Document ID: EPA-HQ-OPP-2006-0181-0010
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2006-11-29T05:00Z

SEQ CHAPTER \h \r 1 Primary Evaluator

Date:  14-SEP-2006

	George F. Kramer, Ph.D., Senior Chemist

Registration Action Branch (RAB1)

Health Effects Division (HED) (7509C)

	Approved by

Date:  14-SEP-2006

	P.V. Shah, Ph.D. Branch Senior Scientist

RAB1/HED (7509C)

	

  SEQ CHAPTER \h \r 1 This DER was originally prepared under contract by
Dynamac Corporation (1910 Sedwick Rd., Building 100, Durham, NC 27713;
submitted 06/23/2006).  The DER has been reviewed by HED and revised to
reflect current Office of Pesticide Programs (OPP) policies.

STUDY REPORT:

46609501.  Corley, J. (2005) Diflubenzuron:  Magnitude of the Residue on
Small Grain.  Lab Project Number:  08024.02-PTR01.  Unpublished study
prepared by Interregional Research Project No. 4 (IR-4).  800 p.

EXECUTIVE SUMMARY:

In a field trial conducted during the 2002 growing season in WA,
diflubenzuron (2 lb/gal flowable concentrate (FlC)) was applied to a
wheat crop (pre-booting growth stage) as one broadcast foliar
application at rates of 0.066 or 0.646 lb ai/A.  Single bulk samples of
control and treated wheat grain were harvested at commercial maturity,
83 days after treatment (DAT).  Wheat grain was processed into aspirated
grain, germ, bran, flour, shorts and middlings using simulated
commercial procedures.  Prior to analysis, wheat grain and processed
products were stored frozen for up to 90-113 days, and one wheat germ
sample was stored for up 321 days prior to analysis of diflubenzuron. 
The storage conditions and intervals of processed samples are partially
supported by adequate storage stability data.  The available storage
stability data for rice bran may be translated to wheat bran; however,
additional storage stability data for wheat flour, middlings, shorts,
and germ are required.

Samples of wheat grain and its processed commodities were analyzed for
residues of diflubenzuron, 4-chlorophenylurea (CPU), and 4-chloroaniline
(PCA), using high-performance liquid chromatography (HPLC)/ultraviolet
(UV), gas chromatography/mass spectroscopy (GC/MS), and GC/MS with
selected-ion monitoring (SIM), respectively.  These methods, which are
similar or based on method submissions previously deemed acceptable by
the Agency, were adequately validated in conjunction with the field
sample analyses.  The lowest limit of method validations (LLMVs) are
0.05 ppm for diflubenzuron and 0.005 ppm for CPU and PCA in all wheat
matrices.

The results show that following one application of the test formulation
at 0.066 lb ai/A (1x), individual residues of diflubenzuron, CPU, and
PCA were each below the respective LOQ in/on the raw agricultural
commodity (RAC) (wheat grain) for a combined total of <0.06 ppm.  The
combined residues were <0.06 ppm in shorts, middlings, flour, bran, and
germ, and were <2.104 ppm in aspirated grain fractions.  These data
indicate that the combined residues did not concentrate in all processed
fractions of wheat but did concentrate in aspirated grain fractions. 
The calculated processing factor for wheat aspirated grain fraction
following processing of 1x-treated wheat grain is >35.1x.

Following one application of the test formulation at 0.646 lb ai/A
(10x), individual residues of diflubenzuron, CPU, and PCA were 0.147
ppm, <0.005 ppm, and <0.005 ppm, respectively in/on the RAC (wheat
grain) for a combined total of <0.157 ppm.  The combined residues were
<0.085 ppm in shorts, <0.06 ppm in middlings, <0.06 ppm in flour, <0.104
ppm in bran, <0.061 ppm in germ, and <28.069 ppm in aspirated grain
fractions.  These data also indicate that the combined residues did not
concentrate in all processed fractions of wheat but did concentrate in
aspirated grain fractions.  The calculated processing factor for wheat
aspirated grain fraction following processing of 10x-treated wheat grain
is 180x.

STUDY/WAIVER ACCEPTABILITY/DEFICIENCIES/CLARIFICATIONS:

Under the conditions and parameters used in the study, the wheat
processing data are classified as scientifically acceptable, pending
submission of additional supporting storage stability data for wheat
processed commodities.  Storage stability data are required
demonstrating the stability of diflubenzuron, CPU, and PCA residues in
all wheat processed commodities, except bran, over the storage duration
of the samples from the subject processing study.  The acceptability of
this study for regulatory purposes is addressed in the forthcoming U.S.
EPA Residue Chemistry Summary Document, DP# 321623.

COMPLIANCE:

Signed and dated Good Laboratory Practice (GLP), Quality Assurance and
Data Confidentiality statements were provided.  The study authors noted
numerous minor deviations from GLP compliance at the trial site,
including the collection of weather data and descriptions of the field
plot.  However, these deviations do not impact the validity of the
study.

A.	BACKGROUND INFORMATION

Diflubenzuron is an insecticide/acaricide (insect growth regulator) that
behaves as a chitin inhibitor to suppress the growth of many leaf-eating
larvae, mosquito larvae, aquatic midges, rust mite, boll weevil, and
flies.  Diflubenzuron was first registered in the United States in 1979
for use as an insecticide.  The Agency issued a Registration Standard
for diflubenzuron in September, 1985, (NTIS #PB86-176500). 
Diflubenzuron was also the subject of a Residue Chemistry Chapter dated
11/16/84, an Addendum to the Registration Standard dated 12/4/84, and a
Reregistration Standard Update dated 6/21/91.  The Reregistration
Eligibility Decision (RED) for diflubenzuron was issued in August, 1997
(EPA 738-R-97-008).  Tolerances for residues of diflubenzuron are
established under 40 CFR §180.377.

TABLE A.1.	Diflubenzuron Nomenclature.

Compound	

Common Name	Diflubenzuron

Trade and other Names	Dimilin, Vigilante, Micromite, Adept

IUPAC Name	1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea

CAS Name	N-[[(4-chlorophenyl)amino]carbonyl]-2,6-difluorobenzamide

CAS Registry Number	35367-38-5

End-Use Product (EP)	2 lb/gal FlC formulation; DIMILIN® 2L (EPA Reg.
No. 400-461)

Regulated Metabolite	

Common name	4-chlorophenylurea (CPU)

Regulated Metabolite	

Common name	4-chloroaniline (PCA)

TABLE A.2.	Physicochemical Properties of Diflubenzuron.

Parameter	Value	Reference

Melting range	230-232 °C	  HYPERLINK
"http://www.arsusda.gov/acsl/services/ppdb/textfiles/DIFLUBENZURON" 
http://www.arsusda.gov/acsl/services/ppdb/textfiles/DIFLUBENZURON 

pH	Not available

	Density	Not available

	Water solubility (25°C)	0.08 ppm

	Solvent solubility (25°C) (ppm)	6.5 x 103     Acetone             

2 x 103        Acetonitrile        

2.4 x 104     Dioxane             

1.04 x 105   Dimethylformamide    

1.2 x 105     Dimethylsulfoxide   

1 x 103        Methanol            

6 x 102        Dichloromethane     

	Vapor pressure (25°C)	1.2 x 10- 4 mPa

	Dissociation constant, pKa	Not available

	Octanol/water partition coefficient, Log(KOW)	3.89

	UV/visible absorption spectrum	Not available

	

B.	EXPERIMENTAL DESIGN

B.1.	Application and Crop Information

TABLE B.1.1.	Trial Site Conditions.

Location:

City, State; Year (Trial ID)	

EP1	Application	

Tank Mix/ Adjuvants

Method; Timing	

Volume2	Single Rate 

(lb ai/A)	RTI3

(days)	Total Rate

(lb ai/A)

	Prosser, WA; 2002 (WA41)	2 lb/gal FlC	One broadcast foliar application:
 Pre-boot stage	13.66	0.066	NA	0.066	None

One broadcast foliar application:  Pre-boot stage	13.66	0.646	NA	0.646

	1 EP = End-use Product; DIMILIN® 2L

2 Gallons per acre 

3 RTI = Retreatment Interval; not applicable (NA) because a single
application was made at each treated plot.

B.2.	Sample Handling and Processing Procedures

Single bulk samples of control and treated wheat grain were harvested at
normal crop maturity, 83 DAT.  Samples were shipped frozen to the
processing facility (Texas A&M University, Bryan, TX), where samples
were processed into aspirated grain, germ, bran, flour, shorts and
middlings using simulated commercial procedures.  After processing,
samples were collected, placed in frozen storage, and shipped frozen to
the analytical laboratory (PTRL West, Inc., Hercules, CA) for analysis.

B.3.	Analytical Methodology

The wheat samples were analyzed for diflubenzuron, CPU, and PCA using a
separate PTRL method for each analyte.  The methods are described in
“Dimilin 25W, Dimilin 80WG, and Dimilin 2L in Almonds:  Magnitude of
Residue Study, J. Rose dated September 1999."

For analysis of diflubenzuron, samples are extracted twice with
acetonitrile (ACN) and centrifuged.  The supernatants are combined and
partitioned with hexane.  The ACN layer is concentrated to dryness,
redissolved in ACN and water, and sequentially cleaned up on a C18
solid-phase extraction (SPE) column and silica-gel SPE column.  After
clean up, residues are analyzed by HPLC on a C8 or C18 column with UV
detection.  The diflubenzuron limits of detection (LODs) for wheat grain
and bran were calculated to be 0.012 ppm and 0.023 ppm, respectively,
and the calculated limits of quantitation (LOQs) were 0.037 ppm and
0.070 ppm, respectively.  The LLMV for diflubenzuron was 0.05 ppm for
each commodity.

For analysis of CPU, samples are dried with sodium sulfate and extracted
with ethyl acetate.  Residues are evaporated to dryness, redissolved in
acetone and petroleum ether and cleaned up on a silica-gel SPE column. 
Again, residues are evaporated to dryness and redissolved in
acetonitrile.  The sample is filtered and derivatized in a glass tube
with heptafluorobutyric anhydride for 10 minutes.  Residues are then
analyzed by GC/MS.  For wheat grain, the LOD for CPU was calculated to
be 0.002 ppm and the calculated LOQ was 0.006 ppm.  The LLMV for CPU was
0.005 ppm for each commodity.

For analysis of PCA, residues are acidified with HCl and sonicated for
30 minutes at 60 °C.  NaOH and NaCl are added, and residues are
extracted three times with hexane.  Residues are then partitioned with
0.1 N HCl, neutralized, and extracted with hexane.  Extracts are dried
and cleaned up with a Florisil column, derivatized with
heptafluorobutyric acid for 10 minutes, after which water, sodium
carbonate and hexane are added.  Residues in the hexane layer are
analyzed by GC/MS with SIM.  The LOD for PCA in each wheat matrix was
calculated to be 0.001 ppm and the calculated LOQ was either 0.003 or
0.004 ppm.  The LLMV for PCA was 0.005 ppm for each commodity.

In conjunction with the analysis of processing samples, the above
methods were validated using control samples of wheat matrices fortified
with diflubenzuron, CPU or CPA at 0.005-0.5 ppm.

C.	RESULTS AND DISCUSSION

The analytical methods used to determine residues of diflubenzuron, CPU,
and PCA in/on wheat grain and its processed commodities were adequately
validated in conjunction with the sample analyses.  Method recoveries
from concurrent analysis of samples as well as from additional method
verification (see Table C.1) were generally within the acceptable range
of 70-120%.  Three low recoveries (58-68%) were obtained for
diflubenzuron in bran and germ, and for CPU in grain, but overall mean
recoveries were acceptable.  Apparent residues of diflubenzuron, CPU,
and PCA were each <LOQ in/on the untreated samples of wheat grain,
shorts, middlings, flour, bran, germ, and aspirated grain fractions. 
Adequate sample calculations and example chromatograms were provided.

Prior to analysis, wheat grain and processed products were stored frozen
for up to 90-113 days, and one wheat germ sample was stored 321 days
prior to analysis for diflubenzuron (Table C.2.1).  A concurrent freezer
storage stability study was conducted with the associated wheat field
trials to validate sample storage conditions and intervals of the RAC
(wheat grain).  The results (Table C.2.2) show that residues of
diflubenzuron are reasonably stable in/on frozen wheat grain (average
corrected stored recovery of 105%) for up to 296 days.  Residues of CPU
were also found to be stable in frozen wheat grain (average corrected
stored recovery of 85%) for up to 348 days.  However, residues of PCA
were unstable (average corrected stored recovery of 32%) after 293 days
of storage.  These data support the storage conditions of samples of
wheat grain and aspirated grain fractions from the wheat processing
study.

No supporting storage stability data were conducted for the wheat
processed commodities.  The available storage stability data (DP#
244487, G. Kramer, 2/17/1999) for processed rice bran indicate that  
SEQ CHAPTER \h \r 1 diflubenzuron and CPU are stable over a ~12-month
period, but that PCA is unstable in rice bran, degrading significantly
after 1 month.  These data may be translated to wheat bran.

The results suggest that residues of PCA should be corrected in order to
determine the residue levels that were present at the time of sample
collection.  However, HED has determined that correction of PCA residues
for degradation during storage would not have a significant effect on
the results of this processing study because individual residues of PCA
in/on wheat grain and bran were all below <0.005 ppm. 

The results of the wheat processing study show that following one
application of the test formulation at 0.066 lb ai/A (1x), individual
residues of diflubenzuron, CPU, and PCA were each below the respective
LLMV in/on the RAC (wheat grain) for a combined total of <0.06 ppm. 
When the 1x-treated samples of wheat grain were processesed, the
combined residues were <0.06 ppm in shorts, middlings, flour, bran, and
germ and were <2.104 ppm in aspirated grain fractions.  These data
indicate that the combined residues did not concentrate in all processed
fractions of wheat but did concentrate in aspirated grain fractions. 
The calculated processing factor for wheat aspirated grain fraction
following processing of 1x-treated wheat grain is >35.1x.

Following one application of the test formulation at 0.646 lb ai/A (1x),
individual residues of diflubenzuron, CPU, and PCA were 0.147 ppm,
<0.005 ppm, and <0.005 ppm, respectively in/on the RAC (wheat grain) for
a combined total of <0.157 ppm.  When the 10x-treated samples of wheat
grain were processesed, the combined residues were <0.085 ppm in shorts,
<0.06 ppm in middlings, <0.06 ppm in flour, <0.104 ppm in bran, <0.061
ppm in germ, and <28.069 ppm in aspirated grain fractions.  These data
also indicate that the combined residues did not concentrate in all
processed fractions of wheat but did concentrate in aspirated grain
fractions.  The calculated processing factor for wheat aspirated grain
fraction following processing of 10x-treated wheat grain is 180x.

TABLE C.1.	Summary of Method Recoveries of Diflubenzuron from Wheat.

Analyte	

Matrix	Spike level (ppm)	

Sample Size(n)	

Recoveries (%)	Mean ± Std Dev (%)

Concurrent

Diflubenzuron	Grain	0.05-0.10	6	94, 95, 100, 106, 108, 116	103 (  8

CPU

0.005	6	80, 80, 80, 100, 100, 100	90 ( 11

PCA

0.005	8	86, 94, 102, 104, 106, 106, 106, 110	102 ( 8

Diflubenzuron	Bran	0.05	2	68, 98	83

CPU

0.01	2	90, 100	95

PCA

0.005	2	96, 114	105

Diflubenzuron	Flour	0.50	2	99, 99	99

CPU

0.01	2	70, 90	80

PCA

0.005	2	96, 102	99

Diflubenzuron	Germ	0.05	2	86, 86	86

CPU

0.01	2	70, 80	75

PCA

0.005	2	96, 98	97

Diflubenzuron	Middlings	0.05	2	88, 100	94

CPU

0.01	2	110, 120	115

PCA

0.005	2	92, 96	94

Method validation

Diflubenzuron	Grain	0.05-0.50	6	87, 94, 94, 96, 98, 101	95 ( 5

CPU

0.005-0.05	6	58, 72, 80, 80, 100, 120	85 ( 22

PCA

0.005-0.10	9	112, 112, 112, 114, 116, 117, 117, 119, 119	115 ( 3

Diflubenzuron	Bran	0.05-0.50	6	70, 74, 82, 84, 85, 91	81 ± 8

CPU

0.005-0.05	6	82, 84, 94, 100, 100, 100	93 ± 8

PCA

0.005-0.05	6	100, 102, 104, 105, 105, 107	104 ± 2

Diflubenzuron	Flour	0.05-0.50	6	84, 96, 98, 98, 98, 102	97 ± 7

CPU

0.005-0.05	6	80, 100, 100, 102, 102, 108	99 ± 10

PCA

0.005-0.05	6	100, 100, 100, 101, 104, 112	103 ± 5

Diflubenzuron	Germ	0.05-0.50	6	62, 70, 75, 83, 83, 84	76 ± 9

CPU

0.005-0.05	6	80, 80, 80, 94, 100, 106	90 ± 12

PCA

0.005-0.05	6	100, 102, 103, 104, 107, 110	104 ± 4

TABLE C.2.1	Summary of Storage Conditions.

Analyte	

Matrix	Storage Temperature

(°C)	Actual Storage Duration (days)	Interval of Demonstrated Storage
Stability (Days)

Diflubenzuron	Wheat, grain	<0	105	296 in/on wheat grain

	Aspirated Grain

98

Shorts

113	None available

	Middlings

113

Flour

106

Germ

321

Bran

98	365 in/on rice bran

CPU	Wheat, grain	<0	100	348 in/on wheat grain

	Aspirated Grain

96

Shorts

101	None available

	Middlings

101

Flour

111

Germ

111

Bran

96	338 in/on rice bran

PCA	Wheat, grain	<0	100	293 in/on wheat grain

	Aspirated Grain

92

Shorts

90	None available

	Middlings

90

Flour

96

Germ

96

Bran

92	336 in/on rice bran with degradation (>45%) after 27 days

1  Storage from processing date to analysis date.  Extracts were stored
1-4 days from extraction to analysis

TABLE C.2.2	Stability of Diflubenzuron and its Metabolites in Frozen
Wheat Matrices.

Matrix	

Analyte	

Spike Level

(ppm)	

Storage Interval (Days)	Freshly Fortified Recoveries (%)

[Average]	

Stored Sample Recoveries (%)

[Average]	

Average Corrected Stored Recoveries (%)

Grain	Diflubenzuron	0.5	296	86, 86 

[86]	93, 88 

[91]	105

	CPU

348	80, 80

[80]	59, 77 

[68]	85

	PCA

293	106, 102

[104]	31, 35

[33]	32



TABLE C.3.	Residue Data from Wheat Processing Study with Diflubenzuron.

RAC	Processed Commodity	Total Rate

(lb ai/A) 	PHI 

(days)	Diflubenzuron + CPU + PCA = 

Combined Residues (ppm)	Processing Factor

(Combined Residues)

Grain	RAC	0.066	83	<0.05 + <0.005 + <0.005 = <0.06	--

	Shorts

	<0.05 + <0.005 + <0.005 = <0.06	1x

	Middlings

	<0.05 + <0.005 + <0.005 = <0.06	1x

	Flour

	<0.05 + <0.005 + <0.005 = <0.06	1x

	Bran

	<0.05 + <0.005 + <0.005 = <0.06	1x

	Germ

	<0.05 + <0.005 + <0.005 = <0.06	1x

	Aspirated grain

	2.094 + <0.005 + <0.005 = <2.104	>35.1

Grain	RAC	0.646	83	0.147 + <0.005 + <0.005 = <0.157	--

	Shorts

	0.075 + <0.005 + <0.005 = <0.085	0.54x

	Middlings

	<0.05 + <0.005 + <0.005 = <0.06	0.38x

	Flour

	<0.05 + <0.005 + <0.005 = <0.06	0.38x

	Bran

	0.094 + <0.005 + <0.005 = <0.104	0.66x

	Germ

	0.051 + <0.005 + <0.005 = <0.061	0.42x

	Aspirated grain

	27.920 + 0.144 + <0.005 = <28.069	180x

D.	CONCLUSION

The wheat processing study is acceptable pending submission of
additional storage stability data for wheat processed commodities.  The
combined residues of diflubenzuron and its CPU and PCA metabolites do
not appear to concentrate in shorts, middlings, flour, bran, and germ
processed from wheat grain treated at 1x and 10x the field rate.  The
combined residues, however, concentrated >35.1x and 180x in aspirated
grain fractions processed from wheat grain treated at 1x and 10x,
respectively.

E.	REFERENCES

DP Barcodes:	  SEQ CHAPTER \h \r 1 D253043, D253041, D244487, D251221
and D251609

Subject:	  SEQ CHAPTER \h \r 1 PP#8F4925.  Diflubenzuron (Dimilin® 2L,
EPA Reg #400-461) on Rice.

Amendments of 8/19/98, 11/20/98, 12/3/98, 1/21/99, 1/27/99 & 2/3/99.

Analytical Method for Metabolites, Revised Label, Additional Residue,
Storage 

Stability and Rotational Crop Data.

From:		G. Kramer

To:		M. Johnson/A. Sibold

Dated:		2/17/1999

MRIDs	44577601, 44689701-02, 44699201, 44692701, 44692703, 4469500102,
and 44707401



F.	DOCUMENT TRACKING

RDI:  RAB1 Chemists (8/23/06)

Petition Number(s):  PP#5E6965

DP#:  321623

PC Code:  108201

Template Version June 2005

	[Name of Active/Active Code/PC Code/Company/Company Code]

	DACO 7.4.5/OPPTS 860.1520/OECD IIA 6.5.4 and IIIA 8.5			

	Processed Food and Feed - [matrices]

	Diflubenzuron/PC Code 108201/IR-4

	DACO 7.4.5/OPPTS 860.1520/OECD IIA 6.5.4 and IIIA 8.5

	Processed Food and Feed – Wheat

PMRA Submission No.     /DP Barcode  D######     /MRID No.  

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