[Federal Register Volume 84, Number 57 (Monday, March 25, 2019)]
[Notices]
[Pages 11109-11110]
[FR Doc No: 2019-05652]

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DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration

[Docket No. FDA-2019-N-0795]

Antimicrobial Drugs Advisory Committee; Notice of Meeting;
Establishment of a Public Docket; Request for Comments

AGENCY: Food and Drug Administration, HHS.

ACTION: Notice; establishment of a public docket; request for comments.

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SUMMARY: The Food and Drug Administration (FDA) announces a forthcoming
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    Dated: March 20, 2019.
Lowell J. Schiller,
Acting Associate Commissioner for Policy.
[FR Doc. 2019-05652 Filed 3-22-19; 8:45 am]
 BILLING CODE 4164-01-P


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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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 Data               DP Barcode:  D340895