Document ID: EPA-HQ-OPP-2006-0181-0008
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:

46609401.  Samoil, K. (2005) Diflubenzuron:  Magnitude of the Residue on
Peanut.  Lab Project Number:  07737.01-PTR02.  Unpublished study
prepared by Interregional Research Project No. 4 (IR-4).  645 p.

EXECUTIVE SUMMARY:

In a field trial conducted during the 2001 growing season in TX,
diflubenzuron (2 lb/gal flowable concentrate (FlC)) was applied to
peanuts as three broadcast foliar applications during the crop’s
developmental stage at ~0.125 lb ai/A/application for a total rate of
0.379 lb ai/A.  Single bulk samples of untreated and treated peanuts
were harvested at commercial maturity, 29 days after the last treatment
(DAT).  The harvested peanuts were dried, shelled, and processed into
meal and refined oil using simulated commercial practices.  Prior to
analysis, peanut nutmeat, meal, and oil were stored frozen for 244-639
days; the storage intervals are supported by the concurrent storage
stability data.

The peanut nutmeat and its processed commodities (meal and refined oil)
were analyzed for residues of diflubenzuron using a high-performance
liquid chromatography (HPLC)/ultraviolet (UV) method, for residues of
4-chlorophenylurea (CPU) using an HPLC/mass spectrometry (MS)/MS or UV
method, and for residues of 4-chloroaniline (PCA) using a gas
chromatography (GC)/MS method.  These methods, which are similar or
based on method submissions previously deemed acceptable by the Agency,
were adequately validated in conjunction with the peanut sample
analyses.

The results show that combined residues of diflubenzuron, CPU, and PCA
were below the combined method LOQ (<0.06 ppm) in/on peanut nutmeat
treated at a seasonal rate of 0.379 lb ai/A.  Following processing of
the treated nutmeat, the combined residues were below the method LOQs
(<0.525 ppm) in peanut meal and <0.066 ppm (below the LOQ for
diflubenzuron and PCA, and 0.011 ppm for CPU) in peanut oil.  Processing
factors for meal and oil could not be reliably calculated due to
differing LOQs and <LOQ residues in all matrices (raw and unprocessed). 
Table 1 of OPPTS 860.1520 reports that the maximum theoretical
concentration factor (by crop) is 3x for peanuts.

STUDY/WAIVER ACCEPTABILITY/DEFICIENCIES/CLARIFICATIONS:

Under the conditions and parameters used in the study, the submitted
peanut processing study is adequate to satisfy the guideline requirement
for processed food/feed (Residue Chemistry Guideline OPPTS 860.1500). 
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)	

EP 1	Application	

Tank Mix/ Adjuvants

Method; Timing	Volume

(GPA) 2	Single Rate 

(lb ai/A)	RTI 3 (days)	Total Rate

(lb ai/A)

	Welasco, TX; 2001 (TX04)	2 lb/gal FlC	Three broadcast foliar
applications; first flower, flowering, nuts from first flowers pink
14-17	0.121-0.132	13, 35	0.379	None

1 EP = End-use Product; DIMILIN® 2L

2 Gallons per acre 

3 RTI = Retreatment Interval

B.2.	Sample Handling and Processing Procedures

Single bulk samples of control and treated peanuts were harvested at
normal crop maturity, 29 DAT, and dried in the field for 7-10 days. 
Collected samples were packed at ambient temperature and transported to
the processing facility (Food Protein Research and Development Center,
Bryan TX), where samples were processed using simulated commercial
procedures.  Samples were stored frozen (ca. -12 ºC) until processing;
processing was initiated 52 days after harvest of peanuts.  

Peanuts were oven dried in the shell until the hull moisture was 7-12%;
then shelled and the kernels oven dried to a moisture content of 7-10%
and moisture-conditioned to 12%; the kernels were then heated to 93-104
°C and pressed to obtain crude oil.  The remaining presscake (meal) was
submerged in heated hexane three times, and then warm air was forced
through to remove residual hexane.  The crude oil mixture was heated to
separate the crude oil and hexane, and the crude oil was refined
separating the refined oil and soapstock.  The refined oil was then
bleached and deodorized.  This process simulated industrial practice
with the exception of a higher dry temperature and batch processing
rather than continuous processing.

After processing, samples of meal and refined oil were collected, placed
in frozen storage, and shipped along with the RAC subsample to PTRL
West, Inc., Hercules CA for residue analysis.  All samples were stored
frozen (<0 ºC) at PTRL until homogenization and analysis.

B.3.	Analytical Methodology

Peanut samples were analyzed for diflubenzuron, CPU, and PCA using
separate methods based on those described in “Dimilin 25W, Dimilin
80WD, and Dimilin 2L in Almonds:  Magnitude of the Residue Study,”
Janine E. Rose, PTRL Study No, 723W, September 1999.  Analysis for CPU
in meal was done based on the method described in “Gas Chromatographic
Determination of Diflubenzuron Metabolite A:  Parachlorophenyl Urea in
Mushrooms,” D. Uhden, S.E. Kane, and M.A Morgenstern, Colorado
Analytical Study No. 1248, November 20, 1995.

Samples of peanut nutmeat, meal, and oil were analyzed for diflubenzuron
using an HPLC/UV method.  Briefly, residues are extracted twice with
ethyl acetate and then filtered.  Residues are evaporated to dryness and
redissolved in hexane.  Residues are partitioned with acetonitrile (ACN)
and again evaporated to dryness.  Residues are redissolved with
dichloromethane, and cleaned up on a Florisil solid-phase extraction
(SPE) column.  After clean up, residues are analyzed by HPLC on a C18
column with UV detection.  The limit of quantitation (LOQ) is 0.05 ppm
from nutmeat and oil, and 0.5 ppm for meal.  The limit of detection
(LOD) is 0.026 ppm for nutmeat, 0.072 ppm for meal and 0.016 ppm for
oil.

Samples of peanut nutmeat and oil were analyzed for CPU using HPLC/MS/MS
or HPLC/UV methods.  Briefly, samples are extracted 3-5 times with ACN,
and centrifuged or phase separated in a separatory funnel (oil).  The
supernatants (or ACN phases) 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 SPE column and silica gel SPE
column.  After clean up, residues in nutmeat and oil are analyzed by
HPLC/MS/MS or HPLC/UV.  The LOQ is 0.005 ppm for nutmeat and oil, and
the LOD was not estimated.

Samples of peanut meal were analyzed for CPU using GC/MS method. 
Briefly, 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 ACN.  The sample
is filtered and derivatized in a glass tube with heptafluorobutyric
anhydride for 10 minutes.  Residues are then analyzed by GC/MS.  The LOQ
is 0.02 ppm, and the LOD is 0.01 ppm for meal.

Samples of peanut nutmeat, meal, and oil were analyzed for PCA using a
GC/MS method.  Briefly, 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.  The LOQ is 0.005 ppm for all matrices and the
calculated LOD is 0.001 ppm.

In conjunction with the analysis of field trial and processing samples,
the above methods were validated using control (or commercially
obtained) samples of peanut matrices fortified with diflubenzuron, CPU
or PCA at 0.005-1 ppm.

C.	RESULTS AND DISCUSSION

The HPLC/UV, HPLC/MS/MS or UV or GC/MS (for meal), and GC/MS methods
used to determine residues of diflubenzuron, CPU, and PCA in/on peanut
nutmeat, meal and oil 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) are
within the acceptable range of 70-120% except for two samples of nutmeat
which reported CPU recoveries of 64% and 68% and one sample of meal
which reported CPU recoveries of 65%.  In addition, method validation
recoveries of diflubenzuron from meal at the 0.05 ppm fortification
level reported only 32 ± 14% average recovery, therefore the method LOQ
was increased to 0.5 ppm for diflubenzuron in meal.  A single low method
validation recovery (48%) was obtained for CPU in refined oil, but all
other recoveries in oil were acceptable.  Apparent residues of
diflubenzuron, CPU, and PCA were each <LOQ in/on the untreated samples
of nutmeat and meal, but residues of CPU in refined oil were 0.008 ppm
in the control sample.  Adequate sample calculations and example
chromatograms were provided.

Prior to analysis, samples of peanut nutmeat, meal, and oil were stored
frozen for 244-639 days (Table C.2.1).  To validate sample storage
conditions and intervals, a freezer storage stability study was
conducted as part of the processing study.  The results of this study
(Table C.2.2) indicate that diflubenzuron (average corrected stored
recoveries of 82-102%) is relatively stable in/on nutmeat for up to 295
days, in meal for up to 643 days, and in oil for up to 365 days.  CPU
and PCA also appear to be relatively stable (average corrected stored
recoveries of 84-99%) in meal stored for up to 645 and 488 days,
respectively, and in oil stored for up to 294 and 286 days,
respectively.  However, CPU (average corrected stored recovery of 67%)
and PCA (average corrected stored recovery of 63%) exhibited 33-37%
reduction in residues in nutmeat after 421 and 289 days of frozen
storage, respectively.

The results suggest that residues of CPU and PCA should be corrected in
nutmeat in order to determine the residue levels that were present at
the time of sample collection.  However, HED has determined that
correction of CPU and PCA residues for degradation during storage would
not have a significant effect on the results of the submitted study
because individual residues of CPU and PCA in/on treated samples were
mostly below the LOQ (<0.005 ppm).  These data are adequate to support
the storage conditions and intervals of samples from the processing
study. 

The results show that combined residues of diflubenzuron, CPU, and PCA
were below the combined method LOQs (<0.06 ppm) in/on peanut nutmeat
treated at a seasonal rate of 0.379 lb ai/A.  Following processing of
the treated nutmeat, the combined residues were below the method LOQs
(<0.525 ppm) in peanut meal and <0.066 ppm (below the LOQ for
diflubenzuron and PCA, and 0.011 ppm for CPU) in peanut oil.  The
petitioner stated that the residue value for CPU in peanut oil is
suspect because apparent residues of CPU in untreated refined oil were
0.008 ppm.  Processing factors for meal and oil could not be reliably
calculated due to differing LOQs and <LOQ residues in all matrices (raw
and unprocessed).  

TABLE C.1.	Summary of Concurrent and Method Recoveries of Diflubenzuron
and its Metabolites from Peanuts and Peanut Processed Commodities.

Analyte	

Matrix	Spike level (ppm)	

Sample size (n)	

Recoveries (%)	Mean std dev (%)

Concurrent

Diflubenzuron	Nutmeat	0.05-0.2	13	74, 76, 76, 80, 80, 86, 86, 87, 90,
92, 94, 95, 102	86 ± 9

	Meal	0.5	2	84, 87	86

	Refined oil	0.05	2	87, 99	93

CPU	Nutmeat	0.01	14	64, 68, 70, 80, 80, 90, 90, 100, 100, 100, 110, 110,
120, 120	93 ± 19

	Meal	0.02	2	65, 85	75

	Refined oil	0.05	2	88, 120	104

PCA	Nutmeat	0.005	14	95, 97, 98, 98, 101, 102, 102, 105, 106, 106, 107,
108, 108, 110	103 ± 5

	Meal	0.005	2	95, 106	101

	Refined oil	0.005	2	93, 97	95

Method Validation

Diflubenzuron	Nutmeat	0.05-1.0	9	81, 84, 85, 85, 97, 98, 102, 106, 116
95 ± 12

	Meal	0.05	3	15, 40, 40	32 ± 14

0.5-1.0	6	78, 79, 79, 82, 84, 85	81 ± 3

	Refined oil	0.05-1.0	9	77, 78, 79, 79, 80, 81, 81, 83, 84	80 ± 2

CPU	Nutmeat	0.005-0.1	9	80, 91, 92, 93, 96, 98, 100, 100, 100	94 ± 6

	Meal	0.02-0.1	9	72, 78, 86, 90, 92, 92, 95, 95, 95 	88 ± 8

	Refined oil	0.005-0.1	9	48, 74, 78, 80, 84, 86, 93, 100, 100	83 ± 16

PCA	Nutmeat	0.005-0.1	9	100, 104, 110, 111, 112, 112, 113, 115, 120	111
± 6

	Meal	0.005-0.1	9	90, 94, 94, 98, 102, 103, 104, 104, 105	99 ± 6

	Refined oil	0.005-0.1	9	96, 96, 98, 101, 102, 102, 104, 106, 111	102 ±
5



TABLE C.2.1	Summary of Storage Conditions.

Analyte	

Matrix	

Storage Temperature (°C)	Actual Storage Duration1 (days)	Interval of
Demonstrated Storage Stability (days)

Diflubenzuron	Nutmeat	<0	244	295

	Meal

629	643

	Oil

276	365

CPU	Nutmeat	<0	408	421

	Meal

639	345

	Oil

270	294

PCA	Nutmeat	<0	266	289

	Meal

254	488

	Oil

252	286

1  From harvest/processing to analysis; extracts were stored for up to 4
days before analysis.  RAC samples were processed 52-56 days after
harvest.

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

Matrix	Analyte	Spike Level

(ppm)	Storage interval (days)	Freshly Fortified Recovery (%)

[Average]	Stored Sample Recovery (%)

[Average]	Average Corrected Stored Recovery (%)

Nutmeat	Diflubenzuron	0.5	295	75, 69

[72]	65, 68

[67]	93

	CPU	0.5	421	79, 84

[81]	54, 53

[54]	67

	PCA	0.1	289	70, 71

[71]	41, 49

[45]	63

Meal	Diflubenzuron	0.5	643	96, 86

[91]	84, 65

[75]	82

	CPU	0.5	645	110, 106

[108]	90, 114

[102]	94

	PCA	0.1	488	108, 106

[107]	91, 88

[90]	84

Oil	Diflubenzuron	0.5	365	115, 113

[114]	114, 117

[116]	102

	CPU	0.5	294	90, 81

[86]	82, 87

[85]	99

	PCA	0.1	286	68, 71

[70]	65, 65

[65]	93

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

RAC	Processed Commodity	Total Rate

(lb ai/A) 	PHI 

(days)	Diflubenzuron + CPU + PCA = 

Combined Residues (ppm)	Processing Factor

(Combined Residues)

Peanut	Nutmeat (RAC)	0.379	29	<0.05 + <0.005 + <0.005 = <0.06	--

	Meal

	<0.5 + <0.02 + <0.005 = <0.525	NC 1

	Refined oil

	<0.05 + 0.011 + <0.005 = <0.066	NC

1  Processing factors could not reliably be calculated (NC) because most
residues were below the respective LOQ for that matrix.

CONCLUSION

The submitted peanut processing study is adequate.  The results show
that combined residues of diflubenzuron, CPU, and PCA were below the
combined method LOQs (<0.06 ppm) in/on peanut nutmeat treated at a
seasonal rate of 0.379 lb ai/A.  Following processing of the treated
nutmeat, the combined residues were below the method LOQs (<0.525 ppm)
in peanut meal and <0.066 ppm (below the LOQ for diflubenzuron and PCA,
and 0.011 ppm for CPU) in peanut oil.  The petitioner stated that the
residue value for CPU in peanut oil is suspect because apparent residues
of CPU in untreated refined oil were 0.008 ppm.  Processing factors for
meal and oil could not be reliably calculated based on the results of
this study.

E.	REFERENCES

None.

F.	DOCUMENT TRACKING

RDI:  RAB1 Chemists (8/23/06)

Petition Number(s):  PP#5E6967

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]

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321623/MRID No. 46609401					Page   PAGE  8  of   NUMPAGES  8