Pyriproxyfen Papaya Draft Protocol

IR-4 NATIONAL PESTICIDE CLEARANCE PROTOCOL Page 1
PYRIPROXYFEN/PAPAYA PR No.: xxxxx

Date: 5/12
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THIS PROTOCOL COPIED ON COLORED PAPER IS AN EXACT COPY OF THE ORIGINAL
1. PROJECT TITLE:

PYRIPROXYFEN: Magnitude of the Residue on PAPAYA

2. JUSTIFICATION AND OBJECTIVES:

IR-4 has received a request from USDA-FAS to assist for the minor use of pyriproxyfen on PAPAYA in Malaysia as part
of the ASEAN tropical fruit residue project

To establish an MRL, it is required that the magnitude of the residue in or on the commodity.Be determines. The
purpose of this study is to collect and analyze treated and untreated residue samples from appropriate field sites
according to the application parameters requested to provide JMPR with residue chemistry data to support a pesticide
tolerance.or CODEX MRL.

To determine the magnitude of residues of total Pyriproxyfen in or on PAPAYA, this protocol will be employed using
appropriate Standard Operating Procedures (SOP's) and will be conducted under provisions outlined in IN
ACCORDANCE WITH EPA GOOD LABORATORY PRACTICE STANDARDS and consistent with the provisions
outlined in the Organization for Economic Cooperation and Development (OECD) Series on Principles of Good
Laboratory Practice and Compliance Monitoring.

3. SPONSOR/TESTING FACILITY NAME, ADDRESS AND PHONE:
Fertilizer and Pesticide Authority of the Philippines, Department of Agriculture, FPA Building B. A. I Visayas Ave.
Dillman, Quezon City 920-8173

4. STUDY DIRECTOR
1
:

Dr. Michael P. Braverman, IR-4 Project Headquarters, 500 College Road East, Suite 201 W, Princeton, NJ 08540, (732)
932-9575 extension 4610, FAX# (609) 514-2612, E-mail: [email protected]

5. PROPOSED DATES: 6. PROPOSED TEST SITES:
Experimental Start : August 2013 Field sites: Refer to Section 23
Experimental Termination: December 2014 Laboratory: Refer to Section 24
Study Completion: June 2015

7. STUDY AUTHORIZATION:

Dr Amelia Tejada, Sponsor/Faciltiy Management / Date
[email protected]

Michael P. Braverman / Study Director / Date
[email protected]

7.1 STUDY DIRECTOR INITIALS: _______________

.
Date: 5/12
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7.2 QUALITY ASSURANCE PROTOCOL /STUDY PLAN REVIEW:

This protocol/study plan has been reviewed by quality assurance.

Ms. Jerolet Sahagun, Quality Assurance / Date
[email protected]

8. GOOD LABORATORY PRACTICE COMPLIANCE:

The appropriate cooperative testing facility (field and laboratory) will be responsible for certifying that its portion of the
study will be conducted in accordance with Good Laboratory Practice (GLP) Standards.

A statement of compliance, together with any GLP deviations will be signed and submitted by the appropriate Research
Directors in their report or data package.

9. QUALITY ASSURANCE:

Quality Assurance duties and responsibilities will be in conformance with Good Laboratory Practices A Quality
Assurance Statement will be submitted in the final report and shall include the date inspections were made and date(s)
the findings were reported to the Study Director and management.

10. TEST SYSTEM/CROP:

PAPAYA - Use a commercial variety. Report: variety if available.

Field trials will be conducted at the appropriate sites to support the establishment/maintenance of a national residue
tolerance, see Section 23 for these assignments. Refer to Section 11.4 for requirements to differentiate multiple trials
by the same field researcher.

11. TEST SYSTEM DESIGN and STATISTICAL METHOD:

11.1 Each test site will consist of one untreated and one treated plot . Each plot will consist of a minimum of 6 trees, 8
trees are preferable. Consider lager plots for the decline study since multiple harvests will be required..

The individual plots shall be of adequate size to ensure that no more than 50% of the harvestable crop in the sampled
area will be needed to provide the necessary plant material. See Parts 17 & 18 for requirements for residue sampling.

11.2 Employ adequate buffer zones (minimum 20 meters) between each of the plots to prevent contamination but a
minimum of 30 meters is strongly preferred. When plants are used as a buffer between the untreated and treated plots,
a lower distance is needed to prevent contamination, but the minimums indicated above must be observed. If another
study using a test substance with the same active ingredient is being conducted at the same research site, the
untreated plot from one study must be separated from the treated plot(s) of the other by the appropriate buffer zone
indicated above.

11.3 If this pesticide use is not registered on this crop, the treated crop must be destroyed or handled in such
a way that it is not consumed as a human food or animal feed in a manner consistent with local government
laws.
Date: 5/12
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11.4 If a Field Research Director is assigned more than one trial in this study, the following requirements
must be met:
An independently prepared tank-mix must be used in each trial. (Not applicable for granular applications)
Also:
Preharvest interval (PHI) <10 days: Choose one option from any 3 of the 5 sets below (for a minimum of 3
criteria met).
Preharvest interval (PHI) 10 days: Choose one option from any 2 of the 5 sets below (for a minimum of 2
criteria met).
(If the protocol specifies a PHI of 101 days, and samples are collected at a 9-day interval, then one option
from any 2 of the 5 sets must be used. This is also true in a decline trial if the standard PHI is at least 10
days.)
Set Option Description
1 A
Spray volume must vary by at least 25% of the lower volume (minimum 10 GPA
difference)
Example 1, Trial A has a volume of 20 GPA and Trial B has a volume 30 GPA
Example 2, Trial A has a volume of 60 GPA and Trial B has a volume 75 GPA
The trial with the lowest spray volume for the first application must remain the lowest for
each application; the trial with the highest must remain the highest for each, and so on
2 A
Different adjuvant class:
Crop oil concentrate or Nonionic surfactant (non-silicone) or Silicone surfactant
(Do not contradict instructions on the label of either the adjuvant or the test substance)
3
A
Different foliar application type: foliar directed or foliar broadcast
(Do not use this option if the label instructions for this commodity will specify one type
or the other)
B
Different types of application equipment be used in each trial (for example, tractor-pulled
boom sprayer, tractor-pulled spreader, airblast sprayer, axial fan orchard sprayer, proptec
sprayer, cannon mist sprayer, tower sprayer, over-row sprayer, tunnel sprayer, backpack
sprayer, waist pack sprayer, hand gun, hand-held spreader, or shaker can)
C
Different people make the applications, using hand-held equipment such as backpack
sprayers, waist pack sprayers, hand guns, hand-held spreaders, or shaker cans
D
Different spray droplet size (fine, medium, coarse, very coarse, or extra coarse)
This may be accomplished by changing nozzles and/or by changing spray pressure
Document in the Field Data Book the droplet size that results from the pressure and
nozzles used in the trial (nozzle catalog may be used as a reference)
E
Different granular application type: broadcast or banded (only if label supports both
types)
F Different incorporation method for soil-applied test substance: mechanical or irrigation
G
Different band width for soil applications: band width must vary by at least 50% of the
lower width
4
A
Different crop variety (different size at maturity, rough vs. smooth surface, different
amount of foliage shielding the commodity, etc.)confirm with Study Director if this
option will be chosen
B
Different irrigation type (drip or furrow or sprinkler/over-the-top)
(Irrigation must be applied at least once after each application, but over-the-top irrigation
must not be applied within one hour of an application, and irrigation is not needed
following the last application if samples are to be collected on the same day)
C
Different planting arrangement for annual crops:
single row beds or multi-row beds (two or more rows on each bed)
Date: 5/12
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D
For test substances that must be applied through drip irrigation: surface drip line or
buried drip line
E One trial shall have trellised plants and the other shall not
F Different training system for fruit trees (for example, central leader or open center)
G
Different maturity of trees or bushes in fruit and nut studiesyoung trees or bushes in
one trial and mature trees or bushes in the other; minimum 5 year age difference
5
A Trial sites must be separated by at least 20 miles (32 km)
B First application in each trial is separated by at least 30 days
C
Different soil series, type, or texture (only in trials in which applications are made to the
soil)
If these criteria cannot be met to separate multiple trials, the Field Research Director should contact the
Study Director to discuss possible alternatives that can be amended to the protocol. Trials conducted in
different calendar years are exempt from these requirements.

11.5 Mark plots with identifiable markers containing at minimum the Field ID number and treatment
number or treatment name that will persist for the duration of the field research trial or that can be readily
replaced.

11.6 This study is not designed for statistical evaluation of field data.

12. TEST SITE PREPARATION:

Select a test site that has been maintained following good local agricultural practices for the production of
PAPAYA including fertilization, irrigation, if necessary and available, and other practices that ensure
commercially acceptable crop production.

The test site will have a known pesticide and crop treatment history of a minimum of 1 year and preferably
3 years.

13. TEST/CONTROL SUBSTANCE:

Use the Admiral 10EW (Pyriproxyfen 10% Japanese Registration No. 19109?????) of pyriproxyfen (EPA
Reg. No. 59639-96, CAS# 95737-68-1) that has been characterized to meet GLP standards. IR-4
Headquarters personnel will arrange procurement of GLP test substance from the Registrant. Upon receipt,
document the lot/batch number, condition, quantity received and if GLP characterized. Temperature
monitoring should begin within 2 days of receipt of the test substance, regardless of where it is held or
stored.

Contact the Study Director if there are any concerns regarding the GLP status, labeled identification, etc.
of the test substance.

The registrant will provide a copy of the Certificate of Analysis to IR-4 Headquarters.

Store the test substance in a secure, clean, dry area and document storage temperatures.

Test substance container(s) must be retained until the final study report is completed.

Study completion can be confirmed by contacting the Study Director. Alternatively, some registrants will
archive the test substance containers. If test substance containers are shipped to another location, the
shipment must be conducted in accordance with local regulations. See shipping documents for directions
Date: 5/12
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for return of the test substance; if none are given, contact the registrant representative: Yoshihiro
Nishimoto, Sumitomo Chemical, Phone: +81-3-5543-5692, Mobil: +81-90-5065-2016, E-mail:
[email protected]

The registrant will archive a retention sample of the test substance.

Control substances are not relevant to this study.

14. TEST SUBSTANCE APPLICATION:

14.1 Simulate commercial application practices by applying the test substance in a manner that
represents a major application technique that is used by area commercial growers, while following the
directions specified in Section 15.
- Use application equipment that will provide uniform application of the test substance and result in
adequate canopy penetration and coverage.
- The test substance, if applied in a mixture, must be applied to the test system within 2 hours of mixing.
- Each field trial requires a unique spray mixture. Do not use the spray mixture from one field trial on
another field trial.
- Agitate the test substance during the application, if practical, to ensure that it is well mixed.

For foliar directed applications (generally used for insecticides and fungicides), do not proportionally
reduce the application rate (the amount of active ingredient applied per acre). Direct the entire per-acre rate
onto the crop. If row widths in the research plots are greater than local commercial practices, then the
application rate should be calculated using a local commercial row width. Contact the Study Director if
guidance is needed.

14.2 Full Calibrations for output and speed must be performed to ensure accurate delivery.
A calibration consists of a minimum of three consecutive, documented checks for nozzle or hopper output
and speed (equipment or walking speed). (When the output of an airblast sprayer is calibrated or rechecked,
it is not necessary to record the outputs of individual nozzles.) The variation of the total output recorded for
any one of the three checks must not be greater than 5% from the mean for the full calibration.

Discharge/Output Calibrations must be performed:
Just prior to the first application of test substance, completely calibrate
2
.

Another complete calibration must be performed and documented when application parameters or
equipment components have changed between applications. Recalibration is required after any of the
following have changed: application type; intended nozzle or hopper output; nozzle size or type, or other
equipment that may affect the output etc. A recalibration is required even if the pressure (intended nozzle
output) has been changed back to the pressure used at the initial calibration. It is not necessary to fully
recalibrate when CO
2
tanks are changed, or when equipment is transported offsite or cleaned, or if nozzles
are removed and then placed back on (even if other nozzles have been used in the interim, unless the
pressure has also been changed); however a recheck must take place prior to the next application. If the
recheck is out of specification (see paragraphs below) a recalibration is required. Use equipment logs to
document changes in the equipment parameters.

2
Just prior includes the day prior to the application, but calibration on the day of use is preferred.
Date: 5/12
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Rechecking the output, at a minimum, is necessary for multiple applications, as long as parameters have
not changed. A single output check may be conducted to confirm consistent delivery (+5% of the last
complete calibration) just prior to subsequent applications.

The equipment must be completely recalibrated if:
- a recheck results in an output that differs from the mean of the complete calibration by greater than 5%
- the variation of any nozzles output from the mean output of all of the nozzles during the same run is
greater than 5% (this statement does not apply to airblast sprayers)

To minimize the occurrence of application rates that fall outside the protocol range, calculations for
the amount of test substance to be applied that are based on the discharge rate should be performed
using mean output calculated from the most recent complete calibration data (mean of three output
checks), not on single-output recheck results. The use of a target output rather than the mean output
may be used in the calculations made prior to the application; however a complete calibration must
be conducted just prior to each use of a target output, and the mean output must be within 5% of the
target output. Using a target output rather than a mean output increases the probability that an
application rate deviation will occur. Verification of the amount of test substance that has been
applied will always be calculated using the most recent complete calibration data.

A speed calibration must also be performed prior to the first test substance application. Conduct speed
calibration in an area adjacent to the test plot, or on similar terrain. Speed rechecks are required for
multiple applications on different days. Speed should be recalibrated if a major equipment change has
been made, such as from a tractor-pulled sprayer to a backpack sprayer. (When a handgun is used to spray
tree fruits or nuts, and each tree is sprayed for a predetermined time from a stationary position, a speed
calibration is not required.)

Complete calibration data from another trial (performed on the day of or day prior to the application in this
trial) may be used. However, a recheck (single output check and speed recheck) must be performed just
prior

to the application in this trial, but subsequent to any other applications with the application equipment.
If more than one field trial in this study has been assigned to the same Field Research Director, it is not
necessary to perform separate output rechecks for applications made on the same day in the respective
trials, and separate speed rechecks are not required unless the treated plots are located on separate farms.

14.3 Actual Application Rate: Record actual application pass-times in the Field Data Book and verify the accuracy of
the application against the protocol rate. The application is considered acceptable if the accuracy is within -5%
and +10% of the target rate specified in Section 15. If the application did not meet this range, the Study
Director must be notified of this deviation before proceeding with this trial.

The submitted Field Data Book shall contain the original calibration data or a true copy of all complete calibrations
referenced, along with the original data from the rechecks performed for this trial.

15. APPLICATION TREATMENTS AND TIMING:

Trt#

Treatment
Target Rate
of active ingredient
Target Rate
of formulated product*
Application
Type
Spray Volume
Range**
01 Untreated Not Applicable Not Applicable Not Applicable Not Applicable
02 PYRIPROXYFEN 50 grams ai/hectare 550ml /hectare Foliar 100 to 200L/Ha)
*The nominal formulation concentration of the test substance will be used in calculating application rates (see Section 13
for the nominal concentration).
**GPA=gallons per acre, L/Ha=liters per hectare
Date: 5/12
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Make 2 applications at 14(+2) day intervals with the last application TBD (+2 days) before harvest.

16. SUPPLEMENTAL CROP TREATMENTS:

Protect the integrity of the field trial by managing pests that may cause significant damage to the test crop. Only
registered maintenance pesticides should be used; apply according to labeled directions. Make identical applications to
the untreated and treated plots.

Consult with Study Director if no registered pesticides are available to control the pests. Document all supplemental
crop treatments. DO NOT USE pesticides that are similar to the test substance or other chemicals that might interfere
with analysis of the test substance. If unsure, contact the Study Director.

17. RESIDUE SAMPLE COLLECTION:

Collect two samples from each plot. Each sample should be representative of the entire plot (except plot ends). At 14
days (2) days after last application, starting with the untreated plot, collect at least 8 fruits per sample from a minimum
of 4 trees. Each sample should be collected during a separate run through the entire plot. Take fruits from all
quadrants of each tree, high and low areas, fruit exposed and sheltered by foliage. Avoid sampling from treated end
trees.

In trials in which treated samples shall be collected on the day of the last application (0-day PHI), the untreated samples
may be collected prior to handling the test substance that day to make the last application.

Follow proper handling practices with clean or gloved hands and clean tools to prevent transfer of pesticide residue
from one sample to another. If practical, complete harvest and sample preparation for the untreated plot(s) before
proceeding to the treated plot(s).

Place all samples in plastic-lined cloth bags. Identify each sample bag** with correct Field ID number, Test Substance
(common chemical name and formulation), complete sample ID (see Section 18) and harvest/sampling dates. See
Section 19 for residue sample handling directions.

**When using IR-4 plastic lined cloth residue sample bags, complete attached sample tag as follows:
Field ID Number; Crop Fraction; Test Substance (enter the chemical name listed in Section 15); Sample ID; Trt#;
Harvest Date; Sample Date; Field Research Director (enter name and telephone number).

18A. FIELD RESIDUE SAMPLE INVENTORY: All trials EXCEPT PH01

SAMPLE
ID
TRT# TREATMENT
DAYS AFTER
LAST APPLIC.
MINIMUM
SAMPLE SIZE
CROP
FRACTION
A 01 Untreated NA 8 fruits / 2 kg. Fruit
B 01 Untreated NA 8 fruits / 2 kg. Fruit
C 02 PYRIPROXYFEN 8 fruits / 2 kg. Fruit
D 02 PYRIPROXYFEN 8 fruits / 2 kg. Fruit

Date: 5/12
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18B. FIELD RESIDUE SAMPLE INVENTORY: For decline trial PH01
SAMPLE
ID
TRT# TREATMENT
DAYS AFTER
LAST APPLIC.
MINIMUM
SAMPLE SIZE
CROP
FRACTION
A 01 Untreated NA 8 fruits / 2 kg. Fruit
B 01 Untreated NA 8 fruits / 2 kg. Fruit
C 02 PYRIPROXYFEN 0 8 fruits / 2 kg. Fruit
D 02 PYRIPROXYFEN 0 8 fruits / 2 kg. Fruit
E 02 PYRIPROXYFEN 1 day 8 fruits / 2 kg. Fruit
E 02 PYRIPROXYFEN 1 day 8 fruits / 2 kg. Fruit
F 02 PYRIPROXYFEN 3 (+1 days) 8 fruits / 2 kg. Fruit
G 02 PYRIPROXYFEN 3 (+1 days) 8 fruits / 2 kg. Fruit
H 02 PYRIPROXYFEN 7 (+1 days) 8 fruits / 2 kg. Fruit
I 02 PYRIPROXYFEN 7 (+1 days) 8 fruits / 2 kg. Fruit
J 02 PYRIPROXYFEN 14 (+2 days) 8 fruits / 2 kg. Fruit
K 02 PYRIPROXYFEN 14 (+2 days) 8 fruits / 2 kg. Fruit
L 02 PYRIPROXYFEN 28 (+2 days) 8 fruits / 2 kg. Fruit
M 02 PYRIPROXYFEN 28 (+2 days) 84 fruits / 2 kg. Fruit

19. RESIDUE SAMPLE HANDLING AND SHIPMENT:

After residue sample collection, store samples in a freezer. If the samples cannot be placed into a freezer within
approximately one hour, use an appropriate method of cooling samples in order to maintain integrity.

Sample handling and storage methods can be outlined generally in SOP's, but describe methods fully in the Field Data
Book.

For pre-shipment storage, the samples will be held frozen at temperatures generally less than -18 C (0

F), allowing
for normal variations of less than 24 hours duration due to freezer cycling, sample movement, etc. If the analytical
laboratory is close enough to the field site to permit delivery of the samples by field personnel on the day of sampling,
then pre-shipment frozen storage is not required.

Freezer logs will be used to document all sample additions to and removals from storage. All on-site storage
temperatures will be monitored and documented.

Shipment of frozen samples will be by freezer truck or express shipment. Shipments sent via express shipment
(overnight carriers such as Federal Express or Airborne) will require the addition of quantities of dry ice sufficient to
maintain sample integrity while in transit to the laboratory. Document the notification made to the sample destination by
use of e-mail, fax, telephone log, Field Data Book communication note, etc.

Insert a true copy of Field Data Book Part 8B and a blank copy of Field Data Book Part 8C (Sample Arrival
Check Sheet) into each box or container used to ship sample bags.

Date: 5/12
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For analysis, send samples to:

Dr. Joyce Masiglat-Sales, Chief, Quality Evaluation Division, Food Development Center [email protected]

20. FIELD DOCUMENTATION AND RECORD KEEPING:

All operations, data and observations appropriate to this study should be recorded directly and promptly into the
IR-4 Field Data Book.

The content of the Field Data Book should be sufficiently detailed to completely reconstruct the field trial. At a
minimum, collect and maintain the following raw data:

20.01- Names of all personnel conducting specific research functions
20.02- Amendments and deviations from protocol and standard operating procedures (including copies of signed
protocol changes received prior to submission of the Field Data Book to the Regional Field Coordinator).
20.03- Test site information
20.04- Plot maps
20.05- Test substance receipt, use and container/substance disposition records
20.06- Test substance storage conditions (including temperatures)
20.07- Data regarding calibration and use of application equipment
20.08- Treatment application data
20.09- Crop maintenance pesticides and cultural practices, test plot history, and soil information. (Reporting soil
information from typical farm service soil analysis labs, or past history for the farm, or from official
documents, such as the SCS Soil Survey for the test plot area is adequate for this study. The nature of this
study is such that soil characteristics do not need to be determined under GLP standards.)
20.10- Residue sample identification, collection, storage conditions and handling (Weight measurements are
considered estimates for the samples collected from field or processing trials, and the scales/balances used
for this purpose do not need to be maintained in strict adherence to GLP.)
20.11- Residue sample shipping information
20.12- Description of crop destruction, or explanation for lack of destruction
20.13- Meteorological/Irrigation records (temperature/humidity records for greenhouse trials)--required from
planting of annual crops or for a minimum of one month prior to the first application onto perennial crops,
until last residue sample collection. These records do not need to be determined under GLP standards.
20.14- Pass times (if applicable) and other data to confirm amount of material applied to plots
20.15- Equipment maintenance records with indication of routine vs. non-routine nature of maintenance
20.16- Other applicable data requested in the IR-4 Field Data Book necessary for confirmation that the study was
conducted in accordance with the protocol.
Date: 5/12
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Compliance with GLPs is not required for the collection of data associated with crop phytotoxicity.

21. PROTOCOL/SOP MODIFICATIONS - FIELD RESEARCH:

Consult with the Study Director to discuss desired changes in the protocol prior to occurrence. If appropriate, an
amendment will be issued.

Any deviations from the protocol will require the Field Research Director to complete a written report outlining the
changes. Provide this report to the Study Director promptly (e.g. within 14 days of occurrence or recognition) for
review and signature.

All deviations from the approved SOP's also require documentation and approval by the Study Director.

22. FIELD RESEARCH REPORT/ARCHIVING:

The Field Research Director will forward the completed originals of the IR-4 Field Data Book and other raw data to
archives of the facility management as soon as possible after the shipment of residue samples. Scan a copy of the field
data notebook and send to the Study Director. The Field data Notebook and all raw data must be reviewed by Quality
Assurance.

The Field Research Director will maintain a complete certified true copy of these field documents.

23. FIELD PERSONNEL / ID NO.

If a Field Research Director is assigned more than one trial in this study, refer to Section 11.4 for requirements to
differentiate the trials.

Field Research
Director
Field ID
NO.
Test Crop
TBD

TBD.13-
PH01*

PAPAYA

TBD.13-
PH02

PAPAYA

TBD.13-
PH03
PAPAYA

TBD.13-
PH04

PAPAYA

TBD.13-
PH05

PAPAYA

TBD.13-
PH06
PAPAYA

*Trial PH01 will be the decline study

Date: 5/12
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24. LABORATORY PERSONNEL/ID NO.:

LABORATORY RESEARCH DIRECTORs/TESTING LABORATORIES

PHILIPPINESE LAB ID NO.: TBD.13-PHR01

25. LABORATORY SAMPLE INVENTORY:

Treated and untreated samples of PAPAYA will be received from each of the field sites in Section 23.

Notify the Field Research Director of sample receipt.

26. LABORATORY SAMPLE IDENTIFICATION:

Each sample (raw commodity, crop fractions, storage stability, method validation, etc.) is to be assigned a unique
laboratory sample number by the laboratory personnel.

A cross-reference must be maintained between the assigned laboratory sample number and the identification utilized in
the Residue Sample Shipping and Identification Sheet.

27. LABORATORY SAMPLE STORAGE/PREPARATION:

Store samples in a limited access area at temperatures that will maintain frozen sample integrity (generally less than -
20C), until extraction.

The samples may be stored whole or ground, depending on the standard procedure of the analytical laboratory.
However, for pyriproxyfen, it is recommended samples not be ground until extraction for analysis.

Do not composite samples.

The entire sample provide from the field must be ground, if sample is too large to be manageable then contact the
Study Director for appropriate subsampling to assure the representative nature of the sample obtained in the field is
maintained by the laboratory procedure. Generally, sample extracts should be stored at < 4C for no longer than 14
days before analysis.

Storage stability of extracts must be demonstrated if extracts are not analyzed on the same day as they are obtained.

Concurrent fortifications may be used to show extract storage stability, as long as the extracts from the concurrent
fortifications have been stored at least as long as the extracts obtained from the weathered samples.

Contact the Study Director if samples extracts are stored greater than 14 days prior to analysis.

All storage temperatures, conditions and location of sample storage are to be monitored and documented.

28. LABORATORY REFERENCE SUBSTANCE:

Date: 5/12
...............................................................................................................................
Obtain the laboratory reference substance(s), Pyriproxyfen, from the Registrant. Contact Yoshihiro Nishimoto,
Sumitomo Chemical, Phone: +81-3-5543-5692, Mobil: +81-90-5065-2016, E-mail: nishimotoy.sc.sumitomo-chem.co.jp
to procure the proper material.

Document the date the analytical standards are received, the source, stated purity, storage conditions, and expiration
date.

Use only reference standards that have been characterized to meet GLP standards.

Archival and characterization of the reference substance (purity, identity, stability and solubility) is the responsibility of
the registrant.

29. ANALYTICAL METHODOLOGY:

29.1 REFERENCE METHOD:

Determination of Pyriproxyfen Residues in Apples, Pears, and Citrus Fruits Method: RM-33P-1-3A, Valent U.S.A.
Corporation, dated Oct. 9, 1998.

The method may be modified to utilize a QuEChERS method S. Lehotay, A. De Kok, M. Hiemstra and P. van
Bodegraven. 2005. Validation of a Fast and Easy Method for the Determination of Residues from 229 Pesticides
in Fruits and Vegetables Using Gas and Liquid Chromatography and Mass Spectrometric Detection. Journal of
AOAC International Vol 88, No. 2, 2005 page 595-614.

29.2 REFERENCE METHOD MODIFICATIONS/METHOD VALIDATION:

The above listed Reference Method(s) may be modified if needed for the test matrix. The Reference Method, along
with any modifications must be validated on each crop fraction prior to residue sample analysis of that crop fraction.

To validate the method, fortify some of the control samples in triplicate with pyriproxyfen at a minimum of 3
concentration levels, lowest level of method validation (0.02 ppm or lower), 0.2 ppm, and 2 ppm).

A minimum of 6 fortification samples (recovery spikes) at the lowest level of method validation (LLMV) is required
prior to completion of the analytical phase of the study. The acceptable recovery range is 70-120%. Documented
approval from the Study Director is needed for recoveries outside of this range.

Document the exact procedures for sample analysis. This validated step-by-step Working Method should
incorporate all changes from the Reference Method. Provide the Study Director with a copy of this Working
Method and results of method validation prior to treated sample analysis.

If the Working Method has been used successfully on the test matrix or a similar matrix, the Study Director may
waive the requirement for method validation. Contact the Study Director for details.

29.3 SAMPLE ANALYSIS:

Samples will be analyzed for the residues of pyriproxyfen following the Working Method. For each field trial
associated with this study, analyze at least one untreated and all treated residue samples for each matrix.

Contact the Study Director if residues above the lowest level of method validation for each matrix are detected in
the untreated samples. Any changes or modifications to the Working Method require Study Director approval.
Date: 5/12
...............................................................................................................................
Whenever possible, notify the Study Director prior to occurrence. Any change or modification to the Working
Method should be documented in the raw data and discussed in the final report.

A typical analytical set (or run) should consist of calibration standards, untreated sample(s), concurrent recovery
sample(s), and treated sample(s). Each analytical set must begin and end with a calibration standard. Additional
calibration standards should be injected with sample analysis to ensure goodness of fit to the standard curve.

Over the course of residue sample analysis, adequate concurrent recovery samples that bracket the actual residues
should be analyzed. [Concurrent recovery samples must be run at a minimum of two levels over the course of the
sample analyses, with at least one concurrent recovery sample below the lowest sample residue (or at the lowest
level of method validation) and at least one concurrent recovery sample higher than the highest sample residue found
in the treated samples.] At least one concurrent fortification sample should be analyzed per analytical set.

The Study Director should be immediately notified if concurrent recoveries deviate from the acceptable recovery
range of 70% to 120%. All efforts will be made to resolve existing recovery problems before continuing forward with
additional analytical sets.

If residues in samples are above the highest Working Method validation concentration, additional recovery samples
at levels above actual residues must be run in triplicate as soon as practical. A minimum of 6 fortification samples
(recovery spikes) at the lowest level of method validation (LLMV) is required prior to completion of the analytical
phase of the study.

Treated samples may be analyzed using a screening run prior to analysis of treated samples using the working
method, if the procedure is covered in the laboratory SOPs and the working method for the study. The peak areas of
the treated samples and highest standard from any screening run will not be quantified or reported. (Any data, such as
chromatograms, generated during screening run(s) will be kept.)

29.4 STORAGE STABILITY ANALYSIS:

As soon as possible after receipt of samples, a minimum of six subsamples of all available crop fractions of the
control shall be fortified with pyriproxyfen at 0.2 ppm.

If samples are analyzed within 2 weeks then SS data may not be needed. If not needed then have lab set up SS
samples but contact SD prior to analysis.

Three samples of each analyte and crop fraction will be analyzed after the appropriate storage period (greater than or
equal to the longest interval that an individual sample was stored between collecting the sample in the
field/processing facility and analysis, unless otherwise specified by the Study Director). The remaining samples
will be retained for long-term storage.

If analysis of treated samples is completed within 30 days of harvest analysis of storage fortification samples may not
be required. If appropriate, contact Study Director.

29.5 STATISTICAL METHOD(S):

Utilize regression analysis to determine the linearity of the standard curve (r
2
) or the goodness of fit if the standard
curve is non-linear. Criteria for acceptance of the standard curve(s) or other statistical methods shall be determined
by Laboratory Research Director and documented in the raw data.

30. DISPOSITION OF SAMPLES:

Date: 5/12
...............................................................................................................................
A minimum of 100 g or all (if less than 100 g) of each of the remaining frozen treated and untreated crop samples is to
be retained for at least 12 months after submission of the report to JMPR.

Long term fortified storage study samples shall be retained for a period of 1 to 5 years, as appropriate, after submission
of the final report.

Sample extracts can be disposed of after data analysis.

The Study Director is to be contacted prior to discarding samples.

31. LABORATORY PROTOCOL/SOP MODIFICATIONS - LABORATORY RESEARCH:

Consult with the Study Director regarding desired changes in the protocol prior to occurrence. If appropriate, an
amendment will be issued. Any unauthorized changes to the protocol will require the Laboratory Research Director to
complete a written report outlining the changes.

This report should be provided to the Study Director promptly (e.g. within 14 days of occurrence) for review and
signature.

All deviations from the approved SOP's also require documentation and approval by the Study Director.

32. LABORATORY DOCUMENTATION AND RECORD KEEPING:

All operations, data and observations shall be recorded in the analyst's notebook and log books, which must be signed
and dated on date of entry.

At a minimum, collect and maintain the following raw data:
32.01 - Analytical standard(s) receipt, use and disposition records
32.02 - Analytical standard(s) storage conditions
32.03 - Analytical standard(s) dilution calculations and preparation records
32.04 - Sample storage conditions and locations
32.05 - Calculation work sheets
32.06 - All chromatograms, including those that are not reported
32.07 - Chain of custody records
32.08 - Deviations from protocol, Working Method and/or standard operating procedures
32.09 - Name of personnel conducting specific research functions
32.10 - Sample analysis worksheets
32.11- Storage stability fortification records
32.12 - Concurrent recovery fortification records
A study file shall be developed and maintained by the Laboratory Research Director in conjunction with the analysis. It
will contain a copy of the protocol, all pertinent raw data, documentation, records, correspondence, and the final
analytical summary report. In addition, records of equipment maintenance and calibrations will be kept and periodically
archived.

33. LABORATORY RESEARCH REPORT:
Date: 5/12
...............................................................................................................................

The analytical summary report sent to IR-4 HQ shall contain, but not be limited to:
33.01 - Applicable method validation data
33.02 - Applicable storage stability data
33.03 - Residue levels for control and treated samples with concurrent fortified recoveries
33.04 - Complete copy of the analytical Working Method
33.05 - Any modifications or deviations from the protocol and/or Working Method
33.06 - Completed IR-4 residue data reporting form or appropriate reporting form which includes information listed on
the IR-4 generic residue data reporting form
33.07 - A minimum of 10 representative chromatograms of treated samples (if fewer than 10 submit all), a minimum of
three chromatograms each of control and fortified control samples, chromatograms (one of each
concentration) for at least one set of calibration standards for each compound analyzed, and any
chromatograms of samples with unusual or inconsistent results
33.08 - Summary of quantitative data associated with samples and spike recovery samples should be provided (e.g.
peak heights, injection volumes, sample sizes, final volumes, etc.)
33.09 - Clearly presented example calculations or statistical evaluations
33.10 - Discussion of results (including purpose of method modifications, sample storage conditions, etc.)
33.11 - Summary data associated with calibration standards (dilution and use records, calibration curves, etc.)

34. LABORATORY ARCHIVES:

When the final analytical summary report is completed and sent to the sponsor representative, all original raw data
including a "true copy" of the final analytical summary report shall be secured in the archives of the Laboratory
Research Director/Testing Facility.

TITLE: PYRIPROXYFEN MAGNITUDE OF THE
RESIDUE ON PAPAYA

PR# TBD

STUDY DIRECTOR
Michael Braverman
732-932-9575 ext 4610
[email protected]

Pyriproxyfen Papaya Draft Protocol

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IR-4 NATIONAL PESTICIDE CLEARANCE PROTOCOL Page 1

PYRIPROXYFEN/PAPAYA PR No.: xxxxx

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