Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode D340895 

Page 1 of 38

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460

Office of Chemical Safety

and Pollution Prevention 

MEMORANDUM

Date: July 21, 2010

Subject: Isoxaben. Petition for Establishment of Tolerances for Residues of Isoxaben in/on 

Grapes and Tree Nuts (including Pistachio). Summary of Analytical Chemistry and 

Residue Data.

PC Code: 125851 DP Barcodes: D340895, D341694, D342083, 

D346398, D352226, D354469,

D355150, D355805, D368803

Decision No.: 379577, 379578,

398238

File Symbol: 62719-LIN

MRID Nos.: 47140006-47140010, 

47140012, 47175101,

47462101, 47492601,

47825501

Regulatory 

Action: Section 3

Petition #: 7F7222 Reregistration 

Case No.: NA

Assessment Type: NA CAS # 82558-50-7

TXR No.: NA 40 CFR 180.xxx (to be determined)

From: Dennis McNeilly, Chemist

William T. Drew, Chemist

Elizabeth Holman, Chemist

Risk Assessment Branch II

Health Effects Division (7509P)

Through: Douglas Dotson, Ph.D., Chemist

 Richard Loranger, Ph.D., Senior Scientist

 Christina Swartz, Chief

 Risk Assessment Branch II

 Health Effects Division (7509P)

To: Kathryn V. Montague, PM-23

 Herbicide Branch

 Registration Division (7505P)

This document was originally prepared under contract by Dynamac Corporation (2275 Research Blvd, Suite 300; 

Rockville, MD 20850; submitted 12/12/2007). The document has been reviewed by the Health Effects Division 

(HED) and revised to reflect current Office of Pesticide Programs (OPP) policies.
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 2 of 38

Executive Summary

Dow AgroSciences has submitted a petition, PP#7F7222, proposing the establishment of 

tolerances for residues of isoxaben [N-[3-(1-ethyl-1-methylpropyl)-5-isoxazolyl]-2,6-

dimethoxybenzamide] in/on the following raw agricultural commodities:

Grape...........................................................................0.01 ppm

Grape, juice .................................................................0.01 ppm

Grape, raisin................................................................0.01 ppm

Nut, tree, group 14 ......................................................0.03 ppm

Pistachio......................................................................0.03 ppm

Almond, hulls..............................................................0.35 ppm

The petitioner has also requested a waiver from the requirement for a tolerance for isoxaben 

residues in or on cattle meat byproducts, meat, and milk.

In conjunction with this petition, Dow AgroSciences has submitted a FIFRA Section 3 request to 

register an end-use product, FN-3133 Speciality Herbicide, a 75% dry flowable (DF) formulation 

of isoxaben with EPA File Symbol 62719-LIN. The product is proposed for use on grapes and 

tree nuts (including pistachio) with two soil applications at application rates ranging from 0.5–

1.0 lb. ai/acre and a maximum of 1.0 lb ai/A per crop per year (harvest to harvest). No minimum 

retreatment interval (RTI) was specified on the proposed label. The proposed pre-harvest 

intervals (PHIs) are 60 days for tree nuts, and 165 days for grapes.

Isoxaben is a benzamide herbicide (Group 21) used for control of broadleaf weeds. Isoxaben 

reportedly acts via inhibition of cell wall synthesis. The herbicide is currently registered for nonfood uses on turf, ornamentals, non-bearing fruit and nut trees, and non-bearing vineyards. 

The petitioner (as the Elanco Products Company) previously applied for temporary tolerances for 

residues of isoxaben in/on barley and wheat grain in conjunction with an experimental use permit 

(PP#5G3216; RCB# 1188, 7/26/85, J. Worthington). At that time, HED recommended in favor 

of the requested temporary tolerances.

For the purposes of this petition only, the nature of the residue in grapes and tree nuts is 

adequately understood based on grape and barley metabolism studies. With respect to future 

uses, HED concludes that the submitted plant metabolism studies are adequate for uses on cereal 

grains, grasses, grapes, and tree nuts. For grapes and tree nuts the grape metabolism study is 

adequate only for early season uses (i.e., prior to or at bloom). Additional radiolabeled studies 

would be needed for later season uses on tree nuts or grapes and for any uses on crops other than 

grains or grasses.

A livestock metabolism study has been submitted to support the proposed uses. Only one 

commodity associated with the proposed uses, almond hulls, is a cattle/dairy cow feed item. 

There are no poultry or swine feed items associated with the proposed uses. HED’s review of 

this livestock metabolism study indicates that a cattle feeding study, and tolerances for livestock 

commodities are not needed for the proposed uses currently under consideration. Although the 

goat metabolism study identified low percentages of the total radioactive residues in most 
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 3 of 38

matrices, it is adequate for the proposed uses, based on the very low dietary burden for cattle. 

Another goat metabolism study with better identification and characterization of the residues will 

be needed to support any additional uses of isoxaben that result in significantly higher dietary

burdens. HED concludes that residues in milk, ruminant and poultry meat, fat, and meat 

byproducts and eggs remain a 40CFR §180.6[a][3] situation (i.e., no expectation of finite 

residues in these commodities).

The petitioner has proposed a high-performance liquid chromatography with tandem mass 

spectromectric detection (LC/MS/MS), method GRM 02.26.S.1, for the enforcement of 

tolerances for residues of isoxaben in plant commodities. The limit of quantitation (LOQ) is 0.01 

ppm for each commodity. An ILV study was submitted with wheat grain and endive leaves 

fortified with isoxaben at 0.01 ppm, 0.02 ppm, and 0.10 ppm. Adequate recoveries were 

obtained for both commodities using the method as written.

HED concludes this LC/MS/MS method is adequate for enforcement of the proposed tolerances. 

In addition, existing data for the FDA Multiresidue Methods indicate that isoxaben is completely

recovered using multiresidue method Section 302.

The petitioner submitted crop field trial studies with grape, almond, and pecan to support the 

proposed uses. All studies were conducted at 1.0x the proposed maximum seasonal rate of 1 lb.

ai/acre. In the submitted crop field trial studies, residues of isoxaben were below the LOQ of 

0.01 ppm in/on all samples of grape and pecan collected 60 days following application at 1 lb.

ai/acre. Residues of isoxaben were <0.01-0.015 ppm in/on almond nutmeat samples collected 60 

days posttreatment. Quantifiable residues of isoxaben (up to 0.30 ppm) were observed in/on 

almond hulls at a PHI of ~60 days.

Dow AgroSciences submitted a storage stability study with isoxaben on grapes, cereal grain,

cereal straw, and endive leaves. Subsamples of fortified samples were collected at 3-, 6-, 12-, 

18- and 24-month storage intervals and analyzed for residues of isoxaben. The storage stability

data indicate that residues of isoxaben are stable under frozen storage conditions for up to 24 

months in/on grapes, cereal grain, and cereal straw, and for up to 12 months in/on endive leaves. 

No storage stability data for livestock sample matrices were provided by the petitioner. The 

dates of sample collection, extraction, and HPLC analysis, indicate that all samples were 

analyzed within 4-6 months of sample collection. Therefore, storage stability data are not 

required.

Because grape vines and tree nuts are not normally rotated, no data pertaining to rotational crops 

are required to support the proposed uses.

There are no Codex, Canadian, or Mexican Maximum Residue Limits (MRLs) established for 

residues of isoxaben. 

Regulatory Recommendations and Residue Chemistry Deficiencies

HED has examined the residue chemistry database for isoxaben and pending the results of the 

forthcoming human health assessment, HED recommends that the requested uses be approved 

and the supporting tolerances as specified in Table 15 be established.
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 4 of 38

860.1300 Nature of the Residue - Plants

 For the purposes of this petition only, the nature of the residue in grapes and tree nuts is 

adequately understood based on grape and barley metabolism studies. With respect to 

future uses, HED concludes that the submitted plant metabolism studies are adequate for 

uses on cereal grains, grasses, grapes and tree nuts. For grapes and tree fruit the grape 

metabolism study is adequate only for early season uses (i.e., prior to or at bloom).

860.1300 Nature of the Residue - Livestock

 The submitted livestock metabolism study is sufficient for the proposed uses currently

under review. However, additional ruminant metabolism data with better identification 

and characterization of the residue will be needed for any additional uses of isoxaben that 

result in significantly higher dietary burdens. 

860.1480 Meat, Milk, Poultry, and Eggs

 HED’s review of the livestock metabolism indicates that a cattle feeding study is not 

needed for the proposed uses currently under consideration. However, a cattle feeding 

study may be needed for any additional uses of isoxaben that result in significantly higher 

dietary burdens.

860.1550 Proposed Tolerances

 HED recommends the isoxaben tolerance expression be established as follows: 

“Tolerances are established for residues of the herbicide isoxaben, including its 

metabolites and degradates, in or on the commodities in the table below. Compliance 

with the tolerance levels specified below is to be determined by measuring only isoxaben 

[N-[3-(1-ethyl-1-methylpropyl)-5-isoxazolyl]-2, 6-dimethoxybenzamide] in or on the 

commodity.

 The proposed tolerances for the tree nut group and pistachios should be revised to 0.02 

ppm. The commodity definition for the tree nut group should be revised to “Nut, tree, 

Group 14.”

 The proposed tolerance for almond hulls should be revised to 0.40 ppm.

 The available processing data indicate that residues of isoxaben do not concentrate in 

grape juice or raisins; therefore, no tolerances are needed for grape processed 

commodities. A revised Section F should be submitted with grape juice and raisins 

removed. 

Background

The chemical structure and nomenclature of isoxaben are presented in Table 1. The 

physiochemical properties of the technical grade of isoxaben are presented in Table. 2. The 
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 5 of 38

chemical names and structures of isoxaben and its transformation products are presented in 

Appendix I.

Table 1. Isoxaben Nomenclature.

Compound

OCH3

N

H

O

O

N

CH3

CH3

CH3

OCH3

Common name Isoxaben

Company experimental 

name

EL-107

IUPAC name N-[3-(1-ethyl-1-methylpropyl)isoxazol-5-yl]-2,6-dimethoxybenzamide

CAS name N-[3-(1-ethyl-1-methylpropyl)-5-isoxazolyl]-2,6-dimethoxybenzamide

CAS registry number 82558-50-7

End-use product (EP) FN-3133 Speciality Herbicide (75% DF formulation; EPA Reg. No. 

62719-LIN)

Table 2. Physicochemical Properties of Isoxaben.

Parameter Value Reference

Melting range 173-176.5oC MRID 47140001

Density (relative) 0.580 g/ml at 22oC MRID 47140001

Water solubility 1.42 mg/L at 20oC MRID 47140001

Solvent solubility, at 20oC Acetone 270 g/L

Methanol 98 g/L

1-Octanol 29 g/L

Ethyl acetate 88 g/L

1,2-Dichloroethane 51 g /L

Xylene 4.0 g/L

Hexane 0.028 g/L

MRID 47140001

Vapor pressure 1.3 x 10-8Pa at 25oC Email for DowAgrosciences

(5/19/10) revising vapor 

pressure

Dissociation constant, pKa 9.78 ± 0.06 at 20oC EFSA1(May 2006 Draft 

Report)

Octanol/water partition coefficient, 

Log(KOW)

3.83 at 20oC MRID 47140001

UV/visible absorption spectrum max1 = 202 nm / 26000 mol-1

cm-1

max2 = 252 nm / 16500 mol-1cm-1

max3 = 275 nm / 20800 mol-1cm-1

EFSA1(May 2006 Draft 

Report)

1- European Food Safety Authority (EFSA)
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 6 of 38

860.1200 Directions for Use

Dow AgroSciences submitted a proposed label for FN-3133 Specialty Herbicide (EPA File 

Symbol No. 62719-LIN, label dated 2/09). The proposed uses include bearing nut trees and 

vineyards and nonbearing nut trees and vineyards. The proposed use directions on bearing crops 

are presented in Table 3 below.

Table 3. Summary of Directions for Use of Isoxaben.

Applic. Timing, 

Type, and Equip.

Formulation

[EPA File 

Symbol No.]

Applic. 

Rate 

(lb ai/A)

Max. No. 

Applic. per 

Season

Max. Seasonal 

Applic. Rate

(lb ai/A)

PHI

(days)

Use Directions and 

Limitations

Grape (American, European, Muscadine)

Soil application,

Equipment type 

not specified

75% DF

[62719-LIN] 0.50-1.0 2

1.0 per crop 

season 

(harvest to 

harvest)

165

Not to be used in NY. 

Application is to be made 

prior to germination of 

target weeds or immediately 

after cultivation. 

Application is to be made in 

a minimum of 10 gal/A.

Almond; Beech Nut; Brazil Nut; Butternut; Cashew; Chestnut; Chinquapin; Filbert (Hazelnut); Hickory Nut; 

Macadamia Nut (Bush Nut); Pecan; Pistachio; and Walnut (Black, English/Persian)

Soil application,

Equipment type 

not specified

75% DF

[62719-LIN] 0.50-1.0 NS

1.0 per crop 

season 

(harvest to 

harvest)

60

Not to be used in NY. 

Application is to be made 

prior to germination of 

target weeds or immediately 

after cultivation. 

Application is to be made in 

a minimum of 10 gal/A.

The product is not to be applied through any type of irrigation system or to crops grown in 

containers. A restricted entry interval of 12 hours has been established on the label.

Conclusions. The proposed use directions are adequate to allow evaluation of the crop field trial 

data submitted with this petition. The proposed use directions represent maximum seasonal rates 

~1x those used in the crop field trials. The available crop field trial data support a maximum 

seasonal rate of 1 lb active ingredient/A.

860.1300 Nature of the Residue - Plants

DER References: 47140006.der.doc (Barley)

47175101.der.doc (Grape)

Residue Chemistry Memo PP#5G3216, 5/17/85, J. Worthington

In conjunction with the temporary tolerance petition reviewed by the Agency in 1985 

(PP#5G3216, J. Worthington, 5/17/85), the petitioner submitted metabolism data for barley and 

wheat, reflecting use of [14C]isoxaben labeled at the carbonyl carbon and in the isoxazole ring. 

Winter wheat and barley plants were grown in small outdoor plots treated at 81-324 g ai/A (0.18-

0.71 lb ai/A) with labeled isoxaben. A greenhouse experiment reflecting 162 g ai/A (0.36 lb 
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 7 of 38

ai/A) was also reported. Grain and straw samples were collected from all of the treated plots 

about 100 days after planting.

The following four metabolites, comprising 33.1% and 14.4% of the activity in wheat and barley

straw, respectively, were identified: (1) and (2) N-[3-(1-ethyl-2-hydroxy-1-methylpropyl)-5-

isoxazolyl]-2,6-dimethoxybenzamide (isoxaben-2-hydroxy) and its diastereomer; (3) N-[3-(1-

ethyl-3-hydroxy-l-methylpropyl)-5-isoxazolyl)-2,6-dimethoxybenzamide (isoxaben-3-hydroxy); 

and (4) 2,6-dimethoxybenzamide. The pattern of residues in wheat and barley grain was similar 

except that more extensive degradation of the radioactivity was shown. About 5% of the 

recovered activity was shown to be parent. Only about 50% of the activity present in the grain 

was solubilized. The same four metabolites were also detected in the grain samples; however, 

quantification of the lower residue levels in the grain samples was more difficult. 

HED concluded that the fate of isoxaben in wheat grain was adequately delineated for the 

purpose of the proposed temporary tolerances, considering the fact that grazing, foraging, and 

cutting crops grown in treated areas was to be prohibited. Isoxaben was determined to be the 

residue of concern. It was also concluded that for any future submission involving feed use of 

treated foliage, characterization of a greater portion of the activity in forage and straw might be 

required.

Barley (Note: This is a new study, not the study reviewed by HED in 1985.)

Dow AgroSciences submitted a study investigating the metabolism of [isoxazole-5-

14C]isoxaben 

(specific activity 5.8 mCi/mmol) and [phenyl14C]isoxaben (specific activity 17.39 mCi/mmol) in 

barley. Each radiolabeled test substance was formulated with blank suspension concentrate (SC)

formulation, diluted with water, and applied as a single foliar application at 0.178 lb ai/A (200 g 

ai/ha) to barley at the BBCH 13 growth stage (3 unfolded leaves) grown outdoors in tubs. The 

application rate used in the study is ~0.18x the proposed maximum seasonal rate of 1 lb ai/A for 

grapes and tree nuts and ~0.18x the maximum rates used in the crop field trials. Immature plant 

samples were taken 3 hours after application (0 days after treatment; 0-DAT), 7 DAT, 14 DAT, 

and 28 DAT. Samples of mature barley grain, straw, and chaff were harvested at 87 DAT and 

allowed to air dry. 

Total radioactive residues (TRR) were generally slightly higher in isoxazole-label samples than 

in phenyl-label samples, and TRR decreased (both labels) with later sampling intervals. TRR in 

mature barley matrices were initially determined by combustion/LSC. However, TRR were 

calculated in immature and mature barley matrices by summing radioactivity in extractable and 

nonextractable fractions, and these values were used for all calculations. Calculated TRR for 

isoxazole-label barley and for phenyl-label barley matrices are provided in Table 4.
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 8 of 38

TABLE 4 Total Radioactive Residues (TRR) in Barley at 0.18X Application Rate. 1

Matrix Application rate

(lb ai/A)

PHI

(days)

Isoxazole label Phenyl label

ppm ppm

Immature plant 0.178 0 5.731 4.718

7 3.129 1.307

14 0.376 0.341

28 0.084 0.052

Grain 87 0.019 (0.019) 0.012 (0.011)

Straw 0.281 (0.298) 0.222 (0.213)

Chaff 0.458 (0.475) 0.279 (0.295)

1

 TRR calculated by summing radioactivity in extractable and nonextractable fractions; TRR in mature grain, straw and chaff determined by

combustion/LSC are reported in parentheses.

Solvent extraction with acetonitrile (ACN) and ACN/water released the majority (92.3-99.9% 

TRR) of the radioactivity (with highest levels in the ACN extract) from immature plants from 

both labels; nonextractable residues accounted for 0.1-7.7% TRR (0.003-0.016 ppm) in immature 

barley. Solvent extraction with ACN and ACN/water released lower levels of radioactivity (with 

highest levels in the ACN/water extract) from mature barley matrices: 63.2-66.6% TRR from 

grain, 73.3-75.3% TRR from chaff, and 73.0-75.4% TRR from straw. Additional radioactivity

(19.2-21.2% TRR) was released from barley straw with acid hydrolysis at reflux. Nonextractable 

residues accounted for 33.3-36.8% TRR (0.004-0.007 ppm) in grain, 4.5-4.6% TRR (0.01-0.013 

ppm) in straw, and 24.7-26.6% TRR (0.069-0.122 ppm) in chaff. Nonextractable residues in 

chaff were not further investigated because chaff represents a small component of barley straw.

The extraction/hydrolysis procedures adequately extracted the majority of residues from barley

matrices. Because TRR in immature barley were determined by summing radioactivity in 

extractable and nonextractable residues, accountabilities for immature plants were 100%. 

Accountabilities for mature barley grain, straw, and chaff were 94.6-109.1% (based on 

comparison of combustion/LSC and calculated TRR values). Residues were identified and 

confirmed by thin layer chromatography (TLC) and HPLC, except for those in the ACN extract 

of grain samples and the enzyme/acid hydrolysates (with low levels) which were characterized by

TLC only. 

The metabolic pattern was similar in isoxazole- and phenyl-label barley matrices. The major and 

only residue identified in 0-, 7- and 14-DAT immature barley was the parent isoxaben, 

accounting for 85.1-94.8% TRR (0.298-5.367 ppm). Isoxaben was also the major residue 

identified in 28-DAT immature barley, at 88.5-89.3% TRR (0.046-0.075 ppm), and mature 

barley straw, at 25.9-29.2% TRR (0.057-0.082 ppm). Isoxaben was identified as a minor residue 

in mature barley grain, at 5.8-6.7% TRR (0.001 ppm), and chaff, at 4.7-5.2% TRR (0.013-0.024 

ppm). 

Major residues identified in mature barley grain, straw, and chaff were Metabolite II (isoxaben-2-

hydroxy) and Metabolite IV (isoxaben-1-hydroxy). Metabolite II accounted for 15.8% TRR 

(0.002-0.003 ppm) in grain, 12.2-16.1% TRR (0.045-0.056 ppm) in chaff, and 13.9-16.2% TRR 

(0.036-0.039 ppm) in straw; Metabolite IV accounted for 9.5-11.7% TRR (0.001-0.002 ppm) in 

grain, 14.0-15.4% TRR (0.043-0.064 ppm) in chaff, and 11.6-11.7% TRR (0.026-0.033 ppm) in 
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 9 of 38

straw. Metabolites II and IV were identified as minor residues in immature 28-DAT barley, each 

at <3% TRR (≤0.002 ppm).

An additional minor residue identified was Metabolite III (isoxaben-3-hydroxy), which 

accounted for <5% TRR (0.001 ppm) in mature barley grain, <7% TRR (≤0.018 ppm) in straw, 

and <5% TRR (≤0.021 ppm) in chaff.

Metabolite I (2,6-dimethoxybenzoic acid) was only identified in phenyl-label straw at 0.9% TRR 

(0.002 ppm) and was suspected by the petitioner to be an artifact of the acid hydrolysis 

procedure. This was confirmed by the detection of Metabolite I and an unknown in the phenyllabel isoxaben standard acid hydrolyzed (as a control) under the same conditions. 

Polar metabolites were a major residue characterized in barley matrices; polar metabolites 

accounted for 10.7-13.5% TRR (0.007-0.009 ppm) in 28-DAT immature barley and 27.5-27.9% 

TRR (0.003-0.005 ppm) in mature barley grain, 12.5-13.5% TRR (0.030-0.035 ppm) in straw, 

and 37.3% TRR (0.104-0.171 ppm) in chaff. Other unknown residues accounted for 2.7% TRR 

(0.006 ppm) in phenyl-label straw. The polar compounds were determined to consist of multiple 

components using HPLC analysis. Polar metabolites in the ACN/water extracts of straw were 

subjected to enzyme and subsequent acid hydrolysis to release conjugated residues. Enzyme 

hydrolysis did not significantly change the polar fraction pattern, but acid hydrolysis after the 

enzyme hydrolysis released small amounts of isoxaben, and Metabolites II, III and IV.

TABLE 5. Summary of Characterization and Identification of Radioactive Residues in Barley Grain, 

Straw, and Chaff Following Application of [Isoxazole-5-

14C] Isoxaben at 0.178 lb ai/A. 

Compound Barley Grain Barley Straw Barley Chaff

TRR = 0.019 ppm TRR = 0.281 ppm TRR = 0.458 ppm

% TRR ppm % TRR ppm % TRR ppm

Isoxaben 5.8 0.0011 29.2 0.082 5.2 0.024

Metabolite II 15.8 0.0030 13.9 0.039 12.2 0.056

Metabolite III 3.4 0.0007 6.4 0.018 4.6 0.021

Metabolite IV 9.5 0.0018 11.7 0.033 14.0 0.064

Polar compounds 27.9 0.0053 12.5 0.035 37.3 0.171

Total identified 34.5 0.0066 61.2 0.172 36.0 0.165

Total characterized 27.9 0.0053 12.5 0.035 37.3 0.171

Total extractable 63.2 0.012 94.7 0.266 73.3 0.336

Unextractable (PES)136.8 0.007 4.6 0.013 26.6 0.122

Accountability2100.0 93.6 96.4

1

 Residues remaining after exhaustive extractions.

2

 Accountability = (Total extractable + Total unextractable)/(TRR from combustion analysis) x 100.

TABLE 6. Summary of Characterization and Identification of Radioactive Residues in Barley Grain, 

Straw, and Chaff Following Application of [Phenyl14C]Isoxaben at 0.178 lb ai/A. 

Compound Barley Grain Barley Straw Barley Chaff

TRR = 0.012 ppm TRR = 0.222 ppm TRR = 0.279 ppm

% TRR ppm % TRR Ppm % TRR ppm

Isoxaben 6.7 0.0008 25.9 0.057 4.7 0.013

Metabolite I -- -- 0.9 0.002 -- --

Metabolite II 15.8 0.0019 16.2 0.036 16.1 0.045
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 10 of 38

TABLE 6. Summary of Characterization and Identification of Radioactive Residues in Barley Grain, 

Straw, and Chaff Following Application of [Phenyl14C]Isoxaben at 0.178 lb ai/A. 

Compound Barley Grain Barley Straw Barley Chaff

TRR = 0.012 ppm TRR = 0.222 ppm TRR = 0.279 ppm

% TRR ppm % TRR Ppm % TRR ppm

Metabolite III 4.4 0.0005 5.4 0.012 2.2 0.006

Metabolite IV 11.7 0.0014 11.6 0.026 15.4 0.043

Polar compounds 27.5 0.0033 13.5 0.030 37.3 0.104

Other Unknowns -- -- 2.7 0.006 -- --

Total identified 38.6 0.0046 60.0 0.133 38.4 0.107

Total characterized 27.5 0.0033 16.2 0.036 37.3 0.104

Total extractable 66.6 0.0080 94.2 0.209 75.3 0.210

Unextractable (PES)133.3 0.0040 4.5 0.010 24.7 0.069

Accountability2109.1 102.8 94.6

1

 Residues remaining after exhaustive extractions.

2

 Accountability = (Total extractable + Total unextractable)/(TRR from combustion analysis) x 100.

The petitioner did not provide any dates of sample combustion, extraction, or analysis. Based on 

the experimental completion date, samples may have been stored for up to ~15 months prior to 

completion of analysis. Extraction and analysis of additional 0-DAT plant samples following 

storage at <-16oC at the end of the experimental stage of the study demonstrated that the parent 

isoxaben (only residue found in 0-DAT barley) was stable in frozen barley plants for the study

duration. Other available storage stability data for grapes, endive, and cereal grains indicate that 

the parent compound is stable in frozen storage for up to two years.

Based on the submitted barley metabolism studies, the petitioner proposed that isoxaben is 

significantly metabolized in summer barley. Hydroxylation of the aliphatic side chain of the 

intact molecule is the major route of metabolism in barley; cleavage of the amide bond was not 

observed. Isoxaben and metabolites were also present as acid-hydrolyzable conjugates. Isoxaben 

and metabolites, as well as conjugates/polar compounds, were partly incorporated into the 

nonextractable residues of barley matrices.

Grape

Dow AgroSciences submitted a study investigating the metabolism of [isoxazole-5-

14C]isoxaben 

(specific activity 9.01 μCi/mg) and [phenyl14C]isoxaben (specific activity 8.55 μCi/mg) in 

grape. The radiolabeled test substances, dissolved in ACN and water, were applied as a single 

broadcast application to the soil surrounding grape vines at the dormant stage at 1.97 lb ai/A for 

the isoxazole label and 1.79 lb ai/A for the phenyl label. The application rates used in the study

are ~2x the proposed maximum seasonal rate of 1 lb ai/A, and ~2x the individual application rate 

used in the grape crop field trials. Samples of grape fruit and foliage were harvested from each 

plot at maturity, 165 days after application. The petitioner reported that the crop did not show 

any sign of phytotoxicity during the course of the study. 

TRR were below the LOQ (<0.008 ppm) in/on grape fruit harvested 165 days following soil 

treatment with isoxazole- or phenyl-labeled isoxaben at 1.79 or 1.97 lb ai/A. TRR were 0.304 

and 0.288 ppm in grape foliage following soil treatment with isoxazole- or phenyl-labeled 
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 11 of 38

isoxaben, respectively. Because TRR were <LOQ in/on grape fruit, no attempts were made to 

characterize or identify metabolites in the fruit.

TABLE 7. Total Radioactive Residues (TRR) in Grape Matrices.

Matrix No. of 

applications

PHI

(days)

Application rate

(lb ai/A)

Isoxazole label Phenyl label

ppm ppm

Grape, fruit 1 165 1.79-1.97 <LOQ (0.0057)1<LOQ (0.0031)1

Grape, foliage 1 165 0.304 0.288

1 The LSC LOQ was 0.008 ppm for grape fruit.

Extraction with ACN and water released the majority of the radioactivity from grape foliage 

samples, 69% TRR for the isoxazole label and 75% TRR for the phenyl label (average of six 

subsamples for each). Additional residues (10-14% TRR) were released from the grape foliage 

with acid hydrolysis. Nonextractable residues in foliage were 11-14% TRR (0.032-0.041 ppm) 

following acid hydrolysis. All extraction results were normalized to 100% for further 

calculations; however, extraction recoveries were reported to be 76-97% (average of 85.1% for 

the isoxazole label and 92.6% for the phenyl label). 

Residues were identified by comparison of HPLC retention times with those of known standards. 

No confirmatory analyses were performed; therefore, all metabolite identifications are considered 

to be tentative. The petitioner reported the dates of sample collection, combustion, extraction, 

and initial HPLC analysis; however, the dates of final analyses were not provided. Initial HPLC 

analyses were conducted within 4 months of sample collection. 

HPLC analysis yielded five distinct metabolite components plus a region of polar metabolites. 

The parent, isoxaben, was not identified in any sample. A metabolite eluting at ~21.2 min, 

tentatively identified as 2-hydroxy-6-methoxy-benzamide, accounted for 2.9% TRR in phenyllabel foliage. A metabolite with a similar retention time was characterized in isoxazole-label 

samples, accounting for 4.7% TRR. The major metabolite detected (retention time of ~18.4 min) 

accounted for 27.8% TRR (0.084 ppm) in isoxazole-label foliage and 23.9% TRR (0.069 ppm) in 

phenyl-label foliage. The metabolite was isolated from the aqueous phase of extractable foliage 

residues (both labels) and purified by partitioning and solid-phase extraction (SPE). Attempts to 

identify the isolated metabolite by LC/MS/MS were unsuccessful; however, the petitioner 

characterized the residue as aqueous-soluble, polar, and containing both sides of the benzamidebridge (the residue was observed in foliage from both labels). 

TABLE 8. Distribution of the Parent and the Metabolites in Grape Foliage Following Application of 

14C-Isoxaben at 1.79-1.97 lb ai/A. 

Metabolite Fraction Isoxazole label Phenyl label

TRR = 0.304 ppm TRR = 0.288 ppm

%TRR ppm %TRR ppm

ACN:H2O

1

75 0.229 69 0.198

Neutral Organic Fraction219 0.057 16 0.045

2-OH-6-OMe benzamide (Rt ~21.2 min) 4.7 30.014 32.9 0.008

Polars (Rt ≤ 5 min) 1.6 0.005 1.6 0.005

Peak Rt ~19.0 min 2.1 0.006 <0.1 0.002

Acidic Organic Fraction25 0.015 4 0.011

Polars (Rt ≤ 5 min) 0.7 0.002 1.5 0.004
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 12 of 38

TABLE 8. Distribution of the Parent and the Metabolites in Grape Foliage Following Application of 

14C-Isoxaben at 1.79-1.97 lb ai/A. 

Metabolite Fraction Isoxazole label Phenyl label

TRR = 0.304 ppm TRR = 0.288 ppm

%TRR ppm %TRR ppm

Peak Rt ~27.0 min 3.7 0.011 2.4 0.007

Aqueous Fraction 252 0.157 49 0.142

Polars (Rt ≤ 5 min) -- -- 17.4 0.050

Peak Rt ~16.5 min 5.1 0.016 4.1 0.012

Peak Rt ~18.4 min 27.8 0.084 23.9 0.069

Peak Rt ~19.0 min 11.3 0.034 4.4 0.013

Post-extracted tissue 2920.08723240.092

Acid hydrolysate 1020.031214 0.039

Organic fraction 1050.03068 0.0236

Aqueous fraction 4

5

0.01266 0.0176

Unextractable 1120.032 14 0.041

1

 Average of 6 subsamples.

2

 Average of 3 subsamples.

3

 

14C-2-OH-6-OMe-benzamide would not be present in isoxazole-label samples, as this compound does not contain the 

isoxazole ring.

4

 Average of 5 subsamples.

5

 Obtained from a single sample; actual result for acid hydrolysis for this sample was 14 %TRR (0.043 ppm).

6

 ppm values were calculated by the study reviewer.

TABLE 9. Summary of Characterization and Identification of Radioactive Residues in Grape Foliage 

Following Application of 14C-Isoxaben at 1.79-1.97 lb ai/A. 

Compound Isoxazole label Phenyl label

TRR = 0.304 ppm TRR = 0.288 ppm

% TRR ppm % TRR ppm

Isoxaben -- -- -- --

2-OH-6-OMe benzamide (RT ~21.2 min) (RT=-retention time) -- -- 2.9 0.008

Peak RT ~21.2 min14.7 0.014 -- --

Polars (RT ≤ 5 min) 2.3 0.007 20.5 0.059

Peak RT ~16.5 min 5.1 0.016 4.1 0.012

Peak RT ~18.4 min 27.8 0.084 23.9 0.069

Peak RT ~19.0 min (neutral organic)22.1 0.006 <0.1 0.002

Peak RT ~19.0 min (aqueous)211.3 0.034 4.4 0.013

Peak RT ~27 min 3.7 0.011 2.4 0.007

Acid hydrolysate 10 0.031 14 0.039

Total identified 0 0 2.9 0.008

Total characterized 67 0.203 69.8 0.201

Total extractable 85 0.260 83 0.237

Unextractable (PES)311 0.032 14 0.041

Accountability496.1 96.5

1

 The petitioner concluded that this peak corresponded to 2-OH-6-OMe benzamide. Because the metabolite identified does not 

contain the isoxazole ring, it could not have been present in the isoxazole-label sample. 

2

 While the retention times are similar for these metabolites, it is expected that they are different as they occur in different 

fractions.

3

 Residues remaining after exhaustive extractions.

4

 Accountability = (Total extractable + Total unextractable)/(TRR from combustion analysis) * 100.
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 13 of 38

Polar unknowns accounted for 20.5% TRR (0.059 ppm) in phenyl-label foliage, but only 2.3% 

TRR in the isoxazole-label samples. Other metabolites characterized by their partitioning 

characteristics and HPLC retention times accounted for ≤5% TRR in grape foliage (both labels), 

except for one unknown peak (retention time ~19 mins) in isoxazole-label foliage which 

accounted for 11% TRR (0.034 ppm).

The petitioner provided example chromatograms which included information indicating that 

additional metabolites may have been identified in grape foliage fractions: (1) 1-hydroxy

isoxaben in the organic phases of isoxazole- and phenyl-label foliage; (2) 5-isoxazolamine in the 

organic phases of isoxazole- and phenyl-label foliage (however, this metabolite would not be 

present in phenyl-label samples because it does not contain the phenyl ring); and, (3) 3-hydroxy

isoxaben in the organic phase of the acid hydrolysates of isoxazole- and phenyl-label foliage. 

These metabolite identifications were not mentioned in the petitioner’s summary discussion or 

tables, and were only reported in the listing of chromatographic data with the representative 

chromatograms. However, these metabolites were included in the petitioner’s proposed 

metabolic pathway for grapes.

Insufficient information was provided with the representative chromatograms to allow the study

reviewer to verify the results reported for the metabolite and peak fractions. The petitioner 

reported peak results in terms of %HPLC; however, as the petitioner did not identify the 

individual fraction that was injected for each representative chromatogram, the study reviewer 

could not convert these results to %TRR or ppm values.

Based on the submitted grape metabolism study, the petitioner proposed that following 

application of isoxaben to the soil, low levels of radioactivity are taken up by the grape vine 

roots, and even lower levels of radioactivity are translocated into the edible fruit. The 

metabolism of isoxaben in grape leaves appears to proceed through hydroxylation of the 

methylpropyl group. Ultimately, the soil degradation/plant metabolism proceeds to low level, 

multiple, polar components, presumably through cleavage of the benzamide bridge. 

Conclusions. For the purposes of this petition only, the nature of the residue in grapes and tree 

nuts is adequately understood based on grape and barley metabolism studies. With respect to 

future uses, HED concludes that the submitted plant metabolism studies are adequate for uses on 

cereal grains, grasses, grapes and tree nuts. For grapes and tree fruit the grape metabolism study

is adequate only for early season uses (i.e., prior to or at bloom). Additional radiolabeled studies 

would be needed for later season uses on tree nuts or grapes and for any uses on other crops not 

mentioned above.

For the grape study, the application rate of the test substance was not sufficient to allow adequate 

characterization/identification of the residues in grapes, and very limited information and data 

were provided in the study report to allow the study reviewer to verify the study results. The 

following additional deficiencies were identified:

 individual sample extracts for which representative chromatograms were provided were 

not identified;

 chromatographic data in figures were not reported in terms of % TRR;

 metabolites were assigned to peaks in chromatograms in the figures but these results were 

not transferred to the petitioner’s summary of results;
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 14 of 38

 metabolites were identified in fractions incorrectly (e.g., 2-hydroxy-6-methoxybenzamide was “identified” in grape foliage treated with isoxazole-label isoxaben; 

however, this metabolite could not possibly be identified radio-chromatographically in 

this fraction because 2-hydroxy-6-methoxy-benzamide does not contain the isoxazole 

ring);

 metabolite identifications were not confirmed using a second chromatographic method;

 dates of final sample/extract analysis were not reported for any sample;

 data were not submitted to support extract stability during storage.

For the barley metabolism study, submission of additional storage stability data/information is 

not required; since storage stability data indicate that the parent compound is stable in frozen 

storage for up to 2 years (samples may have been stored for up to ~15 months). The petitioner 

should have provided the dates of sample collection, combustion, extraction, and analysis for 

each sample. 

860.1300 Nature of the Residue - Livestock

DER Reference: 47825501.der.doc (Goat)

A livestock metabolism study was recently submitted for this petition. The only livestock 

feedstuff associated with the proposed use is almond hulls. Almond hulls are not fed to poultry

or swine. 

Dow AgroSciences has submitted a study investigating the metabolism of [isoxazole-5-

14C]isoxaben (specific activity 5.8 mCi/mmol) and [phenyl14C]isoxaben (specific activity 31.7 

mCi/mmol) in goats. Each radiolabeled test substance was administered to two lactating goats at 

a dose level of 10 mg ai/kg feed/day. The daily oral dose was administered in the morning for 

five consecutive days using a bolus gun. Based on final feed consumption during the dosing 

period, the actual dosing was 12.290 mg ai/kg feed/day for the [isoxazole-5-

14C]isoxaben dosed 

goat and 19.460 mg ai/kg feed/day for the [phenyl14C]isoxaben dosed goat. (The goat dosed 

with [phenyl14C]isoxaben consumed less feed than expected.) Samples of milk, urine, and feces 

were collected daily throughout the study. Approximately 7 hours after the final dose the 

animals were sacrificed and matrices were collected, including liver, kidney, muscle (loin and 

flank), fat (subcutaneous, omental, and renal), bile, and the gastrointestinal tract. The in-life and 

analytical phases of the study were conducted by the Charles River Laboratory (Edinburgh, 

United Kingdom).

Total radioactive residues (TRR) were higher in isoxazole-label samples than in phenyl-label 

samples. TRR in total goat matrices were determined by combustion/LSC. The maximum 

calculated TRR for isoxazole-label goat matrices were 2.026, 0.279, 0.026, 0.024, and 0.021 ppm 

for liver, kidney, milk, muscle, and fat matrices respectively. The maximum calculated TRR for 

phenyl-label goat matrices were 1.080, 0.181, 0.015, 0.015, and 0.012 ppm for liver, kidney, 

milk, muscle, and fat matrices respectively. 

Solvent extraction with acetonitrile (ACN) and ACN/water released the majority (89-98.5% 

TRR) of the radioactivity (with highest levels in the ACN extract) from goat matrices from both 

labels; nonextractable residues accounted for 1.5-11.0% TRR (<0.001-0.051 ppm) in goat 
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 15 of 38

matrices. The extraction/hydrolysis procedures adequately extracted the majority of residues 

from goat matrices. The accountability ranged from 78-100%, with extractions in milk having 

the poorest accountability (78-90%) while other matrices had much higher accountability (97-

100%). Residues were identified and confirmed using HPLC, with comparison to known 

reference standards for identifying specific metabolites. LC/MS analysis was used to analyze the 

putative metabolite of 3-hydroxy isoxaben (using a reference standard) and to evaluate the 

hypothesized fragmentation pathway of this metabolite. 

The metabolic pattern, summarized in Tables 10a-10d was similar in isoxazole- and phenyl-label 

goat matrices. The parent isoxaben was identified as a major residue in fat matrices (11-23% 

TRR; 0.002-0.004 ppm), but was also found to be a minor residue in liver and muscle matrices. 

An oxime isomer metabolite (RT=16.5 min) was identified as a major metabolite in liver (13-

14% TRR; 0.260-0.279 ppm) and kidney (12% TRR; 0.032 ppm) matrices and a minor one in 

muscle in the isoxazole labeled goat. By contrast, this oxime metabolite was only found to be a 

major metabolite in liver and a minor metabolite in kidney and muscle in the phenyl label goat. 

Another oxime metabolite (RT=17.5) was identified as a major metabolite only in kidney (1-22% 

TRR; 0.002-0.069 ppm). Additionally, a carboxylic acid metabolite was found to be a major 

metabolite only in kidney (29-43% TRR; 0.052-0.105 ppm). 

In liver matrices, 60-68% (0.649-1.379 ppm) of the TRR was tentatively identified as being 

either oxime isomer metabolite, 3-hydroxy isoxaben, or hydroxylated isoxaben. In milk, muscle, 

and fat, significant portions of the TRR were identified as being either oxime isomer, 3-hydroxy

isoxaben, hydroxylated isoxaben, or the carboxylic acid metabolite, specifically with the 

following % TRR values observed: Milk – 14-32% (0.001-0.005 ppm); Muscle – 39-59% 

(0.006-0.014 ppm); Fat – 26-35% (0.003-0.007 ppm). In addition, across all matrices there were 

many components <LOQ that were grouped together to form a significant portion of the TRR, 

with the following ranges of % TRR observed: Milk 40-73% (0.005-0.009 ppm); Muscle 18-56% 

(0.003-0.014); Fat 20-36% (0.002-0.007); Liver 4-5% (0.040-0.105 ppm); Kidney 10-23% 

(0.017-0.63 ppm).

Overall, 84% of the administered dose was recovered for the isoxazole-labeled isoxaben and 

69% recovered for the phenyl-labeled isoxaben, with the majority of the dose being recovered in 

the excreta (60% in feces and 14% in urine for the isoxazole label; 53% in feces and 8% in urine 

for the phenyl label). Only a small portion of the administered dose was distributed in milk, with 

0.22% recovered for the isoxazole label and 0.09% for the phenyl label. 

No storage stability data for sample matrices was provided by the petitioner. Based on the dates 

of sample collection, extraction, and HPLC analysis, all samples were analyzed within 4-6 

months of sample collection. 

Isoxaben metabolism in the lactating goat is proposed to occur by hydroxylation to form the 3-

hydoxy isoxaben metabolite and the hydroxylated isoxaben metabolite (with the hydroxylation 

occurring on the methyl pentanyl moiety). Oxidation of these hydroxylated metabolites is 

hypothesized to result in the carboxylic acid metabolite of isoxaben. Two oxime metabolites (of 

unclear stereochemistry) are then formed by hydryolytic cleavage of the isoxazole ring. Finally, 

cleavage of the amide link in isoxaben causes the formation of the 2,6 dimethoxybenzamide, 2,6-

dimethoxybenzoic acid, and 2-hydroxy-6-methoxybenzamide metabolites (minor residues 
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 16 of 38

identified in liver, kidney, and muscle in the phenyl label goat matrices). 

Conclusions. Although the goat metabolism study identified low percentages of the total 

radioactive residues in most matrices, it is adequate for the proposed uses based on the very low 

dietary burden for cattle. Only one commodity associated with the proposed uses, almond hulls, 

is a cattle feed item. There are no poultry or swine feed items associated with the proposed uses. 

HED review of the livestock metabolism study indicates that a cattle feeding study, and 

tolerances for livestock commodities are not needed. Another goat metabolism study with better 

identification and characterization of the residues will be needed for any additional uses of 

isoxaben that result in significantly higher dietary burdens.

HED concludes that residues in milk, ruminant and poultry meat, fat, and meat byproducts and 

eggs remain a 40CFR §180.6[a][3] situation (there is no expectation of finite residues in these 

commodities). No poultry or swine metabolism studies are required to support the proposed 

uses.
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 17 of 38

Table 10a. Summary of Characterization and Identification of Radioactive Residues in Goat Matrices Following the Oral Administration of 

Radiolabeled [

14C]-5-Isoxazole Isoxaben at a Rate of 12.290 mg ai/kg feed/day for Five Consecutive Days

Compound Muscle

(Flank)

Fat (Subcutaneous) Liver

(Replicate 1)

Kidney

(Replicate 1)

Milk – Day 3 Milk – Day 5 

TRRs = 0.024 ppm TRRs = 0.021 ppm TRRs = 2.026 ppm TRRs = 0.279 ppm TRRs = 0.026 ppm TRR=0.020 ppm

% TRR ppm % TRR ppm % TRR ppm % TRR ppm % TRR ppm % TRR ppm

Isoxaben 11.3 0.002 1.4 0.029

Oxime Isomer (RT=16.5) 5.4 0.001 12.9 0.260 11.5 0.032

Oxime Isomer (RT=17.0) 0.6 0.013 0.6 0.002

Oxime Isomer (RT=17.5) 23.4 0.065

Carboxylic Acid 

Metabolite

36.5 0.102

Oxime isomer

3-hydroxy isoxaben

Hydroxylated isoxaben 

68.0 1.378

Oxime isomer

3-hydroxy isoxaben

Hydroxylated isoxaben 

Carboxylic acid 

metabolite

 38.9 0.009 35.1 0.007 20.7 0.004

Unknown (RT=10.5) 1.1 0.022

Unknown (RT=15.0) 2.1 0.043

Unknown (RT=20.5) 22.6 0.005

Unknown (RT=22.0) 5.0 0.101

Acidified aqueous ACN 0.3 0.006 -- <LOQ

Pepsin Labile 0.9 0.018 1.6 0.004

Many components <LOQ 55.7 0.014 20.3 0.005 5.2 0.105 22.5 0.063 73.0 0.019 63.3 0.013

Total identified 5.4 0.001 11.3 0.002 14.9 0.302 72 0.201 0 0 0 0

Total characterized 94.6 0.023 78.0 0.017 82.6 1.673 24.1 0.067 73.0 0.019 84.0 0.017

Total extractable 100.0 0.024 89.3 0.019 97.5 1.975 96.1 0.268 73.0 0.019 84.0 0.017

Unextractable (PES)1-- <LOQ 10.8 0.002 2.5 0.051 2.7 0.008 11.0 0.003 7.1 0.001

Accountability2~100 ~100 ~100 99 85 90

1

Residues remaining after exhaustive extractions.

2

Accountability = (Total extractable + Total unextractable)/(TRRs from combustion analysis) * 100.
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 18 of 38

Table 10b. Summary of Characterization and Identification of Radioactive Residues in Goat Matrices Following the Oral 

Administration of Radiolabeled [

14C]-5-Isoxazole Isoxaben at a Rate of 12.290 mg ai/kg feed/day for Five Consecutive Days

Compound Muscle

(Loin)

Fat 

(Omental)

Fat 

(Renal)

Liver

(Replicate 2)

Kidney

(Replicate 2)

TRRs = 0.023 ppm TRRs = 0.020 ppm TRRs = 0.020 ppm TRRs = 1.979 ppm TRRs = 0.274 ppm

% TRR ppm % TRR ppm % TRR ppm % TRR ppm % TRR ppm

Isoxaben 17.5 0.004 20.6 0.004 1.4 0.027

Oxime Isomer (RT=16.5) 14.1 0.279 11.5 0.032

Oxime Isomer (RT=17.0)

Oxime Isomer (RT=17.5) 25.1 0.069

Carboxylic Acid 

Metabolite

38.4 0.105

Oxime isomer

3-hydroxy isoxaben

Hydroxylated isoxaben 

67.8 1.342

Oxime isomer

3-hydroxy isoxaben

Hydroxylated isoxaben 

Carboxylic acid 

metabolite

58.9 0.014 29.2 0.006 28.9 0.006

Unknown (RT=10.5) 1.3 0.026

Unknown (RT=12.5) 0.6 0.012

Unknown (RT=15.0) 2.4 0.048

Unknown (RT=20.5) 14.7 0.003 17.4 0.004

Unknown (RT=22.0) 5.0 0.098

Acidified aqueous ACN 0.4 0.008 -- <LOQ

Pepsin Labile 1.0 0.020 1.8 0.005

Many components <LOQ 41.1 0.009 36.4 0.007 27.1 0.005 4.6 0.091 18.9 0.051

Total identified 0 0 17.5 0.004 20.6 0.004 15.5 0.306 75.0 0.206

Total characterized 100 0.023 80.3 0.016 73.4 0.015 83.1 1.645 20.7 0.056

Total extractable 100 0.023 97.8 0.020 94.0 0.019 98.6 1.951 95.7 0.262

Unextractable (PES)1-- <LOQ 2.2 <0.001 6.0 0.001 1.5 0.030 2.8 0.008

Accountability2100 100 100 ~100 ~99

1. Residues remaining after exhaustive extractions. 2. Accountability = (Total extractable + Total unextractable)/(TRRs from combustion analysis) * 100.
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 19 of 38

Table 10c. Summary of Characterization and Identification of Radioactive Residues in Goat Matrices Following the Oral Administration of 

Radiolabeled [

14C]-UL-Phenyl Isoxaben at a Rate of 19.460 mg ai/kg feed/day for Five Consecutive Days

Compound Muscle

(Loin)

Fat (Subcutaneous) Liver

(Replicate 2)

Kidney

(Replicate 2)

Milk – Day 3 Milk – Day 5 

TRRs = 0.015 ppm TRRs = 0.012 ppm TRRs = 1.080 ppm TRRs = 0.181 ppm TRRs = 0.009 ppm TRR=0.015 ppm

% TRR ppm % TRR ppm % TRR ppm % TRR ppm % TRR Ppm % TRR ppm

Isoxaben 3.0 <0.001 20.9 0.002 1.9 0.020

Oxime Isomer (RT=16.5) 7.2 0.001 10.8 0.117 9.4 0.017

Oxime Isomer (RT=17.5) 1.0 0.002

2,6-Dimethoxybenzamide 3.4 0.001 4.2 0.045 6.6 0.012 2.5 <0.001 6.2 0.001

2,6-Dimethoxybenzoic 

acid

0.3 0.001

2-Hydroxy-6-methoxy-

 Benzamide

3.8 0.041 8.2 0.015

Desmethyl Isoxaben 2.7 <0.001

Carboxylic Acid 

Metabolite

43.4 0.078

Oxime isomer

3-hydroxy isoxaben

Hydroxylated isoxaben 

60.1 0.649

Oxime isomer

3-hydroxy isoxaben

Hydroxylated isoxaben 

Carboxylic acid metabolite

39.8 0.006 28.8 0.003 13.6 0.001 31.7 0.005

Unknown (RT=3.5) 2.5 <0.001

Unknown (RT=5.0) 0.1 0.001 0.1 <0.001

Unknown (RT=10.5)

Unknown (RT=13.5) 0.3 0.003

Unknown (RT=14.5) 0.4 0.005

Unknown (RT=20.5) 2.7 <0.001 26.4 0.003

Unknown (RT=21.0) 0.1 0.001

Unknown (RT=21.5) 6.9 0.074
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 20 of 38

Table 10c. Summary of Characterization and Identification of Radioactive Residues in Goat Matrices Following the Oral Administration of 

Radiolabeled [

14C]-UL-Phenyl Isoxaben at a Rate of 19.460 mg ai/kg feed/day for Five Consecutive Days

Compound Muscle

(Loin)

Fat (Subcutaneous) Liver

(Replicate 2)

Kidney

(Replicate 2)

Milk – Day 3 Milk – Day 5 

TRRs = 0.015 ppm TRRs = 0.012 ppm TRRs = 1.080 ppm TRRs = 0.181 ppm TRRs = 0.009 ppm TRR=0.015 ppm

% TRR ppm % TRR ppm % TRR ppm % TRR ppm % TRR Ppm % TRR ppm

Unknown (RT=24.0) 1.9 0.020

Unknown (RT=30.0) 9.6 0.001

Unknown (RT=49.5) 0.6 0.007

Acidified aqueous ACN 0.6 0.006 0.7 0.001

Pepsin Labile 1.8 0.019 2.8 0.005

Many components <LOQ 30.6 0.005 21.0 0.003 4.3 0.046 19.7 0.035 53.9 0.005 40.3 0.006

Total identified 13.6 0.002 20.9 0.002 20.7 0.223 68.9 0.125 5.2 <0.002 6.2 0.001

Total characterized 73.1 0.011 76.2 0.009 75.2 0.811 23.3 0.041 77.1 0.007 74.5 0.011

Total extractable 86.7 0.013 97.1 0.011 95.9 1.034 92.2 0.166 82.3 0.007 80.7 0.012

Unextractable (PES)17.7 0.001 2.9 <0.001 4.1 0.044 5.3 0.010 5.2 <0.001 3.8 <0.001

Accountability293 ~100 ~100 97 78 80

1

Residues remaining after exhaustive extractions.

2

Accountability = (Total extractable + Total unextractable)/(TRRs from combustion analysis) * 100.

Table 10d. Summary of Characterization and Identification of Radioactive Residues in Goat Matrices 

Following the Oral Administration of Radiolabeled [

14C]-UL-Phenyl Isoxaben at a Rate of 19.460 mg ai/kg 

feed/day for Five Consecutive Days

Compound Muscle

(Flank)

Fat (Omental) Liver

(Replicate 1)

Kidney

(Replicate 1)

TRRs = 0.013 ppm TRRs = 0.011 ppm TRRs = 1.080 ppm TRRs = 0.177 ppm

% TRR ppm % TRR ppm % TRR ppm % TRR ppm

Isoxaben 5.7 0.001 22.8 0.003 2.0 0.022

Oxime Isomer (RT=16.5) 3.2 <0.001 10.8 0.117 7.8 0.014

Oxime Isomer (RT=17.5) 22.1 0.039

2,6-Dimethoxybenzamide 2.9 <0.001 3.2 0.035 4.5 0.009

2,6-Dimethoxybenzoic 

acid

3.7 0.007

2-Hydroxy-6-methoxy-

 Benzamide

4.2 0.045 8.2 0.015

Desmethyl Isoxaben
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 21 of 38

Table 10d. Summary of Characterization and Identification of Radioactive Residues in Goat Matrices 

Following the Oral Administration of Radiolabeled [

14C]-UL-Phenyl Isoxaben at a Rate of 19.460 mg ai/kg 

feed/day for Five Consecutive Days

Compound Muscle

(Flank)

Fat (Omental) Liver

(Replicate 1)

Kidney

(Replicate 1)

TRRs = 0.013 ppm TRRs = 0.011 ppm TRRs = 1.080 ppm TRRs = 0.177 ppm

% TRR ppm % TRR ppm % TRR ppm % TRR ppm

Carboxylic Acid 

Metabolite

29.1 0.052

Oxime isomer

3-hydroxy isoxaben

Hydroxylated isoxaben 

61.2 0.661

Oxime isomer

3-hydroxy isoxaben

Hydroxylated isoxaben 

Carboxylic acid metabolite

52.0 0.007 25.5 0.003

Unknown (RT=5.0) 0.1 0.001 0.2 <0.001

Unknown (RT=10.0) 0.1 <0.001

Unknown (RT=20.5) 8.5 0.001 24.5 0.003

Unknown (RT=21.5) 8.1 0.087 1.7 0.003

Acidified aqueous ACN 0.6 0.006 0.7 0.001

Pepsin Labile 1.8 0.019 2.8 0.005

Many components <LOQ 18.4 0.003 25.5 0.002 3.7 0.040 10.2 0.017

Total identified 11.8 0.001 22.8 0.003 20.2 0.22 75.4 0.136

Total characterized 78.9 0.011 75.5 0.008 75.5 0.814 15.7 0.026

Total extractable 90.7 0.012 98.3 0.011 95.7 1.034 91.1 0.162

Unextractable (PES)19.3 0.001 1.7 <0.001 4.2 0.045 6.9 0.012

Accountability2100 100 ~100 98

1

Residues remaining after exhaustive extractions.

2

Accountability = (Total extractable + Total unextractable)/(TRRs from combustion analysis) * 100.
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 22 of 38

860.1340 Residue Analytical Methods

DER Reference: 47492601.der.doc Residue Analytical Method

47140007.der.doc Residue Analytical Method and ILV

Enforcement method: Dow AgroSciences has submitted a study of a proposed residue analytical 

method (RAM) utilizing liquid chromatography with tandem mass spectrometric detection 

(LC/MS/MS), GRM 02.26.S.1 (a revision of GRM 02.26). Dow has proposed this method to 

enforce tolerances for residues of isoxaben in or on various crop commodities. The method, 

entitled Determination of Residues of Isoxaben in Cereals, Chicory, Endives, Almonds, Pecans, 

and Grapes by Liquid Chromatography with Tandem Mass Spectrometry, was used for 

supplementary method validation generated during analysis of samples from almond, pecan and 

grape field trial studies associated with DP Barcode D342083. 

Briefly, residues of isoxaben are extracted from crop samples with acetonitrile (ACN)/water (7:3, 

v:v). An aliquot of the extract is diluted with water, and subjected to liquid-liquid extraction on 

a diatomaceous earth column, using dichloromethane to elute the residues. The eluate is 

evaporated to dryness, redissolved in methanol/water (1:1, v:v) containing 5 mM ammonium 

acetate, and analyzed by LC/MS/MS. The validated limit of quantitation (LOQ) is 0.010 ppm for 

isoxaben in each matrix. The limits of detection (LODs) were calculated and reported as ranging 

from 0.0008 ppm (in endives) to 0.0029 ppm (in cereal straw). 

The method includes instructions for the analysis of samples of cereal, chicory, endive, almond, 

pecan and grape commodities. 

The method was adequately validated by the petitioner using fortified samples of barley (forage, 

grain and straw), wheat (forage, grain and straw), chicory (roots and foliage), whole endives, 

almonds (nutmeat and hulls), pecans (nutmeat), and grapes (fruit, juice and raisins). Overall, 

recoveries of isoxaben ranged 63-120% from samples fortified at 0.010 ppm (LOQ), 0.10 ppm, 

0.50 ppm, and 1.00 ppm. 

The revised method, as written, monitors three MS/MS ion transitions for isoxaben (m/z 

333→165 for quantitation, m/z 333→150 for confirmation, and m/z 333→107 for confirmation), 

as shown on page 38 of the report. However, it was noted that the third product ion in the table 

in Section 8.2 (page 19 of the report) is listed as m/z 150 instead of m/z 107. BEAD’s Analytical 

Chemistry Branch (ACB) currently recommends that enforcement methods include information 

for two MS/MS ion transitions per analyte in order to be considered sufficiently specific not to 

require a confirmatory method, so the current revised method exceeds this requirement. 

No radiovalidation data were submitted for method GRM 02.26.S.1. Because the extraction 

solvents used for this method are very similar to those used in the barley and grape metabolism 

studies associated with D342083, HED concludes that radiovalidation of the method will not be 

required at this time. 

An independent laboratory validation (ILV) was not submitted for the enforcement method GRM 

02.26.S.1. However, an ILV study was submitted for the initial Method GRM 02.26 with wheat 

grain and endive leaves fortified with isoxaben at 0.01 ppm, 0.02 ppm, and 0.10 ppm. Adequate 

recoveries were obtained for both commodities using the method as written.
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 23 of 38

Data collection: The related method, LC/MS/MS method GRM 02.26, was used for data 

collection in samples of grape and tree nut commodities from the crop field trial and processing 

studies associated with this petition.

Briefly, residues of isoxaben are extracted from crop samples with acetonitrile (ACN):water 

(70:30, v:v). An aliquot of the extract is diluted with water and subjected to liquid-liquid 

extraction on a diatomaceous column, using dichloromethane to elute the residues. The eluate is 

evaporated to dryness, redissolved in methanol:water (50:50, v:v) containing 5 mM ammonium 

acetate, and analyzed by LC/MS/MS. The validated limit of quantitation (LOQ) is 0.01 ppm for 

isoxaben in each matrix. The LODs were calculated and reported as ranging from 0.00016 ppm 

for endive to 0.0029 ppm for cereal straw.

The method, as written, includes instructions for the analysis of samples of cereals (wheat and 

barley), chicory (roots and leaves) and whole endives.

The method was adequately validated using fortified samples of barley forage, barley grain, 

barley straw, chicory root, chicory foliage, whole endive, wheat forage, wheat grain, and wheat 

straw. Overall, recoveries of isoxaben ranged 71-108%, with an average of 91% (standard 

deviation of 6.5%), from samples fortified at 0.01 ppm (LOQ), 0.10 ppm, and 1.0 ppm. 

The commodities of grapes and tree nuts, in which tolerances have been proposed, were not 

included in the validation study. Concurrent method recovery data for these commodities were 

included with the crop field trial and processing studies submitted in conjunction with DP

Barcode D342083. These concurrent method validation data are sufficiently representative of the 

expected residue levels in the plant commodities.

As stated above, an independent laboratory validation (ILV) study was submitted for Method 

GRM 02.26 (the grape and tree nut data collection method) with wheat grain and endive leaves 

fortified with isoxaben at 0.01 ppm, 0.02 ppm, and 0.10 ppm. Adequate recoveries were 

obtained for both commodities using the method as written.

As with Method GRM 02.26.S.1, no confirmatory analysis procedures were included in the 

method, and no interference study was submitted. The petitioner stated that because of the 

specificity of the LC/MS/MS analyses, no further confirmation was needed. The method as 

written only monitors one transition ion for isoxaben. ACB currently recommends that 

enforcement methods include information for two MS/MS ion transitions per analyte in order to 

be considered sufficiently specific not to require a confirmatory method. When two transitions 

are not available, ACB recommends providing an alternate chromatographic column and/or 

mobile-phase combination to reduce the possibility of false positive residues.

As with Method GRM 02.26.S.1, no radiovalidation data were submitted for method GRM 

02.26. Because the extraction solvents used for the data collection method are very similar to 

those used in the barley and grape metabolism studies associated with D342083, HED concludes 

that radiovalidation of the method will not be required at this time.

Conclusions. The plant commodity analytical method data are adequate to satisfy data 

requirements for both data collection and tolerance enforcement. The petitioner did not submit 
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 24 of 38

an ILV study for the proposed enforcement method using a crop commodity associated with the 

proposed uses. The submitted ILV for Method GRM 02.26 was submitted with wheat grain and 

endive leaves fortified with isoxaben at 0.01 ppm, 0.02 ppm, and 0.10 ppm. Adequate recoveries 

were obtained for both commodities using the method. As a result, an ILV for the proposed 

enforcement method, GRM 02.26.S.1, will not be required. HED notes that the OPPTS 

Guideline 860.1340 specifies that the commodity chosen for ILV should be the commodity the 

petitioner has had the most difficulty analyzing.

Based on the review of the submitted livestock metabolism data, HED concludes that an 

analytical method for livestock commodities is not required to support the proposed uses under 

this petition. HED concludes that residues in milk, ruminant and poultry meat, fat, and meat 

byproducts and eggs remain a 40CFR §180.6[a][3] situation (there is no expectation of finite 

residues in these commodities). However, analytical methods for livestock commodities may be 

needed for any additional uses of isoxaben that result in significantly higher dietary burdens. 

860.1360 Multiresidue Methods

No multiresidue method testing data were submitted with this petition. The FDA PESTDATA 

database (dated 06/05) indicates that isoxaben is completely recovered using multiresidue 

method Section 302. The database did not include any information for any of the other test 

methods. 

860.1380 Storage Stability

DER References: 474621901.der.doc 

The storage durations and conditions of samples from the crop field trials submitted to support 

this petition are presented in Table 11.

Table 11. Summary of Storage Conditions/Durations of Samples from Crop Field Trial and Processing 

Studies.

Matrix Storage 

Temperature

(°C)

Actual Storage Duration Interval of Demonstrated Storage 

Stability

Almond, hulls -20 144-186 days

(4.7-6.1 months)

Stable under frozen storage conditions 

(≤-20°C) for up to 24 months in/on 

grape and cereal matrices, and for up to 

12 months in/on endive leaves.

Almond, nutmeat 149-191 days

(4.9-6.3 months)

Grape (field trials) 106-184 days

(3.5-6.1 months)

Grape (processing study) 128 days

(4.2 months)

Raisins 120 days

(3.9 months)

Juice 124 days

(4.1 months)

Pecan, nutmeat 77-110 days

(2.5-3.6 months)

Dow AgroSciences submitted a storage stability study with isoxaben on grapes, cereal grain,
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 25 of 38

cereal straw, and endive leaves. Untreated samples for all matrices were fortified with isoxaben 

at 0.1 ppm, and then stored frozen (≤-20°C). Subsamples of fortified samples were collected at 

3-, 6-, 12-, 18- and 24-month storage intervals and analyzed for residues of isoxaben using high 

pressure liquid chromatography with positive ion atmospheric pressure chemical ionization mass 

spectrometry (LC/MS). In the submitted storage stability data for isoxaben, residues were

relatively stable under frozen storage conditions (≤-20°C) for up to 24 months in/on grape and 

cereal matrices, and for up to 12 months in/on endive leaves; see Table 12.

TABLE 12. Stability of Isoxaben Residues in Grape, Cereal Grain, Cereal Straw and Endive Leaves 

following Storage at ≤-20°C.

Matrix Spike Level 

(ppm)

Storage Interval 

(days)

Sample Size (n) Mean Recovered 

Residue

(%)

Mean Corrected Recovery

(%)

Grape 0.1 90 3 0.095 113

180 3 0.085 103

365 3 0.079 103

552 3 0.090 114

720 3 0.080 108

Cereal Grain 0.1 85 3 0.097 99.5

179 3 0.100 105

367 3 0.092 100

575 3 0.096 107

725 3 0.091 99.2

Cereal Straw 0.1 85 3 0.088 96.1

179 3 0.096 99.7

367 3 0.087 93.8

575 3 0.094 111

725 3 0.084 98.8

Endive Leaves 0.1 90 3 0.106 112

181 3 0.105 102

365 3 0.104 101

Conclusions. The available storage stability data indicate that residues of isoxaben are stable 

under frozen storage conditions for up to 24 months in/on grapes, cereal grain and cereal straw, 

and for up to 12 months in/on endive leaves.

860.1480 Meat, Milk, Poultry, and Eggs

The only livestock feedstuff associated with the proposed use is almond hulls. Almond hulls are 

not fed to poultry or swine. The worst case dietary burden of isoxaben to beef and dairy cattle is 

presented in Table 13. 

Table 13. Calculation of Dietary Burdens of Isoxaben Residues to Livestock.

Feedstuff Type1 % Dry

Matter2 % Diet2

Recommended 

Tolerance 

(ppm)

Dietary

Contribution (ppm)3

Beef and Dairy Cattle

Almond, hulls R 90 10 0.40 0.044
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 26 of 38

Table 13. Calculation of Dietary Burdens of Isoxaben Residues to Livestock.

Feedstuff Type1 % Dry

Matter2 % Diet2

Recommended 

Tolerance 

(ppm)

Dietary

Contribution (ppm)3

Other R, not treated R -- 20 N/A N/A

CC, not treated CC -- 55 N/A N/A

PC, not treated PC -- 15 N/A N/A

TOTAL BURDEN -- -- 100 -- 0.044

1

 R: Roughage; CC: Carbohydrate concentrate; PC: Protein concentrate.

2

 OPPTS 860.1000 Table 1 Feedstuffs (June 2008). Rarely fed at up to 10%, 5% would reflect a more typical

 feeding level.

3

 Contribution = ([tolerance /% DM] X % diet).

No livestock feeding studies were submitted with this petition. The petitioner has requested a 

waiver from the requirement for a tolerance for isoxaben in or on cattle meat byproducts, meat, 

and milk.

Conclusions. A livestock metabolism study has been submitted to support the proposed uses. 

There are no poultry or swine feed items associated with the proposed uses. HED’s review of 

this goat metabolism study indicates that a cattle feeding study, and tolerances for livestock 

commodities are not needed for the proposed uses currently under consideration. Although the 

goat metabolism study identified low percentages of the total radioactive residues in most 

matrices, it is adequate for the proposed uses, based on the very low dietary burden for cattle. 

Another goat metabolism study with better identification and characterization of the residues will 

be needed to support any additional uses of isoxaben that result in significantly higher dietary

burdens. HED concludes that residues in milk, ruminant and poultry meat, fat, and meat 

byproducts and eggs remain a 40CFR §180.6[a][3] situation (i.e., no expectation of finite 

residues in these commodities).

860.1500 Crop Field Trials

DER Reference: 47140008.der.doc (Almond)

47140009.der.doc (Pecan)

47040010.de1.doc (Grape)

To support the proposed uses on grapes and tree nuts, Dow AgroSciences has submitted crop 

field trial data for grapes, almonds, and pecans. The submitted data are discussed below and 

summarized in Table 14.
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 27 of 38

Table 14. Summary of Residue Data from Crop Field Trials with Isoxaben.

Crop 

matrix

Treat. 

Plot No.1

Total Applic. 

Rate

(lb ai/A)

PHI (days) Residue Levels2

(ppm)

n Min. Max. HAFT3 Median Mean Std. 

Dev.4

GRAPE (proposed use = 1.0 lb ai/A total application rate, PHI = 165 days)

Grape 2 1.0 141-182 24 <0.01 <0.01 0.01 0.01 0.01 NA

3 1.0 60 24 <0.01 <0.01 0.01 0.01 0.01 NA

TREE NUT (proposed use = 1.0 lb ai/A total application rate, PHI = 60 days)

Almond, 

hulls

2 1.0 182-216 10 <0.01 <0.01 0.01 0.01 0.01 NA

3 1.0-1.1 60-61; 68510 <0.01 0.301 0.267 0.039 0.085 0.103

Almond, 

nutmeat

2 1.0 182-216 10 <0.01 <0.01 0.01 0.01 0.01 NA

3 1.0-1.1 60-61; 68510 <0.01 0.015 0.015 0.01 0.011 0.002

Pecan, 

nutmeat

2 0.7, 1.06219-243 10 <0.01 <0.01 0.01 0.01 0.01 NA

3 1.0 57-60 10 <0.01 <0.01 0.01 0.01 0.01 NA

1 Treatment Plot No. 2: single application made just prior to bud break; Treatment Plot No. 3: single application 

made ~60 days prior to harvest.

2

 For the calculation of the HAFT, median, mean, and standard deviation values, the LOQ (0.01 ppm) was used for 

residues reported as <LOQ.

3

 HAFT = Highest Average Field Trial

4

 NA = Not Applicable

5

 In the residue decline trial for Treatment Plot No. 3, residues in hulls and nutmeats were highest in samples from 

the 68-day sampling interval. Therefore, these samples were included in the summary instead of the results from the 

61-day sampling interval.

6

 The application rate was 1.0 lb ai/A at four of the trial sites and 0.7 lb ai/A at one site.

Grape

Dow AgroSciences submitted field trial data for isoxaben on grapes. Twelve trials were 

conducted in Zones 1 (NY; 2 trials), 10 (CA; 8 trials), and 11 (WA; 2 trials) during the 2006 

growing season. 

Each test site included one control plot and two treated plots. Each treated plot received a single 

broadcast application of a 75% DF formulation of isoxaben to the soil at 1.0 lb ai/A. The 

application rate represents 1x the maximum proposed single application rate and 1x the 

maximum proposed seasonal rate to grapes. For one plot (at each site), designated as Treatment 

Plot No. 2, the application was made just prior to bud break. For the other plot, designated as 

Treatment Plot No. 3, the application was made 60 days prior to harvest. Applications were 

made using ground equipment in 23-29 gal/A spray volumes without an adjuvant.

Samples of whole grapes were collected 141-182 days after application from Treatment Plot No. 

2 and 60 days after application from Treatment Plot No. 3. At one trial, additional samples were 

collected at 134, 141, 148, and 155 days after treatment (Treatment Plot No. 2) and 53, 60, 67, 

and 74 days after treatment (Treatment Plot No. 3) for evaluation of residue decline.

Samples of grapes were analyzed for residues of isoxaben using an adequate LC/MS/MS method, 

GRM 02.26, with a validated LOQ of 0.01 ppm. Samples of grapes were stored frozen (<-20C) 

for 106-184 days (3.5-6.1 months) prior to extraction for analysis. Adequate supporting storage 
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 28 of 38

stability data are available for grapes. These data indicate that isoxaben is stable in frozen 

storage for up to 2 years. 

Residues of isoxaben were below the LOQ of 0.01 ppm in/on all grape samples from Treatment 

Plot Nos. 2 and 3. Because isoxaben residues were below the method LOQ in/on all samples of 

grapes from Treatment Plot Nos. 2 and 3, the potential for residue decline could not be assessed.

Tree nut, group 14

Dow AgroSciences submitted crop field trial data for almond and pecan, the representative 

commodities of the tree nut crop group.

Almond: Dow AgroSciences submitted field trial data for isoxaben on almonds. Five field trials 

were conducted in Zone 10 (CA) during the 2006 growing season. 

Each test site included one control plot and two treated plots. Each treated plot received a single 

broadcast application of a 75% DF formulation of isoxaben to the soil at 1.0-1.1 lb ai/A. The 

application rate represents ~1x the maximum proposed single application rate and ~1x the 

maximum proposed seasonal rate to tree nuts. For one plot, designated as Treatment Plot No. 2, 

the application was made just prior to bud break. For the other plot, designated as Treatment 

Plot No. 3, the application was made 60-61 days prior to harvest. Applications were made using 

ground equipment in 22-26 gal/A spray volumes without an adjuvant.

Samples of almonds (hulls and nutmeat) were collected 182-216 days after application from 

Treatment Plot No. 2 and 60-61 days after application from Treatment Plot 3. At one trial, 

additional samples were collected at 175, 182, 189, and 196 days after treatment (Treatment Plot 

No. 2) and 54, 61, 68, and 75 days after treatment (Treatment Plot No. 3) to evaluate residue 

decline.

Samples of almond hulls and nutmeat were analyzed for residues of isoxaben using an adequate 

LC/MS/MS method, GRM 02.26, with a validated LOQ of 0.01 ppm. Samples were stored 

frozen (<-20C) prior to extraction for analysis, for a maximum of 186 days (4.7-6.1 months) for 

almond hulls and a maximum of 191 days (4.9-6.3 months) for almond nutmeat. No supporting 

storage stability data are available for almond commodities. 

Residues of isoxaben were <LOQ in/on all almond nutmeat samples from Treatment Plot Nos. 2 

and 3, with the exception of two samples collected from Treatment Plot No. 3 in the decline trial 

(sampling interval of 68 days) which bore residues of 0.014 and 0.015 ppm. Residues of 

isoxaben were below the LOQ in/on all samples of almond hulls from Treatment Plot No. 2, 

including samples from the decline trials. Residues of isoxaben were <0.01-0.301 ppm in/on 

almond hull samples (including samples from decline trials) from Treatment Plot No. 3. 

In the decline trial for Treatment Plot No. 2, isoxaben residues were below the LOQ in/on all 

samples of almond nutmeat and hulls. In the decline trial from Treatment Plot No. 3, residues of 

isoxaben were <LOQ in/on all samples of nutmeat except those harvested at the 68-day sampling 

interval (as noted above). For almond hulls from Treatment Plot No. 3, residues of isoxaben 

increased from the 54-day sampling interval (average of 0.103 ppm) to the 68-day sampling 
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 29 of 38

interval (average of 0.267 ppm) and then decreased at the 75-day sampling interval (average of 

0.123 ppm). 

Pecan: Dow AgroSciences has submitted field trial data for isoxaben on pecans. Five field trials 

were conducted in Zones 2 (GA; 2 trials), 4 (LA; 1 trial), 6 (TX; 1 trial), and 8 (TX; 1 trial) 

during the 2006 growing season. 

Each test site included one control plot and two treated plots. Each treated plot received a single 

broadcast application of a 75% DF formulation of isoxaben to the soil at 1.0 lb ai/A. The 

application rate represents ~1x the maximum proposed single application rate and ~1x the 

maximum proposed seasonal rate to tree nuts. For one plot, designated as Treatment Plot No. 2, 

the application was made just prior to bud break. For the other plot, designated as Treatment 

Plot No. 3, the application was made 57-60 days prior to harvest. Applications were made using 

ground equipment in 18-30 gal/A spray volumes without an adjuvant. At one site (LA), 

application to Treatment Plot No. 2 was made at 0.7 lb ai/A due to a calculation error. 

Samples of pecans were collected 219-243 days after application from Treatment Plot No. 2 and 

57-60 days after application from Treatment Plot 3. At one trial, additional samples were 

collected at 213, 220, 227, and 234 days after treatment (Treatment Plot No. 2) and 53, 60, 67, 

and 74 days after treatment (Treatment Plot No. 3) to evaluate residue decline.

Samples of pecan nutmeat were analyzed for residues of isoxaben using an adequate LC/MS/MS 

method, GRM 02.26, with a validated LOQ of 0.01 ppm. Samples of pecan nutmeat were stored 

frozen (<-20 C) for 77-110 days (2.5-3.6 months) prior to extraction for analysis. No supporting 

storage stability data are available for pecans.

Residues of isoxaben were <LOQ in/on all pecan nutmeat samples from Treatment Plot Nos. 2 

and 3. Because isoxaben residues were below the method LOQ in/on all samples of pecan 

nutmeat from Treatment Plot Nos. 2 and 3, the potential for residue decline could not be 

assessed.

Conclusions. The submitted field trial data are adequate to satisfy data requirements. Supporting 

storage stability data, while not available for tree nuts, are available for other commodities and 

indicate that isoxaben is stable in frozen storage for up to 2 years. The number and locations of 

field trials are in accordance with OPPTS Residue Chemistry Test Guideline 860.1500 for grapes 

and tree nuts, and samples were analyzed using an adequate method.

The available data support tolerances for residues of isoxaben in/on grapes at 0.01 ppm, the tree 

nut crop group at 0.02 ppm, pistachio at 0.02 ppm, and almond hulls at 0.40 ppm. 

HED notes that the crop field trials did not include the addition of adjuvants to the spray

mixtures. The proposed use of isoxaben involves application to the weeds growing on the 

orchard floor such that very little contact of the crop foliage by spray droplets will occur. As a 

result, adjuvants are not expected to have a significant impact on residues of isoxaben in tree nuts 

or grapes and a prohibition of adjuvants is not needed on the product label.
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 30 of 38

860.1520 Processed Food and Feed

DER Reference: 47140010.de2.doc

Processing data for grapes are required to support the proposed uses; no processing data for tree 

nuts are required.

Dow AgroSciences submitted a processing study with isoxaben on grapes. Samples used for 

processing were generated from one trial conducted in NY during the 2006 growing season. 

Grapes were harvested 60 days following a single broadcast application of the 75% DF 

formulation of isoxaben to the soil at 5.0 lb ai/A (5x the field trial rate and 5x the proposed 

maximum seasonal rate). Application was made using ground equipment in 25 gal/A spray

volumes without an adjuvant. Whole grapes were processed into raisins and juice using 

simulated commercial processes.

Samples of grapes (whole fruit) and the processed commodities (raisins and juice) were analyzed 

for residues of isoxaben using an adequate LC/MS/MS method, GRM 02.26, with a validated 

LOQ of 0.01 ppm. Samples were stored frozen (<-20C) prior to extraction for analysis, for a 

maximum of 128 days (4.2 months) for grape, 120 days (3.9 months) for raisins, and 124 days 

(4.1 months) for grape juice. Supporting storage stability data are available for grapes, and 

indicate that isoxaben is stable in frozen storage for up to 2 years. 

Following a single broadcast application of the 75% DF formulation of isoxaben at 5.0 lb ai/A, 

all residues were below the LOQ in/on all samples of grapes, raisins, and grape juice; therefore, 

processing factors could not be calculated.

The theoretical concentration factors are 1.2x for grape juice (based on separation into 

components; OPPTS 860.1520, Table 3) and 4.7x for raisins (based on loss of water; OPPTS 

860.1520, Table 2).

Conclusions. The submitted processing data are adequate to satisfy data requirements. The data 

indicate that no tolerances are needed for the processed commodities of grapes.

860.1650 Submittal of Analytical Reference Standards

An analytical standard for isoxaben is currently available in the EPA National Pesticide 

Standards Repository with a expiration date of March 2012 (personal communication with 

Theresa M. Cole, ACB, 4/16/08). 

860.1850 and 860.1900 Confined and Field Accumulation in Rotational Crops

Because grapes and tree nuts are not rotated, no data pertaining to rotational crops are required to 

support the proposed use.

860.1550 Proposed Tolerances

The petitioner has proposed that the tolerance expression for isoxaben consist of residues of 

parent isoxaben only. For the purposes of this petition only, the nature of the residue in grapes 
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 31 of 38

and tree nuts is adequately understood (for early season uses only) based on grape and barley

metabolism studies.

HED recommends the isoxaben tolerance expression be established as follows: “Tolerances are 

established for residues of the herbicide isoxaben, including its metabolites and degradates, in or 

on the commodities in the table below. Compliance with the tolerance levels specified below is 

to be determined by measuring only isoxaben [N-[3-(1-ethyl-1-methylpropyl)-5-isoxazolyl]-2, 6-

dimethoxybenzamide] in or on the commodity.”

No Codex, Canadian, or Mexican MRLs have been established for isoxaben. 

A tolerance summary for isoxaben is presented in Table 15. 

The available crop field trial data support tolerances for isoxaben residues in/on grape, the tree 

nut crop group, pistachio, and almond hulls. For all commodities except almond hulls, residues 

were below the LOQ in/on ≥80% of samples; therefore, the tolerance spreadsheet in the 

Agency’s Guidance for Setting Pesticide Tolerances Based on Field Trial Data was not used to 

calculate tolerances for these commodities. 

The tolerance for grape should be set at the LOQ, 0.01 ppm. The maximum residues observed in 

almond and pecan were 0.015 ppm; therefore, the proposed tolerance of 0.03 ppm for the tree nut 

group is too high. A tolerance of 0.02 ppm would be appropriate for the tree nut crop group.

HED has determined that pistachio should be added to the tree nut crop group; almond and pecan 

will remain the representative commodities for this group. Until the crop is officially added to 

the crop group listings in 40 CFR, residue data for almond and pecan may be used to establish a 

tolerance for pistachio, which is to be set at the same level as the tree nut tolerance. Therefore, 

the proposed tolerance of 0.03 ppm for pistachio is too high; a tolerance of 0.02 ppm would be 

appropriate. 

The tolerance calculation for almond hulls is presented in Appendix II. The available data 

indicate that a 0.40 ppm tolerance for almond hulls would be appropriate. 

The available processing data indicate that residues of isoxaben do not concentrate in grape juice 

or raisins. Therefore, no tolerances are needed for grape processed commodities. The proposed 

tolerances should be removed from Section F. 

Although the goat metabolism study identified low percentages of the total radioactive residues 

in most matrices, it is adequate for the proposed uses based on the very low dietary burden for 

cattle. There are no poultry or swine feed items associated with the proposed uses. HED’s

review of the livestock metabolism study indicates that a cattle feeding study and tolerances for 

livestock commodities are not needed. Another goat metabolism study with better identification 

and characterization of the residues will be needed for any additional uses of isoxaben that result 

in significantly higher dietary burdens. HED concludes that residues in milk, ruminant and 

poultry meat, fat, and meat byproducts and eggs remain a 40CFR §180.6[a][3] situation (there is 

no expectation of finite residues in these commodities). Therefore, no tolerances are needed for 

livestock commodities. 
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 32 of 38

Table 15. Tolerance Summary for Isoxaben. 

Commodity Proposed Tolerance 

(ppm)

Recommended 

Tolerance (ppm)1

Comments; Correct Commodity 

Definition

Grape 0.01 0.01

Grape, juice 0.01 Remove A separate tolerance for grape juice 

is not needed.

Grape, raisin 0.01 Remove A separate tolerance for raisin is not 

needed.

Nut, tree, Group 14 0.03 0.02 Nut, tree, Group 14

Pistachio 0.03 0.02

Almond, hulls 0.35 0.40

1

 The recommended tolerance levels are based on the current crop field trial data and assume that the petitioner will 

modify the proposed uses to correspond to the use patterns of the crop field trials.

References

European Food Safety Authority (EFSA), May 2006 Draft Report

 PP #5G3216. EL-107® on wheat and barley. Comments on the analytical method and residue 

data. RCB #591, J. Worthington, 5/17/85. MRIDs: 073291 and 072296

 PP #5G3216. EL-107 on wheat and barley. Comments on the amendment of 6/17/85.

 RCB #1188, J. Worthington, 7/26/85. MRID: 073648

Attachments: 

International Residue Limit Status sheet

Appendix I - Chemical Name and Structure Table

Appendix II - Tolerance Assessment Calculations
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 33 of 38

INTERNATIONAL RESIDUE LIMIT STATUS

Chemical Name: N-[3-(1-

ethyl-1-methylpropyl)-5-

isoxazolyl]-2,6-

dimethoxybenzamide

Common Name:

Isoxaben

X Proposed tolerance

[ ] Reevaluated tolerance

[ ] Other

Date: 7/12/10

Codex Status (Maximum Residue Limits) U. S. Tolerances

X No Codex proposal step 6 or above

[ ] No Codex proposal step 6 or above for the crops 

requested

Petition Number: PP#7E7222

DP#s: 341694, 342083

Other Identifier: 

Residue definition (step 8/CXL): N/A Reviewer/Branch: D. McNeilly/RAB2

Residue definition: Isoxaben

Crop (s) MRL (mg/kg) Crop(s) Tolerance (ppm)

Grape 0.01

Grape, juice N/A

Grape, raisin N/A

Nut, tree, group 0.03

Pistachio 0.03

Almond, hulls 0.40

Limits for Canada Limits for Mexico

X No Limits

[ ] No Limits for the crops requested

X No Limits

[ ] No Limits for the crops requested

Residue definition: N/A Residue definition: N/A

Crop(s) MRL (mg/kg) Crop(s) MRL (mg/kg)

Notes/Special Instructions: S.Funk, 08/16/2007
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 34 of 38

APPENDIX I. Chemical Names and Structures of Isoxaben and Metabolites. 

Common name;

Company code

Chemical name Chemical structure

Isoxaben N-[3-(1-ethyl-1-methylpropyl)-5-

isoxazolyl]-2,6-dimethoxybenzamide

OCH3

N

H

O

O

N

CH3

CH3

CH3

OCH3

Metabolite I12,6-dimethoxybenzoic acid OCH3

OCH3

OH

O

Isoxaben-2-hydroxy/ 

Metabolite II

(mixture of two 

diastereomers)

N-[3-(1-ethyl-2-hydroxy-1-

methylpropyl)-5-isoxazolyl]-2,6-

dimethoxybenzamide

OCH3

OCH3

N

H

O

O

N

CH3

CH3

CH3

OH

Isoxaben-3-hydroxy/ 

Metabolite III

N-[3-(3-hydroxy-1-ethyl-1-

methylpropyl)-5-isoxazolyl]-2,6-

dimethoxybenzamide

OCH3

OCH3

N

H

O

O

N

CH3

CH3

OH

Isoxaben-1-hydroxy/ 

Metabolite IV

N-[3-(1-hydroxy-1-methylpropyl)-5-

isoxazolyl]-2,6-dimethoxybenzamide

OCH3

N

H

O

O

N

HO CH3 CH3

OCH3
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 35 of 38

APPENDIX I. Chemical Names and Structures of Isoxaben and Metabolites. 

Common name;

Company code

Chemical name Chemical structure

2-OH-6-OMe benzamide 2-hydroxy-6-methoxy benzamide OCH3

NH2

O

OH

2,6-dimethoxy benzamide 2,6-dimethoxy benzamide OCH3

OCH3

NH2

O

Oxime metabolite N-[4-ethyl-4-methyl-3-nitroso-1-

hydroxy-1-hexenyl]-2,6-demethoxybenzamide

Carboxylic acid metabolite N-[1-carboxyethyl-1-methylpropyl]-

2,6-dimethoxybenzamide

Hydroxylated isoxaben2

1

 Suspected to be an artifact formed during acid hydrolysis.

2 The 3 possible variations of hydroxylated isoxaben are shown above – Isoxaben-1-hydroxy, Isoxaben-2-hydroxy, 

and Isoxaben-3-hydroxy. Generic version of hydroxylated isoxaben tentatively identified only in livestock 

metabolism study. 
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 36 of 38

Appendix II. Tolerance Assessment Calculations.

The dataset used to establish a tolerance for isoxaben on almond hulls consisted of field trial data 

representing a single application at 1.0 or 1.1 lbs ai/A with 60-61 day PHIs, except for the CA5 

test site, where the PHIs ranged from 54-75 days (decline study data). The application rate was

1x or 1.1x the proposed maximum seasonal use rate. A 60-Day PHI is proposed on the label. 

The residue values used to calculate the tolerance are provided in Table II-1.

The almond hulls dataset was entered into the tolerance spreadsheet. Visual inspection of the 

lognormal probability plot (Figure II-1) provided in the spreadsheet indicates that the dataset is 

not lognormal. The result from the approximate Shapiro-Francia test statistic (Figure II-2) 

indicate that the assumption of lognormality should be rejected. 

Since the data set is flagged as not being reasonable lognormal based on the conclusion of the 

approximate Shapiro-Francia test statistic, and a visual inspection of the lognormal probability

plot indicates the data set are not reasonable lognormal, the “Mean + 3SD method” was used to 

calculate the tolerance for the almond hull data set. As shown in Figure II-3, the recommended 

tolerance is 0.4 ppm.
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 37 of 38

Table II-1. Residue data used to calculate tolerance for isoxaben on almond hulls. 

Chemical: Isoxaben

Crop: Almond hulls (MLE)

PHI: 60-61 days (54-75 Days used for CA5 site)

App. Rate: 1 lb ai/A

MRID Citation: MRID 47140008

Residues of Isoxaben (ppm)

0.042

0.036

0.01 (LOQ)

0.01 (LOQ)

0.01 (LOQ)

0.01 (LOQ)

0.082

0.119

0.087

0.119

0.221 

0.229

0.301

0.232

0.156

0.089

Figure II- 1. Lognormal probability plot of isoxaben field trial data for almond hulls.

Lognormal Probability Plot

0.1 1 2 5 10 20 30 50 70 80 90 95 98 99 99.9

1.0000

0.5000

0.2500

0.1250

0.0625

0.0313

0.0156

0.0078

y = 1.2257x - 2.7523

R

2

 = 0.8845

Percentiles

Concentrations

EPA Isoxaben Almond Hulls 60-61 (54-75 for the CA5 site)
Isoxaben Summary of Analytical Chemistry and Residue Data DP Barcode: D340895

Page 38 of 38

Figure II- 3. Tolerance spreadsheet summary of isoxaben field trial data for almond hulls.

Regulator: EPA

Chemical: Isoxaben

Crop: Almond Hulls

PHI: 60-61 (54-75 for the CA5 site)

App. Rate:

Submitter:

n: 16

min: 0.01

max: 0.30

median: 0.09

average: 0.11

95th Percentile 99th Percentile 99.9th Percentile

0.30 0.35 0.40

(0.35) (0.45) (--)

0.50 1.2 3.5

(1.5) (5.0) (--)

0.45

0.40

0.50

0.8845

0.05 >= p-value > 0.01 : Reject lognormality assumption

Approximate 

Shapiro-Francia 

Normality Test 

EU Method I

Normal

UCLMedian95th

95/99 Rule

EU Method II

Distribution-Free

Mean+3SD