foliar applications to soybean plants grown in a 

planting box, for a total application 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 preemergence 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 procedures were 2.6% TRR (1.304 ppm). 

An additional subsample of forage and samples of 7- and 22-DAT3 hay and 62- and 41-DAT3 

seed were subjected to accelerated solvent extraction (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). In forage 
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DP# 400085/MRID No. 48449801 Page 14 of 26

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. Extraction 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 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 following 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 characterization work was done on 41-DAT3 samples subjected to ASE 

at 40-70 °C, via extraction with hexane and methanol at 40 °C and methanol/water at 70 °C. 

Extraction with hexane, methanol, and methanol/water released ~10.0-10.2%, 11.0-17.0, and 

13.9-19.4% TRR, respectively. Subsequent hydrolysis 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 majority 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 extracts were purified by SPE prior to analysis. Hexane extracts 

from 41-DAT3 seed were also subjected to TLC for residue characterization/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 chromatography with tandem mass spectrometry 

(LC/MS/MS). Metabolite A was thought to be the initial or primary conjugate of 2,4-DCP. 

Metabolites B and C were found to be minor residue components as well as artifacts of the 

extraction/analysis procedures. 

The characterization and identification of residues in soybean matrices 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 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” 

(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 minor residue components as well as artifacts of 

the extraction/analysis procedures was confirmed 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 generally converted to Metabolites B and C on 
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DP# 400085/MRID No. 48449801 Page 15 of 26

concentration under basic conditions. Further acid and base hydrolysis of the methanol extract 

of hay indicated that 2,4-D was 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 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, 

respectively. 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). Most of 

the radioactivity in the 40 °C methanol and 70% methanol/water extracts (~15.6% TRR) was 

comprised of 35 different peaks, with the largest accounting for 5.1% TRR; remaining “other” 

radioactivity in seed was found to be multicomponent 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.

Matrix1 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-analysis 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). Forage and hay were harvested from the planting box only; seed was harvested 62 DAT 

from the planting box and 41 DAT from pots. 

C.2. Identification, Characterization, and Distribution of Residues

TABLE C.2.1. Total Radioactive Residues (TRR) 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 0.415 
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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). Forage and hay were harvested from the planting 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 Metabolites in AAD-12 Soybean (event 416) Forage and 

Hay Following Application of [14C]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 Forage 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 

 Other2 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 

 Other3 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 

 Other4 2.3 0.840 
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DP# 400085/MRID No. 48449801 Page 17 of 26

TABLE C.2.2.1. Distribution of the Parent and the Metabolites in AAD-12 Soybean (event 416) Forage and 

Hay Following Application of [14C]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 Forage 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 

 Other5 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 Extracts6 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 

 Other7 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 characterization analysis was not conducted for the matrix in question. DAT = 

Days after treatment. Forage was harvested 7 days after the second application (DAT2); hay was harvested 7 and 22 days 

following the third application (DAT3). ASE = Accelerated Solvent 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 petitioner based on results from: 40 °C methanol and 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. 
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DP# 400085/MRID No. 48449801 Page 18 of 26

TABLE C.2.2.2. Distribution of the Parent and the Metabolites in AAD-12 Soybean (event 416) Seed 

Following Application of [14C]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 0.82 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 

 Other4 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 

 Other5 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 

 Other6 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) Subsample 
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DP# 400085/MRID No. 48449801 Page 19 of 26

TABLE C.2.2.2. Distribution of the Parent and the Metabolites in AAD-12 Soybean (event 416) Seed 

Following Application of [14C]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 

 Other7 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 characterization analysis was not conducted for the matrix in question. DAT = 

Days after treatment. Seed was harvested following the third application (DAT3) at a 62-day PHI in a planting box and a 41-day

PHI in pots. ASE = Accelerated solvent 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

 The solvent methanol/water (0.02 M EDTA, 1:1; v:v) was accidentally used instead of methanol/ACN/water (2% NH4OH 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 Identification of Radioactive Residues in AAD-12 

Soybean (event 416) Forage and Hay Following Application of [14C]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 Forage 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 extractable2 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 

Accountability4 113 97.9 117 1

 Metabolite fractions were obtained from the summed ASE 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 extractable + Total unextractable)/(TRR from combustion analysis; see TABLE C.2.1) * 100. TRR 

were calculated by summing extractable and nonextractable radioactivity. 
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DP# 400085/MRID No. 48449801 Page 20 of 26

TABLE C.2.3.2. Summary of Characterization and Identification of Radioactive Residues in AAD-12 

Soybean (event 416) Seed Following Application of [14C]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/fatty 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 extractable2 90.9 0.338 

Unextractable (PES)3 2.6 0.010 

Accountability4 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 base hydrolysis of nonextractable residues. 3

 Residues remaining after exhaustive extractions. 

4

 Accountability = (Total extractable + Total unextractable)/(TRR from combustion analysis; see TABLE 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 with AAD-12 soybean (Event 416), the petitioner 

proposed that the metabolism of 2,4-D DMA proceeds rapidly via dissociation to the acid. After 

cleavage of the acid side chain, the resulting dichlorophenol (2,4-DCP) was rapidly conjugated 

with acetylated glucose 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 were 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 CO2 in soil, it was likely that any 2,4-D applied to the soil was 

converted to CO2 and became incorporated into natural plant constituents. Hydrolysis 

experiments conducted with extracted soybean lipids and bound residues indicated that this was 

a likely route of metabolism. Previous 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 incorporation into natural plant constituents. Overall, the 14C-residues in the AAD12 soybeans were similar to those observed on transgenic corn and conventional crops. 

The proposed metabolic pathway (copied without 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 Profile of 2,4-D DMA salt in AAD-12 Soybean.

 ↑ ↑

This compound is Metabolite B This compound is Metabolite C 
 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 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 E 
 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 23 of 26

D. CONCLUSION

The submitted AAD-12 soybean metabolism study is acceptable. Following a single 

preemergence soil application, followed by two foliar applications to soybean plants grown in a 

planting box, for a total application 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 preemergence 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 identified 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 (1.919-2.573 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 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, 

respectively. 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 

found to be multicomponent and polar. Precipitates following base hydrolysis of the 

nonextractable residues accounted for 13.4% TRR. 

Based on the results of the metabolism study with AAD-12 soybean (Event 416), the petitioner 

proposed that the metabolism of 2,4-D DMA proceeds rapidly via dissociation to the acid. After 

cleavage of the acid side chain, the resulting dichlorophenol (2,4-DCP) was rapidly conjugated 

with acetylated glucose 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 were 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 CO2 in soil, it was likely that any 2,4-D applied to the soil was 

converted to CO2 and became incorporated into natural plant constituents. Hydrolysis 

experiments conducted with extracted soybean lipids and bound residues indicated that this was 

a likely route of metabolism. Previous 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 incorporation into natural plant constituents. Overall, the 14C-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 Chemistry and Residue Data for Use of 2,4-D 

Choline in/on Herbicide Tolerant Field Corn Containing 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 Structures 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 Structures of Reference Standards Used in the 

Soybean (AAD-12; Event 416) Metabolism Study (continued). 
2,4-D/PC Code 030001/Dow AgroSciences LLC 

DACO 7.4.5/0PPTS 860.1520/0ECD IIA 6.5.4 and IliA 8.5 

Processed Food and Feed - 2,4-D-To1erant Soybean (AAD-1 2, Event 68416-4) 

Reviewed and 

Approved by --~ -===~~~~2,:::._:::::=~.,.~~-~,..----- Date: 3/4/12 

Danette Drew, Chemist, HED RABV 

Note: This DER was originally prepared under contract by Versar, Inc. (6850 Versar Center, Springfield, VA 22151; submitted 

l/24/12). The DER has been reviewed by the Health Effects Division (HED) and revised to reflect current Office of Pesticide 

Programs (OPP) policies. 

STUDY REPORTS: 

48449802 Culligan, J. (2011) Magnitude ofthe Residue 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. 337 p. 

EXECUTIVE SUMMARY: 

Dow AgroSciences LLC has submitted a processing 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-DCP). Two trials 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 soluble concentrate liquid (SLIL) 

formulation of2,4-D dimethylamine salt (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. Retreatment intervals (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 spray volumes of21.9-32.36 

gall A and included a non-ionic surfactant (NIS) 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 DER for MRID 48449802) due to concerns over the potential for crop 

InJury. 

Samples of soybean seed were processed into aspirated grain fractions (AGF), meal, hulls, and 

alkali-refined oil by GLP Technologies (Navasota, TX) using procedures simulating commercial 

processmg. 

Samples of soybean seed and processed commodities were analyzed for residues of 2,4-D and its 

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 lowest 

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 

DP Barcode 400085/MRID No. 48449802.de2 Page 1 of 13 
 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) 

DP Barcode 400085/MRID No. 48449802.de2 Page 2 of 13 

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 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 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; and rice bran and hulls. 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 available storage stability 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 stability 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 processing 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 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-DCP concentrate in AGF (average processing 

factor of 6.9x) and meal (average processing factor 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 study 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 trial residue data are tentatively 

classified as scientifically acceptable pending submission of confirmatory storage stability data 

demonstrating the stability of 2,4-DCP in/on soybean 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 Summary 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) 

DP Barcode 400085/MRID No. 48449802.de2 Page 3 of 13 

Signed and dated Good Laboratory Practice (GLP), Quality Assurance, and Data Confidentiality 

statements were provided. No deviations from regulatory requirements 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 alkylchlorophenoxy herbicide used to control a 

variety of broadleaf weeds. These data were submitted to support the proposed use for 2,4-D 

choline application to soybeans containing the inserted aryloxyalkanoate dioxygenase-12 (AAD12) 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 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)acetic acid - dimethylamine (1:1) 

CAS name 2-(2,4-dichlorophenoxy)acetic acid 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) 

DP Barcode 400085/MRID No. 48449802.de2 Page 4 of 13 

TABLE A.1. Test Compound Nomenclature.

2,4-D choline 

IUPAC name 2-hydroxy-N,N,N-trimethylethanaminium (2,4-dichlorophenoxy)acetate 

CAS name Ethanaminium, 2-hydroxy-N,N,N-trimethyl-, 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 Technical Grade Test Compound: 2,4-D acid. 

Parameter Value 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 (pKa) Not available 

Octanol/water partition coefficient Log(KOW) 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 
 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) 

DP Barcode 400085/MRID No. 48449802.de2 Page 5 of 13 

B. EXPERIMENTAL DESIGN

B.1. Application and Crop Information 

Two trials were conducted on soybean containing 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 control 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 broadcast 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 the first and second applications and 13 days 

between the second and third applications. Applications were made using ground equipment in 

spray volumes of 21.9-32.36 gal/A and included an NIS 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 concerns 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/ 

Adjuvants3

 Method/Timing Volume 

(gal/A) 

 Rate 

(lb ae./A) 

RTI2

(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 13 

York, 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) 

DP Barcode 400085/MRID No. 48449802.de2 Page 6 of 13 

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 hours 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 shipped frozen via freezer truck to the processing 

facility, GLP Technologies (Navasota, TX). Soybean 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. Samples were stored frozen after processing and 

were shipped frozen overnight with dry ice to Dow AgroSciences, LLC (Indianapolis, IN) for 

residue analysis. Samples were maintained frozen (≤-20 °C) at the analytical laboratory. In 

preparation for analysis, samples (except oil) were ground in the presence of dry ice. 

The petitioner submitted adequate descriptions of the processing procedures including material 

balance summaries. Soybean processing procedures are summarized below in Figures 1 (AGF) 

and 2 (meal, hulls, and alkali-refined oil), which 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) 

DP Barcode 400085/MRID No. 48449802.de2 Page 7 of 13 

FIGURE 1. Processing Flowchart 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) 

DP Barcode 400085/MRID No. 48449802.de2 Page 8 of 13 

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

 Hulls. 

B.3. Analytical Methodology

Samples of soybean and processed commodities were analyzed for residues of 2,4-D and its 

metabolite 2,4-DCP by LC/MS/MS. A complete description of the method was included in the 

submission. 

Briefly, samples were extracted with methanol:1.0 N NaOH (90:10, v:v) and centrifuged. An 

aliquot of the extract was combined with a mixed stable isotope internal standard of 2,4-D(13C6) 

or 2,4-DCP(13C6) and evaporated to near dryness. The sample was mixed with 2 N HCl (at 90 

°C for 1 hour). After cooling, the extract was diluted with methanol, filtered, and analyzed by 
 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) 

DP Barcode 400085/MRID No. 48449802.de2 Page 9 of 13 

on-line solid-phase extraction (SPE; residues eluted with water:methanol 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 durations for samples from the soybean processing study are 

presented in Table C.2. 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 soybean seed (RAC) 

and 276-312 days (9.1-10.3 months) for the processed 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 flour; sugarcane, sugar, 

molasses, and bagasse; and rice bran and hulls (Residue Chemistry 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 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 presented 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 the acceptable range of 70-120% for soybean and 

processed commodities fortified 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 oil. The fortification levels were adequate to 

bracket residues found in treated samples. Apparent residues of 2,4-D were observed in one 

sample of untreated AGF at 0.013 ppm; apparent residues of 2,4-D and 2,4-DCP in/on all 

remaining untreated samples of soybean seed, AGF, meal, hulls, and refined oil. Concurrent 

recoveries were corrected for apparent residues in controls; residues in treated samples were not 

corrected for residues in controls. 

Residue data for the soybean processing study are 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 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 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-DCP concentrate in AGF (average processing factor of 6.9x) and 
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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 study 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 dev2

(%) 

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. 
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TABLE C.2. Summary of Storage Conditions for Soybean Processed Commodities.

Matrix Storage 

Temperature 

 (°C) 

Actual Storage Duration1 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) 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; 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 

months3. 

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 extraction and analysis was not provided. 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 Factor2

2,4-D 2,4-DCP 2,4-D 2,4-DCP 

Sycamore, GA; 2009 (GA-01) 

Soybean RAC3: 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] 

NC4 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 RAC3: 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 <0.8x 
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DP Barcode 400085/MRID No. 48449802.de2 Page 12 of 13 

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 Factor2

2,4-D 2,4-DCP 2,4-D 2,4-DCP 

Average 

Soybean RAC3: 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 matrices; 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 processed fraction] / [Measured residue for analyte in the RAC]. 3

 The residues for RAC: Seed (prior to AGF generation) were used for calculating processing factors. 4

 NC = Not calculated. The processing factor could not be calculated because residues were below the LOQ in the RAC and the 

processed fraction. 

D. CONCLUSION

Pending submission of confirmatory storage stability data for 2,4-DCP, the submitted soybean 

processing study is acceptable. Processing factors for 2,4-D could not be calculated for alkalirefined oil, meal, and hulls because residues were below the LOQ in/on all samples of soybean 

seed and processed commodities. The data indicated that residues of 2,4-D may concentrate in 

AGF (average processing factor of >1.4x). Residues of 2,4-DCP may concentrate 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. Confirmatory 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 for 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): None 
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F. DOCUMENT TRACKING

DP Barcode(s): D400085 

PC Code: 030001 

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