Document ID: EPA-HQ-OPP-2007-0504-0007
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2010-09-15T04:00Z

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.

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 non-food 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
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.  

  SEQ CHAPTER \h \r 1 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.

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

Table 1.		Isoxaben Nomenclature.

Compound	

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 10-8 Pa 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-1 cm-1

(max3 = 275 nm / 20800 mol-1 cm-1	EFSA1 (May 2006 Draft Report)

1-  European Food Safety Authority (EFSA)

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 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-dimethoxybenza
mide (isoxaben-2-hydroxy) and its diastereomer; (3)
N-[3-(1-ethyl-3-hydroxy-l-methylpropyl)-5-isoxazolyl)-2,6-dimethoxybenza
mide (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
[phenyl-14C]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.



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
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
phenyl-label 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)1	36.8	0.007	4.6	0.013	26.6	0.122

Accountability2	100.0	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
[Phenyl-14C]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

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)1	33.3	0.0040	4.5	0.010	24.7	0.069

Accountability2	109.1	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
[phenyl-14C]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,
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 phenyl-label
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 benzamide-bridge (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:H2O1	75	0.229	69	0.198

	Neutral Organic Fraction2	19	0.057	16	0.045

		2-OH-6-OMe benzamide (Rt ~21.2 min)	4.7 3	0.014 3	2.9	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 Fraction2	5	0.015	4	0.011

		Polars (Rt ≤ 5 min)	0.7	0.002	1.5	0.004

		Peak Rt ~27.0 min	3.7	0.011	2.4	0.007

	Aqueous Fraction 2	52	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	292	0.0872	324	0.092

	Acid hydrolysate	102	0.0312	14	0.039

		Organic fraction	105	0.0306	8	0.0236

		Aqueous fraction	45	0.0126	6	0.0176

Unextractable	112	0.032	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.

 ≤ 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)2	2.1	0.006	<0.1	0.002

Peak RT ~19.0 min (aqueous)2	11.3	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)3	11	0.032	14	0.041

Accountability4	96.1	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.

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;

metabolites were identified in fractions incorrectly (e.g.,
2-hydroxy-6-methoxy-benzamide 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
[phenyl-14C]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 [phenyl-14C]isoxaben
dosed goat.  (The goat dosed with [phenyl-14C]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
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 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.

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.

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

Accountability2	100	100	100	~100	~99

1.  Residues remaining after exhaustive extractions.  2.  Accountability
= (Total extractable + Total unextractable)/(TRRs from combustion
analysis) * 100.

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

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)1	7.7	0.001	2.9	<0.001	4.1	0.044	5.3	0.010	5.2	<0.001
3.8	<0.001

Accountability2	93	~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

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)1	9.3	0.001	1.7	<0.001	4.2	0.045	6.9	0.012

Accountability2	100	100	~100	98

1	Residues remaining after exhaustive extractions.

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

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.

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 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, 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

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.

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; 685	10	<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; 685	10	<0.01	0.015	0.015	0.01	0.011	0.002

Pecan, nutmeat	2	0.7, 1.06	219-243	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
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 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.

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.

  SEQ CHAPTER \h \r 1 860.1650 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 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.  



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

  SEQ CHAPTER \h \r 1 Attachments:  

International Residue Limit Status sheet

Appendix I - Chemical Name and Structure Table

Appendix II - Tolerance Assessment Calculations

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



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

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Isoxaben-2-hydroxy/ Metabolite II

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.  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.



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-   SEQ Figure_II- \* ARABIC  1 .  Lognormal probability plot
of isoxaben field trial data for almond hulls.

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

 

Isoxaben                        Summary of Analytical Chemistry and
Residue Data                             DP Barcode D340895             
                                                                        
                          

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Isoxaben                  Summary of Analytical Chemistry and Residue
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